@article {35834, title = {Bio-based production of uroporphyrin in Escherichia coli}, journal = {Synthetic Biology and Engineering}, volume = {2}, year = {2024}, pages = {10002}, author = {B. Arab and A. Westbrook and M. Moo-Young and Y. Liu and C. P. Chou} } @article {35835, title = {High-level bio-based production of coproporphyrin in Escherichia coli}, journal = {Fermentation}, volume = {10}, year = {2024}, pages = {250}, author = {B. Arab and A. Westbrook and M. Moo-Young and Y. Liu and C. P. Chou} } @presentation {31465, title = {Biotechnology, \"Intellectual Properties\", and \"Human Rights\"}, journal = {Contact Dr. Chou for the detailed story}, year = {2023}, author = {C. P. Chou} } @article {35833, title = {A toolkit for effective and successive genome engineering of Escherichia coli}, journal = {Fermentation}, volume = {9}, year = {2023}, pages = {14}, author = {B. Arab and A. Westbrook and M. Moo-Young and C. P. Chou} } @article {31464, title = {Carbon nanogels exert multipronged attack on resistant bacteria and strongly constrain resistance evolution}, journal = {Journal of Colloid and Interface Science}, volume = {608}, year = {2022}, pages = {1813-1826}, author = {J.-Y. Mao and D. Miscevic and B. Unnikrishnana and H.-W. Chua and C. P. Chou and L. Chang and H.-J. Lin and C.-C. Huang} } @article {31462, title = {Bio-based production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with modulated monomeric fraction in Escherichia coli}, journal = {Applied Microbiology and Biotechnology}, volume = {105}, year = {2021}, pages = {1435-1446 }, author = {D. Miscevic and J.-Y. Mao and B. Mozell and K. Srirangan and M. Moo-Young and C. P. Chou} } @article {31463, title = {Strain engineering and bioprocessing strategies for biobased production of porphobilinogen in Escherichia coli}, journal = {Bioresources and Bioprocessing}, volume = {8}, year = {2021}, pages = {122}, author = {D. Lall and D. Miscevic and M. Bruder and A. Westbrook and M. Aucoin and M. Moo-Young and C. P. Chou} } @article {31461, title = {Strain engineering for high-level 5-aminolevulinic acid production in Escherichia coli}, journal = {Biotechnology and Bioengineering}, volume = {118}, year = {2021}, pages = {30-42}, author = {D. Miscevic and J.-Y. Mao and D. Abedi and M. Moo-Young and C. P. Chou} } @article {22078, title = {Heterologous production of 3-hydroxyvalerate in engineered Escherichia coli}, journal = {Metabolic Engineering}, volume = {61}, year = {2020}, pages = {141-151}, abstract = {3-Hydroxyacids are a group of valuable fine chemicals with numerous applications, and 3-hydroxybutyrate (3-HB) represents the most common species with acetyl-CoA as a precursor. Due to the lack of propionyl-CoA in most, if not all, microorganisms, bio-based production of 3-hydroxyvalerate (3-HV), a longer-chain 3-hydroxyacid member with both acetyl-CoA and propionyl-CoA as two precursors, is often hindered by high costs associated with the supplementation of related carbon sources, such as propionate or valerate. Here, we report the derivation of engineered Escherichia coli strains for the production of 3-HV from unrelated cheap carbon sources, in particular glucose and glycerol. Activation of the sleeping beauty mutase (Sbm) pathway in E. coli enabled the intracellular formation of non-native propionyl-CoA. A selection of enzymes involved in 3-HV biosynthetic pathway from various microorganisms were explored for investigating their effects on 3-HV biosynthesis in E. coli. Glycerol outperformed glucose as the carbon source, and glycerol dissimilation for 3-HV biosynthesis was primarily mediated through the aerobic GlpK-GlpD route. To further enhance 3-HV production, we developed metabolic engineering strategies to redirect more dissimilated carbon flux from the tricarboxylic acid (TCA) cycle to the Sbm pathway, resulting in an enlarged intracellular pool of propionyl-CoA. Both the presence of succinate/succinyl-CoA and their interconversion step in the TCA cycle were identified to critically limit the carbon flux redirection into the Sbm pathway and, therefore, 3-HV biosynthesis. A selection of E. coli host TCA genes encoding enzymes near the succinate node were targeted for manipulation to evaluate the contribution of the three TCA routes (i.e. oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt) to the redirected carbon flux into the Sbm pathway. Finally, the carbon flux redirection into the Sbm pathway was enhanced by simultaneously deregulating glyoxylate shunt and blocking the oxidative TCA cycle, significantly improving 3-HV biosynthesis. With the implementation of these biotechnological and bioprocessing strategies, our engineered E. coli strains can effectively produce 3-HV up to 3.71 g l-1 with a yield of 24.1\% based on the consumed glycerol in shake-flask cultures.}, author = {Miscevic, D. and Srirangan, K. and Kilpatrick, S. and Chung, D. A. and Moo-Young, M and Chou, C. P.} } @article {24031, title = {High-level heterologous production of propionate in engineered Escherichia coli}, journal = {Biotechnology and Bioengineering}, volume = {117}, year = {2020}, pages = {1304-1315 }, abstract = {Previously, we derived a propanologenic (i.e., 1-propanol-producing) bacterium Escherichia coli strain by activating the genomic Sleeping beauty mutase (Sbm) operon. The activated Sbm pathway branches out of the tricarboxylic acid (TCA) cycle at the succinyl-CoA node to form propionyl-CoA and its derived metabolites of 1-propanol and propionate. In this study, we targeted several TCA cycle genes encoding enzymes near the succinyl-CoA node for genetic manipulation in order to identify the individual contribution of the carbon flux into the Sbm pathway from the three TCA metabolic routes, i.e., oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt. For the control strain CPC-Sbm in which propionate biosynthesis occurred under relatively anaerobic conditions, the carbon flux into the Sbm pathway were primarily derived from the reductive TCA branch; and both succinate availability and the SucCD-mediated interconversion of succinate/succinyl-CoA were critical for such carbon flux redirection. While the oxidative TCA cycle normally had a minimal contribution to the carbon flux redirection, the glyoxylate shunt could be an alternative and effective carbon flux contributor under aerobic conditions. With mechanistic understanding of such carbon flux redirection, metabolic strategies based on blocking the oxidative TCA cycle (via ∆sdhA mutation) and deregulating the glyoxylate shunt (via ∆iclR mutation) were developed to enhance the carbon flux redirection and, therefore, propionate biosynthesis, achieving a high propionate titer of 30.9 g l-1 with an overall propionate yield of 49.7\% upon fed-batch cultivation of the double mutant strain CPC-Sbm∆sdhAiclR under aerobic conditions. The results also suggest that the Sbm pathway could be metabolically active under both aerobic and anaerobic conditions.}, author = {D. Miscevic and J.-Y. Mao and M. Moo-Young and C. P. Chou} } @article {24032, title = {Integrated strain engineering and bioprocessing strategies for high-level bio-based production of 3-hydroxyvalerate in Escherichia coli}, journal = {Applied Microbiology and Biotechnology}, volume = {104}, year = {2020}, pages = {5259-5272}, abstract = {As petro-based production generates numerous environmental impacts and their associated technological concerns, bio-based production has been well recognized these days as a modern alternative to manufacture chemical products in a more renewable, environmentally friendly, and sustainable manner. Herein, we report the development of a microbial bioprocess for high-level and potentially economical production of 3-hydroxyvalerate (3-HV), a valuable special chemical with multiple applications in chemical, biopolymer, and pharmaceutical industries, from glycerol, which can be cheaply and renewably refined as a byproduct from biodiesel production. We used our recently derived 3-HV-producing Escherichia coli strains for bioreactor characterization under various culture conditions. In the parental strain, 3-HV biosynthesis was limited by the intracellular availability of propionyl-CoA, whose formation was favored by anaerobic conditions, which often compromised cell growth. With appropriate strain engineering, we demonstrated that 3-HV can be effectively produced under both microaerobic (close to anaerobic) and aerobic conditions, which determine the direction of dissimilated carbon flux toward the succinate node in the tricarboxylic acid (TCA) cycle. We first used the ∆sdhA single mutant strain, in which the dissimilated carbon flux was primarily directed to the Sleeping beauty mutase (Sbm) pathway (via the reductive TCA branch, with enhanced cell growth under microaerobic conditions, achieving 3.08 g l-1 3-HV in a fed-batch culture. In addition, we used the ∆sdhA-∆iclR double mutant strain, in which the dissimilated carbon flux was directed from the TCA cycle to the Sbm pathway via the deregulated glyoxylate shunt, for cultivation under rather aerobic conditions. In addition to demonstrating effective cell growth, this strain has shown impressive 3-HV biosynthesis (up to 10.6 g l-1), equivalent to an overall yield of 18.8\% based on consumed glycerol, in aerobic fed-batch culture. This study not only represents one of the most effective bio-based production of 3-HV from structurally unrelated carbons to date, but also highlights the importance of integrated strain engineering and bioprocessing strategies to enhance bio-based production.}, author = {D. Miscevic and J.-Y. Mao and D. Abedi and C.-C. Huang and M. Moo-Young and C. P. Chou} } @article {31460, title = {New insights into affinity proteins for HER2-targeted therapy: beyond trastuzumab}, journal = {BBA - Reviews on Cancer}, volume = {1874}, year = {2020}, pages = {188448}, author = {V. Akbari and C. P. Chou and D. Abedi} } @article {21885, title = {Graphene oxide and carbon dots as broad-spectrum antimicrobial agents - a minireview}, journal = {Nanoscale Horizons}, volume = {4}, number = {1}, year = {2019}, note = {Anand, Anisha Unnikrishnan, Binesh Wei, Shih-Chun Chou, C. Perry Zhang, Li-Zhi Huang, Chih-Ching}, pages = {117-137}, abstract = {Due to the increasing global population, growing contamination of water and air, and wide spread of infectious diseases, antibiotics are extensively used as a major antibacterial drug. However, many microbes have developed resistance to antibiotics through mutation over time. As an alternative to antibiotics, antimicrobial nanomaterials have attracted great attention due to their advantageous properties and unique mechanisms of action toward microbes. They inhibit bacterial growth and destroy cells through complex mechanisms, making it difficult for bacteria to develop drug resistance, though some health concerns related to biocompatibility remain for practical applications. Among various antibacterial nanomaterials, carbon-based materials, especially graphene oxide (GO) and carbon dots (C-Dots), are promising candidates due to the ease of production and functionalization, high dispersibility in aqueous media, and promising biocompatibility. The antibacterial properties of these nanomaterials can be easily adjusted by surface modification. They are promising materials for future applications against multidrug-resistant bacteria based on their strong capacity in disruption of microbial membranes. Though many studies have reported excellent antibacterial activity of carbon nanomaterials, their impact on the environment and living organisms is of concern due to the accumulatory and cytotoxic effects. In this review, we discuss antimicrobial applications of the functional carbon nanomaterials (GO and C-Dots), their antibacterial mechanisms, factors affecting antibacterial activity, and concerns regarding cytotoxicity.}, author = {Anand, A. and Unnikrishnan, B. and Wei, S. C. and Chou, C. P. and Zhang, L. Z. and Huang, C. C.} } @article {21891, title = {Production of cellulosic butyrate and 3-hydroxybutyrate in engineered Escherichia coli}, journal = {Applied Microbiology and Biotechnology}, volume = {103}, number = {13}, year = {2019}, note = {Miscevic, Dragan Srirangan, Kajan Kefale, Teshager Abedi, Daryoush Moo-Young, Murray Chou, C. Perry}, pages = {5215-5230}, abstract = {Being the most abundant renewable organic substance on Earth, lignocellulosic biomass has acted as an attractive and cost-effective feedstock for biobased production of value-added products. However, lignocellulosic biomass should be properly treated for its effective utilization during biotransformation. The current work aimed to demonstrate biobased production of butyrate and 3-hydroxybutyrate (3-HB) in engineered Escherichia coli using pretreated and detoxified aspen tree (Populus tremuloides) wood chips as the feedstock. Various bioprocessing and genetic/metabolic factors limiting the production of cellulosic butyrate and 3-HB were identified. With these developed bioprocessing strategies and strain engineering approaches, major carbons in the hydrolysate, including glucose, xylose, and even acetate, could be completely dissimilated during shake-flask cultivation with up to 1.68 g L-1 butyrate, 8.95 g L-1 3-HB, and minimal side metabolites (i.e., acetate and ethanol) being obtained. Our results highlight the importance of consolidating bioprocess and genetic engineering strategies for effective biobased production from lignocellulosic biomass.}, author = {Miscevic, D. and Srirangan, K. and Abedi, D. and Moo-Young, M. and Chou, C. P.} } @article {21921, title = {Strain engineering for microbial production of value-added chemicals and fuels from glycerol}, journal = {Biotechnology Advances}, volume = {37}, number = {4}, year = {2019}, note = {Westbrook, Adam W. Miscevic, Dragan Kilpatrick, Shane Bruder, Mark R. Moo-Young, Murray Chou, C. Perry Si}, pages = {538-568}, abstract = {While the widespread reliance on fossil fuels is driven by their low cost and relative abundance, this fossil-based economy has been deemed unsustainable and, therefore, the adoption of sustainable and environmentally compatible energy sources is on the horizon. Biorefinery is an emerging approach that integrates metabolic engineering, synthetic biology, and systems biology principles for the development of whole-cell catalytic platforms for biomanufacturing. Due to the high degree of reduction and low cost, glycerol, either refined or crude, has been recognized as an ideal feedstock for the production of value-added biologicals, though microbial dissimilation of glycerol sometimes can be difficult particularly under anaerobic conditions. While strain development for glycerol biorefinery is widely reported in the literature, few, if any, commercialized bioprocesses have been developed as a result, such that engineering of glycerol metabolism in microbial hosts remains an untapped opportunity in biomanufacturing. Here we review the recent progress made in engineering microbial hosts for the production of biofuels, diols, organic acids, biopolymers, and specialty chemicals from glycerol. We begin with a broad outline of the major pathways for fermentative and respiratory glycerol dissimilation and key end metabolites, and then focus our analysis on four key genera of bacteria known to naturally dissimilate glycerol, i.e. Klebsiella, Citrobacter, Clostridium, and Lactobacillus, in addition to Escherichia colt, and systematically review the progress made toward engineering these microorganisms for glycerol biorefinery. We also identify the major biotechnological and bioprocessing advantages and disadvantages of each genus, and bottlenecks limiting the production of target metabolites from glycerol in engineered strains. Our analysis culminates in the development of potential strategies to overcome the current technical limitations identified for commonly employed strains, with an outlook on the suitability of different hosts for the production of key metabolites and avenues for their future development into biomanufacturing platforms.}, author = {Westbrook, A. W. and Miscevic, D. and Kilpatrick, S. and Bruder, M. R. and Moo-Young, M. and Chou, C. P.} } @article {21923, title = {Application of hydrocarbon and perfluorocarbon oxygen vectors to enhance heterologous production of hyaluronic acid in engineered Bacillus subtilis}, journal = {Biotechnology and Bioengineering}, volume = {115}, number = {5}, year = {2018}, note = {Westbrook, Adam W. Ren, Xiang Moo-Young, Murray Chou, C. Perry}, pages = {1239-1252}, abstract = {In microbial cultivations for hyaluronic acid (HA) production, oxygen can be a limiting substrate due to its poor solubility in aqueous medium and the substantial increase in culture viscosity at relatively low HA titers. Shear stress due to the high agitation and aeration rates required to overcome oxygen limitation may reduce the quality (i.e., molecular weight) of HA, and production costs associated with power consumption and supplemental oxygen may be excessive. Here, we report the application of oxygen vectors to the heterologous production of HA in engineered Bacillus subtilis, leading to significantly improved culture performance. We first derived an improved HA-producing strain of B. subtilis through engineering of the promoter driving coexpression of seHas and tuaD, leading to high-level HA production. Out of seven potential oxygen vectors evaluated in a preliminary screening, significant improvements to the HA titer and/or cell density were observed in cultures containing n-heptane, n-hexadecane, perfluoromethyldecalin, and perfluoro-1,3-dimethylcyclohexane. Adjustments to the vector concentration, timing of vector addition, and the agitation rate resulted in further enhancements, with the HA titer reaching up to 4.5g/L after only 10hr cultivation. Moreover, our results indicate that certain vectors may alter the functional expression of Class I hyaluronan synthase (HAS) in B. subtilis, and that higher shear rates may drive more carbon flux through the HA biosynthetic pathway without negatively affecting the MW. Our study demonstrates the efficacy of oxygen vectors to enhance heterologous HA production in B. subtilis, and provides valuable insight for future bioprocess development in microbial HA production.}, author = {Westbrook, A. W. and Ren, X. and Moo-Young, M. and Chou, C. P.} } @article {21924, title = {Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis}, journal = {Biotechnology and Bioengineering}, volume = {115}, number = {1}, year = {2018}, note = {Westbrook, Adam W. Ren, Xiang Moo-Young, Murray Chou, C. Perry}, pages = {216-231}, abstract = {Hyaluronic acid (HA) is a high-value biopolymer used in the biomedical, pharmaceutical, cosmetic, and food industries. Current methods of HA production, including extraction from animal sources and streptococcal cultivations, are associated with high costs and health risks. Accordingly, the development of bioprocesses for HA production centered on robust Generally Recognized as Safe (GRAS) organisms such as Bacillus subtilis is highly attractive. Here, we report the development of novel strains of B. subtilis in which the membrane cardiolipin (CL) content and distribution has been engineered to enhance the functional expression of heterologously expressed hyaluronan synthase (HAS) of Streptococcus equisimilis (SeHAS), in turn, improving the culture performance for HA production. Elevation of membrane CL levels via overexpressing components involved in the CL biosynthesis pathway, and redistribution of CL along the lateral membrane via repression of the cell division initiator protein FtsZ resulted in increases to the HA titer of up to 204\% and peak molecular weight of up to 2.2MDa. Moreover, removal of phosphatidylethanolamine and neutral glycolipids from the membrane of HA-producing B. subtilis via inactivation of pssA and ugtP, respectively, has suggested the lipid dependence for functional expression of SeHAS. Our study demonstrates successful application of membrane engineering strategies to develop an effective platform for biomanufacturing of HA with B. subtilis strains expressing Class I streptococcal HAS.}, author = {Westbrook, A. W. and Ren, X. and Moo-Young, M. and Chou, C. P.} } @article {21925, title = {Metabolic engineering of Bacillus subtilis for l-valine overproduction}, journal = {Biotechnology and Bioengineering}, volume = {115}, number = {11}, year = {2018}, note = {Westbrook, Adam W. Ren, Xiang Moo-Young, Murray Chou, C. Perry}, pages = {2778-2792}, abstract = {Bacillus subtilis has been commonly applied to industrial enzyme production due to its genetic tractability, generally recognized as safe (GRAS) status, and robust growth characteristics. In spite of its ideal attributes as a biomanufacturing platform, B. subtilis has seen limited use in the production of other value-added biochemicals. Here, we report the derivation of engineered strains of B. subtilis for l-valine overproduction using our recently developed CRISPR (clustered regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated [protein] 9) toolkit. We first manipulate the native l-valine biosynthetic pathway by relieving transcriptional and allosteric regulation, resulting in a \>14-fold increase in the l-valine titer, compared to the wild-type strain. We subsequently identify and eliminate factors limiting l-valine overproduction, specifically increasing pyruvate availability and blocking the competing l-leucine and l-isoleucine biosynthetic pathways. By inactivating (a) pdhA, encoding the E1 subunit of the pyruvate dehydrogenase complex, to increase the intracellular pyruvate pool, and (b) leuA and ilvA, respectively encoding 2-isopropylmalate synthase and l-threonine dehydratase, to abolish the competing pathways, the l-valine titer reached 4.61g/L in shake flask cultures. Our engineered l-valine-overproducing strains of B. subtilis are devoid of plasmids and do not sporulate due to the inactivation of sigF, encoding the sporulation-specific transcription factor sigma(F), making them attractive for large-scale l-valine production. However, acetate dissimilation was identified as limiting l-valine overproduction in pdhA B. subtilis strains, and improving acetate dissimilation or identifying alternate modes of increasing pyruvate pools to enhance l-valine-overproduction should be explored.}, author = {Westbrook, A. W. and Ren, X. and Moo-Young, M. and Chou, C. P.} } @article {21926, title = {Metabolic engineering to enhance heterologous production of hyaluronic acid in Bacillus subtilis}, journal = {Metabolic Engineering}, volume = {47}, year = {2018}, note = {Westbrook, Adam W. Ren, Xiang Oh, Jaewon Moo-Young, Murray Chou, C. Perry}, pages = {401-413}, abstract = {Hyaluronic acid (HA) is a high-value biopolymer that is produced in large scales using attenuated strains of group C streptococci. However, due to the pathogenicity and fastidious nature of these bacteria, the development of bioprocesses for HA production centered on robust {\textquoteright}Generally Recognized as Safe (GRAS){\textquoteright} organisms, such as Bacillus subtilis, is of increased interest. Here, we report metabolic engineering of novel B. subtilis strains in which the carbon flux has been partially diverted from central metabolism, i.e. the pentose phosphate pathway (PPP) and glycolysis, into HA biosynthesis. First, an improved base strain of B. subtilis was engineered for more effective HA production with less susceptibility to catabolite repression when expressing genes from a xylose-inducible promoter. Subsequently, Clustered Regularly Interspaced Palindromic Repeats interference (CRISPRi) was applied to reduce the expression of individual pfkA or zwf in the base strain, leading to substantial improvements to the HA titer with a concomitant decrease in the molecular weight (MW). On the other hand, multiplexed repression of both pfkA and zwf expression resulted in increases to the HA titer of up to 108\% and enhancements to the MW, compared to the base strain. Moreover, the addition of exogenous HA monomers, i.e. glucuronic acid (GlcUA) and N-acetyl-glucosamine (GlcNAc), to B. subtilis cultures markedly improved the HA MW but decreased the HA titer, providing insights into the mechanism of HA biosynthesis by streptococcal hyaluronan synthase (SeHAS) in B. subtilis. Our study demonstrates the successful application of metabolic engineering strategies to establish B. subtilis as an effective platform for high-level HA production.}, author = {Westbrook, A. W. and Ren, X. and Oh, J. and Moo-Young, M. and Chou, C. P.} } @article {21913, title = {Recent advances in engineering propionyl-CoA metabolism for microbial production of value-added chemicals and biofuels}, journal = {Critical Reviews in Biotechnology}, volume = {37}, number = {6}, year = {2017}, note = {Srirangan, Kajan Bruder, Mark Akawi, Lamees Miscevic, Dragan Kilpatrick, Shane Moo-Young, Murray Chou, C. Perry}, pages = {701-722}, abstract = {Diminishing fossil fuel reserves and mounting environmental concerns associated with petrochemical manufacturing practices have generated significant interests in developing whole-cell biocatalytic systems for the production of value-added chemicals and biofuels. Although acetylCoA is a common natural biogenic precursor for the biosynthesis of numerous metabolites, propionyl-CoA is unpopular and non-native to most organisms. Nevertheless, with its C3-acyl moiety as a discrete building block, propionyl-CoA can serve as another key biogenic precursor to several biological products of industrial importance. As a result, engineering propionyl-CoA metabolism, particularly in genetically tractable hosts with the use of inexpensive feedstocks, has paved an avenue for novel biomanufacturing. Herein, we present a systematic review on manipulation of propionyl-CoA metabolism as well as relevant genetic and metabolic engineering strategies for microbial production of value-added chemicals and biofuels, including odd-chain alcohols and organic acids, bio(co) polymers and polyketides. [GRAPHICS] .}, author = {Srirangan, K. and Bruder, M. and Akawi, L. and Miscevic, D. and Kilpatrick, S. and Moo-Young, M. and Chou, C. P.} } @article {21912, title = {Use of a Case on Metabolically Engineered Escherichia coli to Develop a Framework for the Design and Analysis of Bioprocesses}, journal = {International Journal of Engineering Education}, volume = {33}, number = {2}, year = {2017}, note = {Srirangan, Kajan Akawi, Lamees K. Chou, C. Perry Aucoin, Marc G. Stacey, Lyndia E. Newton, Cheryl A}, pages = {751-760}, abstract = {Thematic case-based studies can be used by instructors to enhance critical thinking and knowledge in a holistic fashion, as well as improve students{\textquoteright} cognitive and metacognitive processes. While case-based learning approaches have been long used in the teaching of business, law and medicine, they have yet to see widespread use in many engineering disciplines. Furthermore, empirical research on the effectiveness of case-based learning within engineering is still in its infancy. Herein, we describe the use and impact of case-based instruction implemented in a third-year undergraduate bioprocess engineering course at the University of Waterloo. The overall objective of our study was to link key concepts related to bioprocess engineering to "real-world{\textquoteright}{\textquoteright} bioprocesses through a series of tutorial modules and research projects tied together by a common thematic case. The developed case focuses on genetically engineered Escherichia coli for the production of biofuels, specifically 1-propanol. The intent was to provide the students (n = 94) with the opportunity to gain a deeper understanding of biological systems by linking traditional fields of bioprocessing to an emerging field such as synthetic biology while also immersing students into situations that they could encounter while working in industry. Our results suggest that a majority of the students felt that the case was engaging and that the small-group based problem-solving exercises helped their understanding of design principles relevant to bioprocess engineering.}, author = {Srirangan, K. and Akawi, L. K. and Chou, C. P. and Aucoin, M. G. and Stacey, L. E. and Newton, C.} } @article {21922, title = {Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis}, journal = {Applied and Environmental Microbiology}, volume = {82}, number = {16}, year = {2016}, note = {Westbrook, Adam W. Moo-Young, Murray Chou, C. Perry}, pages = {4876-4895}, abstract = {The establishment of a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system for strain construction in Bacillus subtilis is essential for its progression toward industrial utility. Here we outline the development of a CRISPR-Cas9 tool kit for comprehensive genetic engineering in B. subtilis. In addition to site-specific mutation and gene insertion, our approach enables continuous genome editing and multiplexing and is extended to CRISPR interference (CRISPRi) for transcriptional modulation. Our tool kit employs chromosomal expression of Cas9 and chromosomal transcription of guide RNAs (gRNAs) using a gRNA transcription cassette and counterselectable gRNA delivery vectors. Our design obviates the need for multicopy plasmids, which can be unstable and impede cell viability. Efficiencies of up to 100\% and 85\% were obtained for single and double gene mutations, respectively. Also, a 2.9-kb hyaluronic acid (HA) biosynthetic operon was chromosomally inserted with an efficiency of 69\%. Furthermore, repression of a heterologous reporter gene was achieved, demonstrating the versatility of the tool kit. The performance of our tool kit is comparable with those of systems developed for Escherichia coli and Saccharomyces cerevisiae, which rely on replicating vectors to implement CRISPR-Cas9 machinery. IMPORTANCE In this paper, as the first approach, we report implementation of the CRISPR-Cas9 system in Bacillus subtilis, which is recognized as a valuable host system for biomanufacturing. The study enables comprehensive engineering of B. subtilis strains with virtually any desired genotypes/phenotypes and biochemical properties for extensive industrial application.}, author = {Westbrook, A. W. and Moo-Young, M. and Chou, C. P.} } @article {21909, title = {Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum}, journal = {Applied and Environmental Microbiology}, volume = {82}, number = {17}, year = {2016}, note = {Pyne, Michael E. Sokolenko, Stanislav Liu, Xuejia Srirangan, Kajan Bruder, Mark R. Aucoin, Marc G. Moo-Young, Murray Chung, Duane A. Chou, C. Perry}, pages = {5375-5388}, abstract = {Crude glycerol, the major by-product of biodiesel production, is an attractive bioprocessing feedstock owing to its abundance, low cost, and high degree of reduction. In line with the advent of the biodiesel industry, Clostridium pasteurianum has gained prominence as a result of its unique capacity to convert waste glycerol into n-butanol, a high-energy biofuel. However, no efforts have been directed at abolishing the production of 1,3-propanediol (1,3-PDO), the chief competing product of C. pasteurianum glycerol fermentation. Here, we report rational metabolic engineering of C. pasteurianum for enhanced n-butanol production through inactivation of the gene encoding 1,3-PDO dehydrogenase (dhaT). In spite of current models of anaerobic glycerol dissimilation, culture growth and glycerol utilization were unaffected in the dhaT disruption mutant (dhaT:: Ll.LtrB). Metabolite characterization of the dhaT:: Ll.LtrB mutant revealed an 83\% decrease in 1,3-PDO production, encompassing the lowest C. pasteurianum 1,3-PDO titer reported to date (0.58 g liter(-1)). With 1,3-PDO formation nearly abolished, glycerol was converted almost exclusively to n-butanol (8.6 g liter(-1)), yielding a high n-butanol selectivity of 0.83 g n-butanol g (-1) of solvents compared to 0.51 g n-butanol g (-1) of solvents for the wild-type strain. Unexpectedly, high-performance liquid chromatography (HPLC) analysis of dhaT:: Ll.LtrB mutant culture supernatants identified a metabolite peak consistent with 1,2-propanediol (1,2-PDO), which was confirmed by nuclear magnetic resonance (NMR). Based on these findings, we propose a new model for glycerol dissimilation by C. pasteurianum, whereby the production of 1,3-PDO by the wild-type strain and low levels of both 1,3-PDO and 1,2-PDO by the engineered mutant balance the reducing equivalents generated during cell mass synthesis from glycerol. IMPORTANCE Organisms from the genus Clostridium are perhaps the most notable native cellular factories, owing to their vast substrate utilization range and equally diverse variety of metabolites produced. The ability of C. pasteurianum to sustain redox balance and glycerol fermentation despite inactivation of the 1,3-PDO pathway is a testament to the exceptional metabolic flexibility exhibited by clostridia. Moreover, identification of a previously unknown 1,2-PDO-formation pathway, as detailed herein, provides a deeper understanding of fermentative glycerol utilization in clostridia and will inform future metabolic engineering endeavors involving C. pasteurianum. To our knowledge, the C. pasteurianum dhaT disruption mutant derived in this study is the only organism that produces both 1,2- and 1,3-PDOs. Most importantly, the engineered strain provides an excellent platform for highly selective production of n-butanol from waste glycerol. IMPORTANCE Organisms from the genus Clostridium are perhaps the most notable native cellular factories, owing to their vast substrate utilization range and equally diverse variety of metabolites produced. The ability of C. pasteurianum to sustain redox balance and glycerol fermentation despite inactivation of the 1,3-PDO pathway is a testament to the exceptional metabolic flexibility exhibited by clostridia. Moreover, identification of a previously unknown 1,2-PDO-formation pathway, as detailed herein, provides a deeper understanding of fermentative glycerol utilization in clostridia and will inform future metabolic engineering endeavors involving C. pasteurianum. To our knowledge, the C. pasteurianum dhaT disruption mutant derived in this study is the only organism that produces both 1,2-and 1,3-PDOs. Most importantly, the engineered strain provides an excellent platform for highly selective production of n-butanol from waste glycerol.}, author = {Pyne, M. E. and Sokolenko, S. and Liu, X. J. and Srirangan, K. and Bruder, M. R. and Aucoin, M. G. and Moo-Young, M. and Chung, D. A. and Chou, C. P.} } @article {21914, title = {Engineering Escherichia coli for Microbial Production of Butanone}, journal = {Applied and Environmental Microbiology}, volume = {82}, number = {9}, year = {2016}, note = {Srirangan, Kajan Liu, Xuejia Akawi, Lamees Bruder, Mark Moo-Young, Murray Chou, C. Perry}, pages = {2574-2584}, abstract = {To expand the chemical and molecular diversity of biotransformation using whole-cell biocatalysts, we genetically engineered a pathway in Escherichia coli for heterologous production of butanone, an important commodity ketone. First, a 1-propanol-producing E. coli host strain with its sleeping beauty mutase (Sbm) operon being activated was used to increase the pool of propionyl-coenzyme A (propionyl-CoA). Subsequently, molecular heterofusion of propionyl-CoA and acetyl-CoA was conducted to yield 3-ketovaleryl-CoA via a CoA-dependent elongation pathway. Lastly, 3-ketovaleryl-CoA was channeled into the clostridial acetone formation pathway for thioester hydrolysis and subsequent decarboxylation to form butanone. Biochemical, genetic, and metabolic factors affecting relative levels of ketogenesis, acidogenesis, and alcohologenesis under selected fermentative culture conditions were investigated. Using the engineered E. coli strain for batch cultivation with 30 g liter(-1) glycerol as the carbon source, we achieved coproduction of 1.3 g liter(-1) butanone and 2.9 g liter(-1) acetone. The results suggest that approximately 42\% of spent glycerol was utilized for ketone biosynthesis, and thus they demonstrate potential industrial applicability of this microbial platform.}, author = {Srirangan, K. and Liu, X. J. and Akawi, L. and Bruder, M. and Moo-Young, M. and Chou, C. P.} } @article {21915, title = {Engineering of Escherichia coli for direct and modulated biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer using unrelated carbon sources}, journal = {Scientific Reports}, volume = {6}, year = {2016}, note = {Srirangan, Kajan Liu, Xuejia Tran, Tam T. Charles, Trevor C. Moo-Young, Murray Chou, C. Perry}, abstract = {While poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] is a biodegradable commodity plastic with broad applications, its microbial synthesis is hindered by high production costs primarily associated with the supplementation of related carbon substrates (e.g. propionate or valerate). Here we report construction of engineered Escherichia coli strains for direct synthesis of P(3HB-co-3HV) from an unrelated carbon source (e.g. glucose or glycerol). First, an E. coli strain with an activated sleeping beauty mutase (Sbm) operon was used to generate propionyl-CoA as a precursor. Next, two acetyl-CoA moieties or acetyl-CoA and propionyl-CoA were condensed to form acetoacetyl-CoA and 3-ketovaleryl-CoA, respectively, by functional expression of beta-ketothiolases from Cupriavidus necator (i.e. PhaA and BktB). The resulting thioester intermediates were channeled into the polyhydroxyalkanoate (PHA) biosynthetic pathway through functional expression of acetoacetyl-CoA reductase (PhaB) for thioester reduction and PHA synthase (PhaC) for subsequent polymerization. Metabolic engineering of E. coli host strains was further conducted to enhance total PHA content and the 3-hydroxyvaleryl (3HV) monomer fraction in the copolymer. Using a selection of engineered E. coli strains for batch cultivation with an unrelated carbon source, we achieved high-level P(3HB-co-3HV) production with the 3HV monomer fraction ranging from 3 to 19 mol\%, demonstrating the potential industrial applicability of these whole-cell biocatalysts.}, author = {Srirangan, K. and Liu, X. J. and Tran, T. T. and Charles, T. C. and Moo-Young, M. and Chou, C. P.} } @article {21888, title = {Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium}, journal = {Applied and Environmental Microbiology}, volume = {82}, number = {20}, year = {2016}, note = {Bruder, Mark R. Pyne, Michael E. Moo-Young, Murray Chung, Duane A. Chou, C. Perry}, pages = {6109-6119}, abstract = {The discovery and exploitation of the prokaryotic adaptive immunity system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins have revolutionized genetic engineering. CRISPR-Cas tools have enabled extensive genome editing as well as efficient modulation of the transcriptional program in a multitude of organisms. Progress in the development of genetic engineering tools for the genus Clostridium has lagged behind that of many other prokaryotes, presenting the CRISPR-Cas technology an opportunity to resolve a long-existing issue. Here, we applied the Streptococcus pyogenes type II CRISPR-Cas9 (SpCRISPR-Cas9) system for genome editing in Clostridium acetobutylicum DSM792. We further explored the utility of the SpCRISPR-Cas9 machinery for gene-specific transcriptional repression. For proof-of-concept demonstration, a plasmid-encoded fluorescent protein gene was used for transcriptional repression in C. acetobutylicum. Subsequently, we targeted the carbon catabolite repression (CCR) system of C. acetobutylicum through transcriptional repression of the hprK gene encoding HPr kinase/phosphorylase, leading to the coutilization of glucose and xylose, which are two abundant carbon sources from lignocellulosic feedstocks. Similar approaches based on SpCRISPR-Cas9 for genome editing and transcriptional repression were also demonstrated in Clostridium pasteurianum ATCC 6013. As such, this work lays a foundation for the derivation of clostridial strains for industrial purposes. IMPORTANCE After recognizing the industrial potential of Clostridium for decades, methods for the genetic manipulation of these anaerobic bacteria are still underdeveloped. This study reports the implementation of CRISPR-Cas technology for genome editing and transcriptional regulation in Clostridium acetobutylicum, which is arguably the most common industrial clostridial strain. The developed genetic tools enable simpler, more reliable, and more extensive derivation of C. acetobutylicum mutant strains for industrial purposes. Similar approaches were also demonstrated in Clostridium pasteurianum, another clostridial strain that is capable of utilizing glycerol as the carbon source for butanol fermentation, and therefore can be arguably applied in other clostridial strains.}, author = {Bruder, M. R. and Pyne, M. E. and Moo-Young, M. and Chung, D. A. and Chou, C. P.} } @article {21905, title = {Genome-directed analysis of prophage excision, host defence systems, and central fermentative metabolism in Clostridium pasteurianum}, journal = {Scientific Reports}, volume = {6}, year = {2016}, note = {Pyne, Michael E. Liu, Xuejia Moo-Young, Murray Chung, Duane A. Chou, C. Perry}, abstract = {Clostridium pasteurianum is emerging as a prospective host for the production of biofuels and chemicals, and has recently been shown to directly consume electric current. Despite this growing biotechnological appeal, the organism{\textquoteright}s genetics and central metabolism remain poorly understood. Here we present a concurrent genome sequence for the C. pasteurianum type strain and provide extensive genomic analysis of the organism{\textquoteright}s defence mechanisms and central fermentative metabolism. Next generation genome sequencing produced reads corresponding to spontaneous excision of a novel phage, designated phi 6013, which could be induced using mitomycin C and detected using PCR and transmission electron microscopy. Methylome analysis of sequencing reads provided a near-complete glimpse into the organism{\textquoteright}s restriction-modification systems. We also unveiled the chief C. pasteurianum Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) locus, which was found to exemplify a Type I-B system. Finally, we show that C. pasteurianum possesses a highly complex fermentative metabolism whereby the metabolic pathways enlisted by the cell is governed by the degree of reductance of the substrate. Four distinct fermentation profiles, ranging from exclusively acidogenic to predominantly alcohologenic, were observed through redox consideration of the substrate. A detailed discussion of the organism{\textquoteright}s central metabolism within the context of metabolic engineering is provided.}, author = {Pyne, M. E. and Liu, X. J. and Moo-Young, M. and Chung, D. E. A. and Chou, C. P.} } @article {21904, title = {Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium}, journal = {Scientific Reports}, volume = {6}, year = {2016}, note = {Pyne, Michael E. Bruder, Mark R. Moo-Young, Murray Chung, Duane A. Chou, C. Perry}, abstract = {Application of CRISPR-Cas9 systems has revolutionized genome editing across all domains of life. Here we report implementation of the heterologous Type II CRISPR-Cas9 system in Clostridium pasteurianum for markerless genome editing. Since 74\% of species harbor CRISPR-Cas loci in Clostridium, we also explored the prospect of co-opting host-encoded CRISPR-Cas machinery for genome editing. Motivation for this work was bolstered from the observation that plasmids expressing heterologous cas9 result in poor transformation of Clostridium. To address this barrier and establish proof-of-concept, we focus on characterization and exploitation of the C. pasteurianum Type I-B CRISPR-Cas system. In silico spacer analysis and in vivo interference assays revealed three protospacer adjacent motif (PAM) sequences required for site-specific nucleolytic attack. Introduction of a synthetic CRISPR array and cpaAIR gene deletion template yielded an editing efficiency of 100\%. In contrast, the heterologous Type II CRISPR-Cas9 system generated only 25\% of the total yield of edited cells, suggesting that native machinery provides a superior foundation for genome editing by precluding expression of cas9 in trans. To broaden our approach, we also identified putative PAM sequences in three key species of Clostridium. This is the first report of genome editing through harnessing native CRISPR-Cas machinery in Clostridium.}, author = {Pyne, M. E. and Bruder, M. R. and Moo-Young, M. and Chung, D. A. and Chou, C. P.} } @article {21886, title = {Twin arginine translocation system in secretory expression of recombinant human growth hormone}, journal = {Research in Pharmaceutical Sciences}, volume = {11}, number = {6}, year = {2016}, note = {Bagherinejad, Mohammad Reza Sadeghi, Hamid Mir-Mohammad Abedi, Daryoush Chou, C. Perry Moazen, Fatemeh Rabbani, Mohammad}, pages = {461-469}, abstract = {Recombinant protein production in E. coli has several advantages over other expression systems. Misfolding, inclusion body formation, and lack of eukaryotic post translational modification are the most disadvantages of this system. Exporting of correctly folded proteins to the outside of reductive cytoplasmic environment through twin-arginine system could help to pass these limiting steps. Two signal sequences, TorA and SufI are used at N-terminal of human growth hormone (hGH) bearing DsbA gene sequence at C-terminal to enhance folding. The synthetic cassettes including the signal sequence, hGH and DsbA were transformed into E. coli BL21 (DE3) to study the effect of signal sequence and DsbA chaperone on translocation and folding of the protein. The results confirmed using signal sequence at N-terminal of targeted protein and coexpression with DsbA could transport proteins to the periplasmic space and culture media compared to control groups. Although there is no protein band of somatropin in SDS-Page of culture media samples when using SufI as signaling sequence, the study demonstrated TorA signal sequence could transport the target protein to the culture media. However, there was a considerable amount of hGH in periplasmic space when using SufI compared to control.}, author = {Bagherinejad, M. R. and Sadeghi, H. M. M. and Abedi, D. and Chou, C. P. and Moazen, F. and Rabbani, M.} } @article {21908, title = {Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli}, journal = {Applied and Environmental Microbiology}, volume = {81}, number = {15}, year = {2015}, note = {Pyne, Michael E. Moo-Young, Murray Chung, Duane A. Chou, C. Perry}, pages = {5103-5114}, abstract = {To date, most genetic engineering approaches coupling the type II Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements in Escherichia coli can be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome-encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient production of gene replacement mutants that are both markerless and "scar"-less.}, author = {Pyne, M. E. and Moo-Young, M. and Chung, D. E. A. and Chou, C. P.} } @article {21887, title = {Elimination of carbon catabolite repression in Clostridium acetobutylicum-a journey toward simultaneous use of xylose and glucose}, journal = {Applied Microbiology and Biotechnology}, volume = {99}, number = {18}, year = {2015}, note = {Bruder, Mark Moo-Young, Murray Chung, Duane A. Chou, C. Perry}, pages = {7579-7588}, abstract = {The industrial Gram-positive anaerobe Clostridium acetobutylicum is a valued acetone, butanol, and ethanol (ABE) solvent producer that is able to utilize a vast array of carbon sources in fermentation. When glucose is present in the growth medium, however, C. acetobutylicum, like many Gram-positive organisms, exhibits biphasic growth characteristics in which glucose is used preferentially over secondary carbon sources, a phenomenon known as carbon catabolite repression (CCR). The secondary carbon source is only utilized when the supply of glucose is exhausted, resulting in inefficient use of complex carbon sources. As biofuel production is sought from cheap feedstock, attention has turned to lignocellulosic biomass. Growth of C. acetobutylicum on lignocellulose, however, can be limited by CCR. Here, we present a method to relieve the inhibitory effect of CCR and allow simultaneous utilization of the lignocellulosic sugars of glucose and xylose by C. acetobutylicum. First, we utilized an in vivo gene reporter assay to demonstrate that an identified 14-nucleotide catabolite responsive element (CRE) sequence was sufficient to introduce CCR-mediated transcriptional inhibition, while subsequent mutation of the CRE sequence relieved the inhibitory effect. Next, we demonstrated that C. acetobutylicum harboring a CRE-less plasmid-borne xylose and pentose phosphate pathway operon afforded a 7.5-fold increase in xylose utilization in the presence of glucose as compared to a wild-type CRE plasmid-borne operon, effectively overcoming native CCR effects. The methodology presented here should translate to other members of Clostridium that exhibit CCR to enable simultaneous utilization of a vast array of carbon sources.}, author = {Bruder, M. and Moo-Young, M. and Chung, D. A. and Chou, C. P.} } @article {21881, title = {Engineering Escherichia coli for high-level production of propionate}, journal = {Journal of Industrial Microbiology \& Biotechnology}, volume = {42}, number = {7}, year = {2015}, note = {Akawi, Lamees Srirangan, Kajan Liu, Xuejia Moo-Young, Murray Chou, C. Perry}, pages = {1057-1072}, abstract = {Mounting environmental concerns associated with the use of petroleum-based chemical manufacturing practices has generated significant interest in the development of biological alternatives for the production of propionate. However, biological platforms for propionate production have been limited to strict anaerobes, such as Propionibacteria and select Clostridia. In this work, we demonstrated high-level heterologous production of propionate under microaerobic conditions in engineered Escherichia coli. Activation of the native Sleeping beauty mutase (Sbm) operon not only transformed E. coli to be propionogenic (i.e., propionate-producing) but also introduced an intracellular "flux competition" between the traditional C2-fermentative pathway and the novel C3-fermentative pathway. Dissimilation of the major carbon source of glycerol was identified to critically affect such "flux competition" and, therefore, propionate synthesis. As a result, the propionogenic E. coli was further engineered by inactivation or overexpression of various genes involved in the glycerol dissimilation pathways and their individual genetic effects on propionate production were investigated. Generally, knocking out genes involved in glycerol dissimilation (except glpA) can minimize levels of solventogenesis and shift more dissimilated carbon flux toward the C3-fermentative pathway. For optimal propionate production with high C3:C2-fermentative product ratios, glycerol dissimilation should be channeled through the respiratory pathway and, upon suppressed solventogenesis with minimal production of highly reduced alcohols, the alternative NADH-consuming route associated with propionate synthesis can be critical for more flexible redox balancing. With the implementation of various biochemical and genetic strategies, high propionate titers of more than 11 g/L with high yields up to 0.4 g-propionate/g-glycerol (accounting for similar to 50 \% of dissimilated glycerol) were achieved, demonstrating the potential for industrial application. To our knowledge, this represents the most effective engineered microbial system for propionate production with titers and yields comparable to those achieved by anaerobic batch cultivation of various native propionate-producing strains of Propionibacteria.}, author = {Akawi, L. and Srirangan, K. and Liu, X. J. and Moo-Young, M. and Chou, C. P.} } @article {21882, title = {Improved biological activity of a single chain antibody fragment against human epidermal growth factor receptor 2 (HER2) expressed in the periplasm of Escherichia coli}, journal = {Protein Expression and Purification}, volume = {116}, year = {2015}, note = {Akbari, Vajihe Sadeghi, Hamid Mir Mohammad Jafarian-Dehkordi, Abbas Abedi, Daryoush Chou, C. Perry}, pages = {66-74}, abstract = {A novel monoclonal antibody against human epidermal growth factor receptor 2 (HER2), i.e., pertuzumab (Perjeta (R)) developed by Genentech, has been verified to be effective in treating metastatic HER2-overexpressing breast cancer. The fact that the presence of the Fc region of the anti-HER2 is uncritical for growth inhibition of tumor cells suggests the potential biological activity of the associated antibody fragments. In the present study, we report functional expression of anti-HER2his-scFv, a single-chain variable fragment (scFv) derived from pertuzumab, in the periplasm of Escherichia coli and its purification. Biological activity of the soluble scFv produced in this manner was characterized using immunofluorescent staining, immunocytochemistry, flow cytometry and cytotoxicity assay. The effect of anti-HER2his-scFv on HER2 dimerization was also assessed by tyrosine kinase assay. It was observed that the purified scFv had a high specificity and affinity to HER2 receptors expressed on the surface of tumor cells with a selective cytotoxic effect on HER2-overexpressing SK-OV-3 cells. In addition, anti-HER2his-scFy was able to suppress phosphorylation of HER2 in the presence of heregulin. The results suggest that anti-HER2his-scFv can be a potential candidate for various therapeutic and diagnosis applications. (C) 2015 Elsevier Inc. All rights reserved.}, author = {Akbari, V. and Sadeghi, H. M. M. and Jafarian-Dehkordi, A. and Abedi, D. and Chou, C. P.} } @article {21883, title = {Optimization of a single-chain antibody fragment overexpression in Escherichia coli using response surface methodology}, journal = {Research in Pharmaceutical Sciences}, volume = {10}, number = {1}, year = {2015}, note = {Akbari, V. Sadeghi, H. Mir Mohammad Jafarian-Dehkordi, A. Chou, C. Perry Abedi, D.}, pages = {75-83}, abstract = {Human epidermal growth factor receptor (HER) family plays an important role in various types of cancers. As a result, antibodies against HER and the mechanism of antigen-antibody binding action are under active investigation. We previously constructed a single-chain variable fragment (ScFv) against HER2, i.e. anti-Her2 ScFv, for expressing in the Escherichia coli. In the present study, we report the optimization of anti-Her2 ScFv expression in an E. coli host of BL21 (DE3) pLysS using response surface methodology based on tuning of three cultivation variables, including isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration, temperature and post-induction time. A model for protein expression according to the Box-Behnken design predicted a maximal anti-Her2 ScFv expression at 37 degrees C, a post-induction time of 10.45 h and 0.75 mM IPTG. In addition, strategies based on inclusion body isolation and affinity chromatography were applied to purify anti-Her2 ScFv. The purity of the final product for inclusion bodies isolation and purification by Ni-NTA resin were 70 \% and 95 \%, respectively. The solubilization of the inclusion bodies was carried out using two denaturant agents, guanidine hydrochloride and urea. The present study showed that guanidine hydrochloride was more effective than urea in solubilizing the inclusion bodies.}, author = {Akbari, V. and Sadeghi, H. M. M. and Jafarian-Dehkordi, A. and Chou, C. P. and Abedi, D.} } @article {21920, title = {Application of a two-dimensional disposable rocking bioreactor to bacterial cultivation for recombinant protein production}, journal = {Biochemical Engineering Journal}, volume = {88}, year = {2014}, note = {Westbrook, Adam Scharer, Jeno Moo-Young, Murray Oosterhuis, Nico Chou, C. Perry}, pages = {154-161}, abstract = {Disposable rocking bioreactors (RBs) are widely employed for cultivation of recombinant mammalian and insect cell lines, although the perception of inadequate mass transfer has prevented their application to bioprocesses based on microbial platforms. In this study, one-dimensional (1D) and two-dimensional (2D) RBs were assessed and compared with the conventional stirred tank reactor (STR) for recombinant therapeutic protein production in Escherichia coll. The comparison involved: (1) physical characterization of oxygen mass transfer efficiency and mixing intensity, (2) growth characteristics in batch cultivation, and (3) culture performance for the production of recombinant protein. Our results show that oxygen mass transfer was comparable between the 1D RB and STR at low working volume (WV), declining linearly with increasing WV, and was highest in the 2D RB for all tested WVs with the maximum mass transfer coefficient (kLa) at 3 L WV. Well mixing behavior was observed in all three systems for water and aqueous carboxymethylcellulose (CMC) solutions. Batch growth characteristics were similar in all bioreactor systems, although metabolite accumulation was significant in the I D RB. Culture performance for the production of recombinant GST-hCD83ext (glutathione S-transferase-hCD83ext fusion protein) was similar in terms of soluble protein yield and inclusion body formation for all bioreactor systems. (C) 2014 Elsevier B.V. All rights reserved.}, author = {Westbrook, A. and Scharer, J. and Moo-Young, M. and Oosterhuis, N. and Chou, C. P.} } @article {21916, title = {Biochemical, genetic, and metabolic engineering strategies to enhance coproduction of 1-propanol and ethanol in engineered Escherichia coli}, journal = {Applied Microbiology and Biotechnology}, volume = {98}, number = {22}, year = {2014}, note = {Srirangan, Kajan Liu, Xuejia Westbrook, Adam Akawi, Lamees Pyne, Michael E. Moo-Young, Murray Chou, C. Perry}, pages = {9499-9515}, abstract = {We recently reported the heterologous production of 1-propanol in Escherichia coli via extended dissimilation of succinate under anaerobic conditions through expression of the endogenous sleeping beauty mutase (Sbm) operon. In the present work, we demonstrate high-level coproduction of 1-propanol and ethanol by developing novel engineered E. coli strains with effective cultivation strategies. Various biochemical, genetic, metabolic, and physiological factors affecting relative levels of acidogenesis and solventogenesis during anaerobic fermentation were investigated. In particular, CPC-PrOH3, a plasmid-free propanogenic E. coli strain derived by activating the Sbm operon on the genome, showed high levels of solventogenesis accounting for up to 85 \% of dissimilated carbon. Anaerobic fed-batch cultivation of CPC-PrOH3 with glycerol as the major carbon source produced high titers of nearly 7 g/L 1-propanol and 31 g/L ethanol, implying its potential industrial applicability. The activated Sbm pathway served as an ancillary channel for consuming reducing equivalents upon anaerobic dissimilation of glycerol, resulting in an enhanced glycerol dissimilation and a major metabolic shift from acidogenesis to solventogenesis.}, author = {Srirangan, K. and Liu, X. J. and Westbrook, A. and Akawi, L. and Pyne, M. E. and Moo-Young, M. and Chou, C. P.} } @article {21907, title = {Expansion of the genetic toolkit for metabolic engineering of Clostridium pasteurianum: chromosomal gene disruption of the endogenous CpaAI restriction enzyme}, journal = {Biotechnology for Biofuels}, volume = {7}, year = {2014}, note = {Pyne, Michael E. Moo-Young, Murray Chung, Duane A. Chou, C. Perry}, abstract = {Background: Clostridium pasteurianum is one of the most promising biofuel producers within the genus Clostridium owing to its unique metabolic ability to ferment glycerol into butanol. Although an efficient means is available for introducing foreign DNA to C. pasteurianum, major genetic tools, such as gene knockout, knockdown, or genome editing, are lacking, preventing metabolic engineering of C. pasteurianum. Results: Here we present a methodology for performing chromosomal gene disruption in C. pasteurianum using the programmable lactococcus Ll.ltrB group II intron. Gene disruption was initially found to be impeded by inefficient electrotransformation of Escherichia coli-C. pasteurianum shuttle vectors, presumably due to host restriction. By assessing the ability of various vector deletion derivatives to electrotransform C. pasteurianum and probing the microorganism{\textquoteright}s methylome using next-generation sequence data, we identified a new C. pasteurianum Type I restriction-methylation system, CpaAII, with a predicted recognition sequence of 5{\textquoteright}-AAGNNNNNCTCC-3{\textquoteright} (N = A, C, G, or T). Following rescue of high-level electrotransformation via mutation of the sole CpaAII site within the shuttle vectors, we retargeted the intron to the cpaAIR gene encoding the CpaAI Type II restriction endonuclease (recognition site of 5{\textquoteright}-CGCG-3{\textquoteright}). Intron insertion was potentially hindered by low retrohoming efficiency, yet this limitation could be overcome by a procedure for enrichment of the intron insertion. The resulting Delta cpaAIR mutant strain was efficiently electrotransformed with M. FnuDII-unmethylated plasmid DNA. Conclusions: The markerless and plasmidless Delta cpaAIR mutant strain of C. pasteurianum developed in this study can serve as a general host strain for future genetic and metabolic manipulation. Further, the associated gene disruption protocol should not only serve as a guide for chromosomal gene inactivation studies involving mobile group II introns, but also prove invaluable for applying metabolic engineering strategies to C. pasteurianum.}, author = {Pyne, M. E. and Moo-Young, M. and Chung, D. A. and Chou, C. P.} } @article {21884, title = {Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli}, journal = {Journal of Industrial Microbiology \& Biotechnology}, volume = {41}, number = {6}, year = {2014}, note = {Akbari, Vajihe Sadeghi, Hamid Mir Mohammad Jafrian-Dehkordi, Abbas Abedi, Daryoush Chou, C. Perry}, pages = {947-956}, abstract = {The human epidermal growth factor receptor (HER) family plays an important role in cell growth and signaling and alteration of its function has been demonstrated in many different kinds of cancer. Receptor dimerization is necessary for the HER signal transduction pathway and tyrosine kinase activity. Recently, several monoclonal antibodies have been developed to directly interfere with ligand-HER receptor binding and receptor dimerization. A single chain variable fragment (ScFv) is a valuable alternative to an intact antibody. This report describes the production and purification of an ScFv specific for domain II of the HER2 receptor in Escherichia coli BL21 (DE3) cytoplasm. The majority of expressed of anti-her2his-ScFv protein was produced as inclusion bodies. A Ni-NTA affinity column was used to purify the anti-her2his-ScFv protein. The molecular weight of anti-her2his-ScFv protein was estimated to be approximately 27 kDa, as confirmed by SDS-PAGE and Western blotting assay. The anti-her2his-ScFv showed near 95 \% purity and reached a yield of approximately 29 mg/l in flask fermentation. The purified anti-her2his-ScFv showed its biological activity by binding to HER2 receptor on the surface of BT-474 cells. This ScFv may be a potential pharmaceutical candidate for targeting tumour cells overexpressing HER2 receptor.}, author = {Akbari, V. and Sadeghi, H. M. M. and Jafrian-Dehkordi, A. and Abedi, D. and Chou, C. P.} } @article {22006, title = {Improved Draft Genome Sequence of Clostridium pasteurianum Strain ATCC 6013 (DSM 525) Using a Hybrid Next-Generation Sequencing Approach}, journal = {Genome announcements}, volume = {2}, number = {4}, year = {2014}, note = {25103768[pmid] PMC4125779[pmcid]}, pages = {e00790-14}, abstract = {We present an improved draft genome sequence for Clostridium pasteurianum strain ATCC 6013 (DSM 525), the type strain of the species and an important solventogenic bacterium with industrial potential. Availability of a near-complete genome sequence will enable strain engineering of this promising bacterium.}, author = {Pyne, Michael E. and Utturkar, Sagar and Brown, Steven D. and Moo-Young, Murray and Chung, Duane A. and Chou, C. Perry} } @article {21903, title = {Technical guide for genetic advancement of underdeveloped and intractable Clostridium}, journal = {Biotechnology Advances}, volume = {32}, number = {3}, year = {2014}, note = {Pyne, Michael E. Bruder, Mark Moo-Young, Murray Chung, Duane A. Chou, C. Perry}, pages = {623-641}, abstract = {In recent years, the genus Clostridium has risen to the forefront of both medical biotechnology and industrial biotechnology owing to its potential in applications as diverse as anticancer therapy and production of commodity chemicals and biofuels. The prevalence of hyper-virulent strains of C. difficile within medical institutions has also led to a global epidemic that demands a more thorough understanding of clostridial genetics, physiology, and pathogenicity. Unfortunately. Clostridium suffers from a lack of sophisticated genetic tools and techniques which has hindered the biotechnological exploitation of this important bacterial genus. This review provides a comprehensive summary of biotechnological progress made in clostridial genetic tool development, while also aiming to serve as a technical guide for the advancement of underdeveloped clostridial strains, including recalcitrant species, novel environmental samples, and non-type strains. Relevant strain engineering techniques, from genome sequencing and establishment of a gene transfer methodology through to deployment of advanced genome editing procedures, are discussed in detail to provide a blueprint for future clostridial strain construction endeavors. It is expected that a more thorough and rounded-out genetic toolkit available for use in the clostridia will bring about the construction of superior bioprocessing strains and a more complete understanding of clostridial genetics, physiology, and pathogenicity. (C) 2014 Elsevier Inc. All rights reserved.}, author = {Pyne, M. E. and Bruder, M. and Moo-Young, M. and Chung, D. A. and Chou, C. P.} } @article {21917, title = {Biotechnological advances on Penicillin G acylase: Pharmaceutical implications, unique expression mechanism and production strategies}, journal = {Biotechnology Advances}, volume = {31}, number = {8}, year = {2013}, note = {Srirangan, Kajan Orr, Valerie Akawi, Lamees Westbrook, Adam Moo-Young, Murray Chou, C. Perry}, pages = {1319-1332}, abstract = {In light of unrestricted use of first-generation penicillins, these antibiotics are now superseded by their semisynthetic counterparts for augmented antibiosis. Traditional penicillin chemistry involves the use of hazardous chemicals and harsh reaction conditions for the production of semisynthetic derivatives and, therefore, is being displaced by the biosynthetic platform using enzymatic transformations. Penicillin G acylase (PGA) is one of the most relevant and widely used biocatalysts for the industrial production of beta-lactam semisynthetic antibiotics. Accordingly, considerable genetic and biochemical engineering strategies have been devoted towards PGA applications. This article provides a state-of-the-art review in recent biotechnological advances associated with PGA, particularly in the production technologies with an emphasis on using the Escherichia coli expression platform. (C) 2013 Elsevier Inc. All rights reserved.}, author = {Srirangan, K. and Orr, V. and Akawi, L. and Westbrook, A. and Moo-Young, M. and Chou, C. P.} } @article {21906, title = {Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum}, journal = {Biotechnology for Biofuels}, volume = {6}, year = {2013}, note = {Pyne, Michael E. Moo-Young, Murray Chung, Duane A. Chou, C. Perry}, abstract = {Background: Reducing the production cost of, and increasing revenues from, industrial biofuels will greatly facilitate their proliferation and co-integration with fossil fuels. The cost of feedstock is the largest cost in most fermentation bioprocesses and therefore represents an important target for cost reduction. Meanwhile, the biorefinery concept advocates revenue growth through complete utilization of by-products generated during biofuel production. Taken together, the production of biofuels from low-cost crude glycerol, available in oversupply as a by-product of bioethanol production, in the form of thin stillage, and biodiesel production, embodies a remarkable opportunity to advance affordable biofuel development. However, few bacterial species possess the natural capacity to convert glycerol as a sole source of carbon and energy into value-added bioproducts. Of particular interest is the anaerobe Clostridium pasteurianum, the only microorganism known to convert glycerol alone directly into butanol, which currently holds immense promise as a high-energy biofuel and bulk chemical. Unfortunately, genetic and metabolic engineering of C. pasteurianum has been fundamentally impeded due to lack of an efficient method for deoxyribonucleic acid (DNA) transfer. Results: This work reports the development of an electrotransformation protocol permitting high-level DNA transfer to C. pasteurianum ATCC 6013 together with accompanying selection markers and vector components. The CpaAI restriction-modification system was found to be a major barrier to DNA delivery into C. pasteurianum which we overcame by in vivo methylation of the recognition site (5{\textquoteright}-CGCG-3{\textquoteright}) using the M. FnuDII methyltransferase. With proper selection of the replication origin and antibiotic-resistance marker, we initially electroporated methylated DNA into C. pasteurianum at a low efficiency of 2.4 x 10(1) transformants mu g(-1) DNA by utilizing conditions common to other clostridial electroporations. Systematic investigation of various parameters involved in the cell growth, washing and pulse delivery, and outgrowth phases of the electrotransformation procedure significantly elevated the electrotransformation efficiency, up to 7.5 x 10(4) transformants mu g(-1) DNA, an increase of approximately three order of magnitude. Key factors affecting the electrotransformation efficiency include cell-wall-weakening using glycine, ethanol-mediated membrane solubilization, field strength of the electric pulse, and sucrose osmoprotection. Conclusions: C. pasteurianum ATCC 6013 can be electrotransformed at a high efficiency using appropriately methylated plasmid DNA. The electrotransformation method and tools reported here should promote extensive genetic manipulation and metabolic engineering of this biotechnologically important bacterium.}, author = {Pyne, M. E. and Moo-Young, M. and Chung, D. A. and Chou, C. P.} } @article {21910, title = {Manipulating the sleeping beauty mutase operon for the production of 1-propanol in engineered Escherichia coli}, journal = {Biotechnology for Biofuels}, volume = {6}, year = {2013}, note = {Srirangan, Kajan Akawi, Lamees Liu, Xuejia Westbrook, Adam Blondeel, Eric J. M. Aucoin, Marc G. Moo-Young, Murray Chou, C. Perry}, abstract = {Background: While most resources in biofuels were directed towards implementing bioethanol programs, 1-propanol has recently received attention as a promising alternative biofuel. Nevertheless, no microorganism has been identified as a natural 1-propanol producer. In this study, we manipulated a novel metabolic pathway for the synthesis of 1-propanol in the genetically tractable bacterium Escherichia coli. Results: E. coli strains capable of producing heterologous 1-propanol were engineered by extending the dissimilation of succinate via propionyl-CoA. This was accomplished by expressing a selection of key genes, i.e. (1) three native genes in the sleeping beauty mutase (Sbm) operon, i.e. sbm-ygfD-ygfG from E. coli, (2) the genes encoding bifunctional aldehyde/alcohol dehydrogenases (ADHs) from several microbial sources, and (3) the sucCD gene encoding succinyl-CoA synthetase from E. coli. Using the developed whole-cell biocatalyst under anaerobic conditions, production titers up to 150 mg/L of 1-propanol were obtained. In addition, several genetic and chemical effects on the production of 1-propanol were investigated, indicating that certain host-gene deletions could abolish 1-propanol production as well as that the expression of a putative protein kinase (encoded by ygfD/argK) was crucial for 1-propanol biosynthesis. Conclusions: The study has provided a novel route for 1-propanol production in E. coli, which is subjected to further improvement by identifying limiting conversion steps, shifting major carbon flux to the productive pathway, and optimizing gene expression and culture conditions.}, author = {Srirangan, K. and Akawi, L. and Liu, X. J. and Westbrook, A. and Blondeel, E. J. M. and Aucoin, M. G. and Moo-Young, M. and Chou, C. P.} } @article {21902, title = {Recent advances in bioprocessing application of membrane chromatography}, journal = {Biotechnology Advances}, volume = {31}, number = {4}, year = {2013}, note = {Orr, Valerie Zhong, Luyang Moo-Young, Murray Chou, C. Perry}, pages = {450-465}, abstract = {Compared to traditional chromatography using resins in packed-bed columns, membrane chromatography is a relatively new and immature bioseparation technology based on the integration of membrane filtration and liquid chromatography into a single-stage operation. Over the past decades, advances in membrane chemistry have yielded novel membrane devices with high binding capacities and improved mass transfer properties, significantly increasing the bioprocessing efficiency for purification of biomolecules. Due to the disposable nature, low buffer consumption, and reduced equipment costs, membrane chromatography can significantly reduce downstream bioprocessing costs. In this review, we discuss technological merits and disadvantages associated with membrane chromatography as well as recent bioseparation applications with a particular attention on purification of large biomolecules. (C) 2013 Elsevier Inc. All rights reserved.}, author = {Orr, V. and Zhong, L. Y. and Moo-Young, M. and Chou, C. P.} } @article {21919, title = {Developing an Extended Genomic Engineering Approach Based on Recombineering to Knock-in Heterologous Genes to Escherichia coli Genome}, journal = {Molecular Biotechnology}, volume = {51}, number = {2}, year = {2012}, note = {Sukhija, Karan Pyne, Michael Ali, Saad Orr, Valerie Abedi, Daryoush Moo-Young, Murray Chou, C. Perry}, pages = {109-118}, abstract = {Most existing genomic engineering protocols for manipulation of Escherichia coli are primarily focused on chromosomal gene knockout. In this study, a simple but systematic chromosomal gene knock-in method was proposed based on a previously developed protocol using bacteriophage lambda (lambda Red) and flippase-flippase recognition targets (FLP-FRT) recombinations. For demonstration purposes, DNA operons containing heterologous genes (i.e., pac encoding E. coli penicillin acylase and palB2 encoding Pseudozyma antarctica lipase B mutant) engineered with regulatory elements, such as strong/inducible promoters (i.e., P (trc) and P (araB) ), operators, and ribosomal binding sites, were integrated into the E. coli genome at designated locations (i.e., lacZYA, dbpA, and lacI-mhpR loci) either as a gene replacement or gene insertion using various antibiotic selection markers (i.e., kanamycin and chloramphenicol) under various genetic backgrounds (i.e., HB101 and DH5 alpha). The expression of the inserted foreign genes was subjected to regulation using appropriate inducers [isopropyl beta-d-1-thiogalactopyranoside (IPTG) and arabinose] at tunable concentrations. The developed approach not only enables more extensive genomic engineering of E. coli, but also paves an effective way to "tailor" plasmid-free E. coli strains with desired genotypes suitable for various biotechnological applications, such as biomanufacturing and metabolic engineering.}, author = {Sukhija, K. and Pyne, M. and Ali, S. and Orr, V. and Abedi, D. and Moo-Young, M. and Chou, C. P.} } @article {21900, title = {Integrated development of an effective bioprocess for extracellular production of penicillin G acylase in Escherichia coli and its subsequent one-step purification}, journal = {Journal of Biotechnology}, volume = {161}, number = {1}, year = {2012}, note = {Orr, Valerie Scharer, Jeno Moo-Young, Murray Honeyman, C. Howie Fenner, Drew Crossley, Lisa Suen, Shing-Yi Chou, C. Perry}, pages = {19-26}, abstract = {An integrated bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed in this study had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography column while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange media, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level at 871 U/g DCW, of which more than 90\% was localized in the extracellular medium. In addition, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. The formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. Clarified culture medium was applied to a strong anion-exchange (Q) column and PAC was purified by non-retentive separation, where most contaminant proteins bound to the chromatographic media with PAC being collected as the major component in the flow-through fraction. After removing the contaminant oligopeptides using ultrafiltration, purified PAC with a specific activity of 16.3 U/mg was obtained and the overall purification factor for this one-step downstream purification process was up to 3 fold. (C) 2012 Elsevier B. V. All rights reserved.}, author = {Orr, V. and Scharer, J. and Moo-Young, M. and Honeyman, C. H. and Fenner, D. and Crossley, L. and Suen, S. Y. and Chou, C. P.} } @article {21901, title = {Simultaneous clarification of Escherichia coli culture and purification of extracellularly produced penicillin G acylase using tangential flow filtration and anion-exchange membrane chromatography (TFF-AEMC)}, journal = {Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences}, volume = {900}, year = {2012}, note = {Orr, Valerie Scharer, Jeno Moo-Young, Murray Honeyman, C. Howie Fenner, Drew Crossley, Lisa Suen, Shing-Yi Chou, C. Perry}, pages = {71-78}, abstract = {Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins since conventional purification processes are lengthy, technically complicated, and time-consuming. To address this issue, herein we demonstrated the simultaneous clarification and purification of the extracellularly produced recombinant protein by Escherichia coli using an integrated system of tangential flow filtration and anion exchange membrane chromatography (TFF-AEMC). After cultivation in a bench-top bioreactor with 1 L working volume using the developed host/vector system for high-level expression and effective secretion of recombinant penicillin G acylase (PAC), the whole culture broth was applied directly to the established system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry) and cross-flow operations. Most contaminant proteins in the extracellular medium were captured by the anion-exchange membrane and cells remained in the retentate, whereas extracellular PAC was purified and collected in the filtrate. The batch time for both cultivation and purification was less than 2411 and recombinant PAC with high purity (19 U/mg), yield (72\% recovery), and productivity (41 mg of purified PAC per liter of culture) was obtained. Due to the nature of the non-selective protein secretion system and the versatility of ion-exchange membrane chromatography, the developed system can be widely applied for effective production and purification of recombinant proteins. (C) 2012 Elsevier B.V. All rights reserved.}, author = {Orr, V. and Scharer, J. and Moo-Young, M. and Honeyman, C. H. and Fenner, D. and Crossley, L. and Suen, S. Y. and Chou, C. P.} } @article {21911, title = {Towards sustainable production of clean energy carriers from biomass resources}, journal = {Applied Energy}, volume = {100}, year = {2012}, note = {Srirangan, Kajan Akawi, Lamees Moo-Young, Murray Chou, C. Perry}, pages = {172-186}, abstract = {A great fraction of the world{\textquoteright}s energy requirements are presently met through the unfettered use of fossil-derived fuels. However, due to the anticipated demise of these energy sources and the environmental and socioeconomic concerns associated with their use, a recent paradigm shift is to displace conventional fuels with renewable energy sources. Among various alternatives, biomasses have garnered tremendous interests as potential feedstock for clean energy production. While numerous biorefinery schemes and conversion technologies exist for the transformation of biomass into usable energy forms, they are not cost-efficient and economically viable to compete with the existing petroleum-refinery technologies. In particular, the recalcitrant nature of several feedstock presents a major technological obstacle for their processing and transformation. Providentially, the synergistic integration of various biochemical and bioprocessing technologies is aiding in the establishment of future biomass energy programs. This article reviews the state of the art and future challenges in the recent development of biomass and associated transformation technologies for clean production of biofuels. (C) 2012 Elsevier Ltd. All rights reserved.}, author = {Srirangan, K. and Akawi, L. and Moo-Young, M. and Chou, C. P.} } @article {21918, title = {Biochemical and genetic engineering strategies to enhance hydrogen production in photosynthetic algae and cyanobacteria}, journal = {Bioresource Technology}, volume = {102}, number = {18}, year = {2011}, note = {Srirangan, Kajan Pyne, Michael E. Chou, C. Perry Si}, pages = {8589-8604}, abstract = {As an energy carrier, hydrogen gas is a promising substitute to carbonaceous fuels owing to its superb conversion efficiency, non-polluting nature, and high energy content. At present, hydrogen is predominately synthesized via chemical reformation of fossil fuels. While various biological methods have been extensively explored, none of them is justified as economically feasible. A sustainable platform for biological production of hydrogen will certainly impact the biofuel market. Among a selection of biological systems, algae and cyanobacteria have garnered major interests as potential cell factories for hydrogen production. In conjunction with photosynthesis, these organisms utilize inexpensive inorganic substrates and solar energy for simultaneous biosynthesis and hydrogen evolution. However, the hydrogen yield associated with these organisms remains far too low to compete with the existing chemical systems. This article reviews recent advances of biochemical, bioprocess, and genetic engineering strategies in circumventing technological limitations to hopefully improve the applicative potential of these photosynthetic hydrogen production systems. (C) 2011 Elsevier Ltd. All rights reserved.}, author = {Srirangan, K. and Pyne, M. E. and Chou, C. P.} } @article {21940, title = {Developing an RNase-free bioprocess to produce pharmaceutical-grade plasmid DNA using selective precipitation and membrane chromatography}, journal = {Separation and Purification Technology}, volume = {83}, year = {2011}, note = {Zhong, Luyang Srirangan, Kajan Scharer, Jeno Moo-Young, Murray Fenner, Drew Crossley, Lisa Honeyman, C. Howie Suen, Shing-Yi Chou, C. Perry}, pages = {121-129}, abstract = {A novel, RNase-free, and potentially scalable bioprocess was developed for the production of pharmaceutical-grade plasmid DNA. High bioprocess recovery and product quality were primarily associated with the optimal integration of impurity removal by calcium chloride precipitation and anion-exchange membrane chromatography and the implementation of isopropanol precipitation as a coupling step between the two impurity-removing steps. Complete removal of total cellular RNA was demonstrated without the use of animal-derived RNase. High-molecular-weight (HMW) RNA and genomic DNA (gDNA) were removed by selective precipitation using calcium chloride. Complete removal of the remaining low-molecular-weight (LMW) RNA was achieved by membrane chromatography using a high-capacity hydrogel-based strong anion-exchange membrane. The simultaneous achievement of desalting, concentrating and buffer exchange by the coupling step of isopropanol precipitation and the high efficiency and resolution of DNA RNA separation by anion-exchange membrane chromatography significantly reduced the operating complexity of the overall bioprocess, increased the overall recovery of plasmid DNA, and enhanced product quality by removing trace amounts of major impurities of concern for biomedical applications, such as gDNA, proteins, and endotoxin. (C) 2011 Elsevier B.V. All rights reserved.}, author = {Zhong, L. Y. and Srirangan, K. and Scharer, J. and Moo-Young, M. and Fenner, D. and Crossley, L. and Honeyman, C. H. and Suen, S. Y. and Chou, C. P.} } @article {21935, title = {Disulfide bond formation and its impact on the biological activity and stability of recombinant therapeutic proteins produced by Escherichia coli expression system}, journal = {Biotechnology Advances}, volume = {29}, number = {6}, year = {2011}, note = {Zhang, Lin Chou, C. Perry Moo-Young, Murray}, pages = {923-929}, abstract = {Therapeutic proteins require correct disulfide bond formation for biological activity and stability. This makes their manufacturing and storage inherently challenging since disulfide bonds can be aberrantly formed and/or undergo significant structural changes. In this paper the mechanisms of disulfide bond formation and scrambling are reviewed, with a focus on their impact on the biological activity and storage stability of recombinant proteins. After assessing the research progress in detecting disulfide bond scrambling, strategies for preventing this phenomenon are proposed. (C) 2011 Elsevier Inc. All rights reserved.}, author = {Zhang, L. and Chou, C. P. and Moo-Young, M.} } @article {21898, title = {Enhancing Functional Expression of Heterologous Burkholderia Lipase in Escherichia coli}, journal = {Molecular Biotechnology}, volume = {47}, number = {2}, year = {2011}, note = {Narayanan, Niju Khan, Manal Chou, C. Perry}, pages = {130-143}, abstract = {Functional expression of lipase from Burkholderia sp. C20 (Lip) in various cellular compartments of Escherichia coli was explored. The poor expression in the cytoplasm of E. coli was improved by several strategies, including coexpression of the cytoplasmic chaperone GroEL/ES, using a mutant E. coli host strain with an oxidative cytoplasm, and protein fusion technology. Fusing Lip with the N-terminal peptide tags of T7PK, DsbA, and DsbC was effective in enhancing the solubility and biological activity. Non-fused Lip or Lip fusions heterologously expressed in the periplasm of E. coli formed insoluble aggregates with a minimum activity. Biologically active and intact Lip was obtained upon the secretion into the extracellular medium using the native signal peptide and the expression performance was further improved by coexpression of the periplasmic chaperon Skp. The extracellular expression was even more effective when Lip was secreted as a Lip-HlyA fusion via the a-hemolysin transporter. Finally, Lip could be functionally displayed on the E. coli cell surface when fused with the carrier EstA.}, author = {Narayanan, N. and Khan, M. and Chou, C. P.} } @article {21937, title = {Molecular manipulation associated with disulfide bond formation to enhance the stability of recombinant therapeutic protein}, journal = {Protein Expression and Purification}, volume = {75}, number = {1}, year = {2011}, note = {Zhang, Lin Moo-Young, Murray Chou, C. Perry}, pages = {28-39}, abstract = {Cys(27) in the extracellular domain of human CD83 (hCD83ext) a potential therapeutic protein was identified as a target for molecular manipulation Two Escherichia coli strains of BL21(DE3) and Origami B(DE3) respectively with a reducing and an oxidative cytoplasm were used as the expression host to produce the Cys(27) mutants It was observed that Cys(27) was involved in the in vivo formation of intramolecular disulfide bonds when hCD83ext was expressed in Origami B(DE3) The Origami-derived protein products had a higher tendency than the BL21-derived counterparts for multimerization via the in vitro formation of Intermolecular disulfide bonds Various analyses were conducted to identify the structural differences among these mutant variants Most importantly molecular stability was enhanced by the Cys(27) mutations since the Cys(27) mutants derived from either BL21 or Origami were much less susceptible to degradation compared to wild-type hCD83ext This study highlights the implications of aberrant disulfide bond formation on the production of therapeutic proteins (C) 2010 Elsevier Inc All rights reserved}, author = {Zhang, L. and Moo-Young, M. and Chou, C. P.} } @article {21939, title = {Potential application of hydrogel-based strong anion-exchange membrane for plasmid DNA purification}, journal = {Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences}, volume = {879}, number = {9-10}, year = {2011}, note = {Zhong, Luyang Scharer, Jeno Moo-Young, Murray Fenner, Drew Crossley, Lisa Honeyman, C. Howie Suen, Shing-Yi Chou, C. Perry}, pages = {564-572}, abstract = {The potential application of a hydrogel-based strong anion-exchange (Q) membrane to purify plasmid DNAs was evaluated. The maximum binding capacity of plasmid DNA was estimated to be 12.4 mg/ml of membrane volume with a plasmid recovery yield of similar to 90\%. The effect of the inherent properties of plasmid DNA, membrane adsorbent, and the ionic environment on membrane performance was systematically investigated. Plasmid DNAs with smaller tertiary structure tended to have a better recovery than those with larger tertiary structure. Environmental Scanning Electron Microscopy (ESEM) revealed that the hydrogel structure is more porous on one side of membrane than the other. Membrane pre-treatment significantly improved pore distribution and increased membrane porosity resulting in a better adsorption, recovery, and higher flux. The selection of proper operating pH led to further improvement. The relative contribution of these factors to improve membrane chromatography of plasmid DNAs was analyzed using statistical modeling. It was found that the adsorption of plasmid DNA was mainly affected by the available adsorptive area associated with membrane porosity, whereas the recovery of plasmid DNAs was mainly affected by the environmental pH. (c) 2011 Elsevier BM. All rights reserved.}, author = {Zhong, L. Y. and Scharer, J. and Moo-Young, M. and Fenner, D. and Crossley, L. and Honeyman, C. H. and Suen, S. Y. and Chou, C. P.} } @article {21936, title = {Effect of aberrant disulfide bond formation on protein conformation and molecular property of recombinant therapeutics}, journal = {Pure and Applied Chemistry}, volume = {82}, number = {1}, year = {2010}, note = {Zhang, Lin Moo-Young, Murray Chou, C. Perry}, pages = {149-159}, abstract = {As a comparative study, the extracytoplasmic domain of human CD83 (hCD83ext) was expressed as a glutathione.-S-transferase (GST) fusion in two Escherichia coli B strains, i.e., BL21 and Orioami B, respectively, with a reductive and oxidative cytoplasm. The final therapeutic products of hCD83ext produced by the two expression hosts exhibited significant differences in protein coil formation and molecular property, which presumably resulted from different disulfide patterns. The Study highlights the importance of developing proper host/vector systems and biomanufacturing conditions for the production of recombinant therapeutic proteins with a consistent product quality.}, author = {Zhang, L. and Moo-Young, M. and Chou, C. P.} } @article {21897, title = {Enhancing functional expression of heterologous lipase B in Escherichia coli by extracellular secretion}, journal = {Journal of Industrial Microbiology \& Biotechnology}, volume = {37}, number = {4}, year = {2010}, note = {Narayanan, Niju Khan, Manal Chou, C. Perry}, pages = {349-361}, abstract = {Functional expression of recombinant Pseudozyma antarctica lipase B (PalB) in Escherichia coli has been technically problematic due to protein misfolding, ineffective disulfide bond formation, and protein instability associated with intracellular proteolysis. To overcome these problems, an alternative approach was explored in this study by extracellular secretion of PalB via two Sec-independent secretion systems, i.e., the alpha-hemolysin (type I) and the modified flagellar (type III) secretion systems, which can export proteins of interest from the cytoplasm directly to the exterior of the cell. Both shaker flask and bioreactor cultivations were performed to characterize the developed PalB expression/secretion systems. Bioactive PalB was expressed and secreted extracellularly either as a HlyA fusion (i.e., PalB-HlyA via type I system) or an intact protein (via type III system). However, the secretion intermediates in the intracellular fraction of culture samples were non-bioactive even though they were soluble, suggesting that the extracellular secretion did mediate the development of PalB activity. Also importantly, the secretion strategy appeared to have a minimum impact on cell physiology. PalB secretion via the type I system was fast with higher specific PalB activities but poor cell growth. On the other hand, the secretion via the type III system was slow with lower specific PalB activities but effective cell growth.}, author = {Narayanan, N. and Khan, M. and Chou, C. P.} } @article {22005, title = {Immunosuppression Involving Soluble CD83 Induces Tolerogenic Dendritic Cells That Prevent Cardiac Allograft Rejection}, journal = {Transplantation}, volume = {90}, number = {11}, year = {2010}, note = {Ge, Wei Arp, Jacqueline Lian, Dameng Liu, Weihua Baroja, Miren L. Jiang, Jifu Ramcharran, Siobhan ElDeen, Firas Zahr Zinser, Elisabeth Steinkasserer, Alexander Chou, Perry Brand, Stephen Nicolette, Charles Garcia, Bertha Wang, Hao}, pages = {1145-1156}, abstract = {Background. Dendritic cells (DCs) are crucial regulators of immunity and important in inducing and maintaining tolerance. Here, we investigated the potential of a novel DC-immunomodulating agent, soluble CD83 (sCD83), in inducing transplant tolerance. Methods. We used the C3H-to-C57BL/6 mouse cardiac transplantation model that exhibits a combination of severe cell-mediated rejection and moderate antibody-mediated rejection and investigated whether sCD83 could augment a combination therapy consisting of Rapamycin (Rapa) and anti-CD45RB monoclonal antibody (alpha-CD45) to prolong allograft survival. Results. Monotherapies consisting of Rapa and alpha-CD45 were incapable of preventing rejection. However, all treatments involving sCD83 were capable of (1) down-modulating expression of various DC surface molecules, such as major histocompatibility complex class II and costimulatory molecules, (2) reducing the allogeneic stimulatory capacity of the DCs, and (3) significantly inhibiting antidonor antibody responses. Most striking results were observed in the triple therapy-treated group, sCD83(+)Rapa(+) alpha-CD45, where cell-mediated rejection and antibody-mediated rejection were abrogated for over 100 days. Donor-specific tolerance was achieved in long-term surviving recipients, because donor skin transplants were readily accepted for an additional 100 days, whereas third-party skin grafts were rejected. Success of triple therapy treatment was accompanied by enhancement of tolerogenic-DCs that conferred antigen-specific protection on adoptive transfer to recipients of an allogeneic heart graft. Conclusions. Our study revealed that sCD83 is capable of attenuating DC maturation and function, and inducing donor-specific allograft tolerance, in the absence of toxicity. Thus, sCD83 seems to be a safe and valuable counterpart to current DC-modulating agents.}, author = {Ge, W. and Arp, J. and Lian, D. M. and Liu, W. H. and Baroja, M. L. and Jiang, J. F. and Ramcharran, S. and ElDeen, F. Z. and Zinser, E. and Steinkasserer, A. and Chou, P. and Brand, S. and Nicolette, C. and Garcia, B. and Wang, H.} } @inbook {21984, title = {Strain improvement of Escherichia coli to enhance recombinant protein production}, booktitle = {Manual of Industrial Microbiology and Biotechnology}, year = {2010}, pages = {273-286}, publisher = {ASM Press}, organization = {ASM Press}, edition = {3}, author = {Pyne, M. E. and Sukhija, K. S. and Chou, C. P.} } @article {21938, title = {Structural identification of recombinant human CD83 mutant variant as a potent therapeutic protein}, journal = {Protein Expression and Purification}, volume = {73}, number = {2}, year = {2010}, note = {Zhang, Lin Narayanan, Niju Brand, Stephen R. Nicolette, Charles A. Baroja, Miren Arp, Jacqueline Wang, Hao Moo-Young, Murray Chou, C. Perry}, pages = {140-146}, abstract = {The formation of aberrant disulfide bonds is a structural consideration for the manufacturing of the extracellular domain of human CD83 (hCD83ext), a potential therapeutic protein. In certain instances, hCD83ext protein products, even when stored frozen, tended to dimerize or even multimerize through the formation of aberrant intermolecular disulfide bonds. Herein, we discovered an analytical inconsistency and applied a modified sample preparation protocol for proper structural analysis of hCD83ext products which are heterologously expressed in Escherichia coli and subsequently purified. In addition, a mutant derivative with the Cys100Ser mutation was identified as an improved version which did not form dimers or multimers. The identification of this mutant variant as a more potent therapeutic protein than other hCD83ext species demonstrated that the structural variation associated with disulfide bond formation can be a critical issue for rigorous control of the quality and bioactivity of therapeutic proteins. The application of this mutant variant for protein therapeutics is currently under exploration. (C) 2010 Elsevier Inc. All rights reserved.}, author = {Zhang, L. and Narayanan, N. and Brand, S. R. and Nicolette, C. A. and Baroja, M. and Arp, J. and Wang, H. and Moo-Young, M. and Chou, C. P.} } @article {21894, title = {Alleviation of Proteolytic Sensitivity To Enhance Recombinant Lipase Production in Escherichia coli}, journal = {Applied and Environmental Microbiology}, volume = {75}, number = {16}, year = {2009}, note = {Narayanan, Niju Chou, C. Perry}, pages = {5424-5427}, abstract = {Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression.}, author = {Narayanan, N. and Chou, C. P.} } @article {21928, title = {Bioprocess development for production, purification, and structural characterization of recombinant hCD83ext as a potential therapeutic protein}, journal = {Protein Expression and Purification}, volume = {65}, number = {1}, year = {2009}, note = {Xu, Yali Zhang, Lin Yao, Weifang Yedahalli, Shreyas S. Brand, Stephen Moo-Young, Murray Chou, C. Perry}, pages = {92-99}, abstract = {An effective bioprocess for the production of hCD83ext (i.e. the extracytoplasmic domain of human CD83) as a potential therapeutic protein was developed. It primarily consists of (I) cell cultivation for the production of recombinant glutathione-S-transferase-hCD83ext (GST-hCD83ext) fusion protein and (2) downstream processing for purification of hCD83ext. The developed bioprocess is robust, reproducible, easy to operate, and, most importantly, can generate hCD83ext with a high yield and purity. For cell cultivation, a high GST-hCD83ext expression level, estimated to be more than 10\% of total cellular protein, with a cell density of 8 OD600 was obtained by tuning several culture parameters, including medium recipe, host/vector system, induction condition, temperature, and aeration. For downstream processing, milligrams of very pure and low-endotoxin hCD83ext was obtained through simultaneous binding and cleavage of GST-hCD83ext in a GST affinity chromatographic column followed by a polishing step using anion exchange chromatography. To identify potential factors associated with bioactivity consistency, structural changes for the final product of hCD83ext were characterized and monitored. Formation of various hCD83ext multimeric forms, including dimer, trimer, and tetramer, via intermolecular disulfide bonds was observed. (C) 2008 Elsevier Inc. All rights reserved.}, author = {Xu, Y. and Zhang, L. and Yao, W. and Yedahalli, S. S. and Brand, S. and Moo-Young, M. and Chou, C. P.} } @article {21890, title = {Metabolic pathways of clostridia for producing butanol}, journal = {Biotechnology Advances}, volume = {27}, number = {6}, year = {2009}, note = {Gheshlaghi, R. Scharer, J. M. Moo-Young, M. Chou, C. P.}, pages = {764-781}, abstract = {Worldwide demand for energy has been the impetus for research to produce alcohol biofuels from renewable resources. This review focuses on the biosynthesis of butanol, which is regarded to be Superior to ethanol as a fuel. Although acetone/butanol fermentation is one of the oldest large-scale fermentation processes. butanol yield by anaerobic fermentation remains sub-optimal Metabolic engineering provides a means for fermentation improvements Consequently, a comprehensive assessment of the intermediary enzymes involved in butanol formation from carbohydrates by the saccharolytic bacterium. Clostridium acetobutylicum and other closely allied clostridia was performed to provide guidelines for potentially enhancing butanol productivity. The activity of the enzymes, their regulation and contribution to the metabolic pathways was reviewed Published kinetic data for each important enzymatic reaction were assessed For most enzymatic reactions, the systematic investigation of the kinetic data and the properties of the enzymes led to the development of rate equations that were able to describe activity as the function of the substrates, products, and allosteric effectors. (C) 2009 Published by Elsevier Inc}, author = {Gheshlaghi, R. and Scharer, J. M. and Moo-Young, M. and Chou, C. P.} } @article {21930, title = {Effect of folding factors in rescuing unstable heterologous lipase B to enhance its overexpression in the periplasm of Escherichia coli}, journal = {Applied Microbiology and Biotechnology}, volume = {79}, number = {6}, year = {2008}, note = {Xu, Yali Lewis, Darrell Chou, C. Perry}, pages = {1035-1044}, abstract = {Functional expression of recombinant Pseudozyma antarctica lipase B (PalB) in Escherichia coli was explored. While PalB was stably expressed in the cytoplasm, most of the expressed gene product aggregated in cells as inactive inclusion bodies. In contrast, PalB was extremely unstable when expressed in the periplasm, also leading to poor expression performance. Such unstable PalB can be rescued by coexpression of several periplasmic folding factors, such as DegP, FkpA, DsbA, and DsbC but not cytoplasmic ones. As a result, the performance for functional PalB expression in the periplasm was significantly improved. To our knowledge, this is the first report demonstrating the use of folding factors to rescue the extremely unstable gene product that is otherwise completely degradable.}, author = {Xu, Y. L. and Lewis, D. and Chou, C. P.} } @article {21934, title = {Enhancing functional expression of heterologous lipase in the periplasm of Escherichia coli}, journal = {World Journal of Microbiology \& Biotechnology}, volume = {24}, number = {12}, year = {2008}, note = {Xu, Yali Yasin, Amrita Wucherpfennig, Thomas Chou, C. Perry}, pages = {2827-2835}, abstract = {Functional expression of heterologous Pseudozyma antarctica lipase B (PalB) in the periplasm of Escherichia coli was explored using four fusion tags, i.e. DsbC, DsbA, maltose-binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of PalB, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegP(S210A)), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for all the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/L.}, author = {Xu, Y. L. and Yasin, A. and Wucherpfennig, T. and Chou, C. P.} } @article {21933, title = {Heterologous expression of lipase in Escherichia coli is limited by folding and disulfide bond formation}, journal = {Applied Microbiology and Biotechnology}, volume = {81}, number = {1}, year = {2008}, note = {Xu, Yali Yasin, Amrita Tang, Raymond Scharer, Jeno M. Moo-Young, Murray Chou, C. Perry}, pages = {79-87}, abstract = {Functional expression of lipase B from Pseudozyma antarctica (PalB) in the cytoplasm of Escherichia coli BL21(DE3) and its mutant derivative Origami B(DE3) was explored. Coexpression of DsbA was found to be effective in enhancing PalB expression. The improvement was particularly pronounced with Origami B(DE3) as a host, suggesting that both folding and disulfide bond formation may be major factors limiting PalB expression. Fusion tag technique was also explored by constructing several PalB fusions for the evaluation of their expression performance. While the solubility was enhanced for most PalB fusions, only the DsbA tag was effective in boosting PalB activity, possibly by both enhanced solubility and correct disulfide bond formation. Our results suggest that PalB activity is closely associated with correct disulfide bond formation, and increased solubilization by PalB fusions does not necessarily result in activity enhancement.}, author = {Xu, Y. L. and Yasin, A. and Tang, R. and Scharer, J. M. and Moo-Young, M. and Chou, C. P.} } @article {21895, title = {In vivo monitoring and alleviation of extracytoplasmic stress to recombinant protein overproduction in the periplasm of Escherichia coli}, journal = {Biochemical Engineering Journal}, volume = {42}, number = {1}, year = {2008}, note = {Narayanan, Niju Follonier, Stephanie Chou, C. Perry}, pages = {13-19}, abstract = {In Escherichia coli, there are two major pathways, i.e. Cpx and sigma(E), for dealing with the extracytoplasmic stress in the cell envelope. Due to the unique periplasmic processing steps and the tendency to form periplasmic inclusion bodies, penicillin acylase (PAC) offers a model system for studying he induction of extracytoplasmic stress associated with recombinant proteins overproduction in the periplasm of E. coli. In this study, E. coli strains carrying the lacZ reporter gene fusion with the promoters of three stress-responsive genes, i.e. degP, cpxP, and rpoH, were constructed in the JM 109 background for characterization. We demonstrate that pac overexpression induced the extracytoplasmic stress primarily via the Cpx pathway. The upregulated cpxP promoter activity can be a suitable sensor for in vivo monitoring of the extracytoplasmic stress upon pac overexpression. However, such physiological challenge was not observed and all the three promoter activities were reduced when arabinose was used to induce pac overexpression. This result suggests that the physiological impact observed for the IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced cultures could be overcome by the use of arabinose for induction. The extracytoplasmic stress response associated with pac overexpression could be significantly alleviated by the exogenous presence of DegP, but only partially alleviated by its mutant derivative of DegP(S210A.) (C) 2008 Elsevier B.V. All rights reserved.}, author = {Narayanan, N. and Follonier, S. and Chou, C. P.} } @article {21892, title = {Periplasmic chaperone FkpA reduces extracytoplasmic stress response and improves cell-surface display on Escherichia coli}, journal = {Enzyme and Microbial Technology}, volume = {42}, number = {6}, year = {2008}, note = {Narayanan, Niju Chou, C. Perry}, pages = {506-513}, abstract = {When enhanced yellow fluorescence protein (EYFP) was displayed on the cell surface of Escherichia coli using EstA* as a carrier, the integrity of the cell envelope was compromised and cell physiology was severely impaired, resulting in poor display performance. Coexpression of fkpA, a gene encoding periplasmic chaperone, was demonstrated to be effective for restoring cell physiology and enhancing display performance. On the basis of monitoring the promoter activities of degP, rpoH, and cpxP under various culture conditions, we also showed that cell-surface display using EstA* as a carrier induced the extracytoplasmic stress response and the stress could be partially alleviated by fkpA coexpression. (C) 2008 Elsevier Inc. All rights reserved.}, author = {Narayanan, N. and Chou, C. P.} } @article {21893, title = {Physiological improvement to enhance Escherichia coli cell-surface display via reducing extracytoplasmic stress}, journal = {Biotechnology Progress}, volume = {24}, number = {2}, year = {2008}, note = {Narayanan, Niju Chou, C. Perry}, pages = {293-301}, abstract = {Cell physiology was impaired when enhanced yellow fluorescence protein (EYFP) was displayed on the Escherichia coli cell surface, resulting in growth arrest and poor display performance. Coexpression of Skp, a periplasmic chaperone known to interact with several outer membrane proteins for their transport and insertion in the outer membrane, was demonstrated to be effective to restore cell physiology. When Skp was coexpressed with EYFP display, host cells became less sensitive to ethylenediaminetetraacetic acid and sodium dodecyl sulfate, implying that cell physiology was improved. Most importantly, the display performance was highly enhanced as a result of the increased specific fluorescence intensity without growth arrest. The results of transmission electron microscopy indicate that the density of surface-displayed EYFP was highly increased upon Skp coexpression. Cells with EYFP display experienced extracytoplasmic stress, as reflected by the induced promoter activities of three stress-responsive genes, degP, CPXP, and rpoH. The extracytoplasmic stress reflected by the degP promoter activity appears to be consistent with the cell physiology observed phenotypically under various culture conditions for cell-surface display. Therefore, the P-degP::lacZ allele was proposed to be a suitable "sensor" for monitoring the extracytoplasmic stress and cell physiology during the course of E. coli cell-surface display.}, author = {Narayanan, N. and Chou, C. P.} } @article {22003, title = {Preparation of inorganic-organic anion-exchange membranes and their application in plasmid DNA and RNA separation}, journal = {Journal of Membrane Science}, volume = {311}, number = {1-2}, year = {2008}, note = {Chang, Chao-Shuan Ni, Ho-Sheng Suen, Shing-Yi Tseng, Wen-Chi Chiu, Hsin-Cheng Chou, C. Perry}, pages = {336-348}, abstract = {Inorganic-organic anion-exchange membranes were prepared in this study by coating a precursor, the product of N-[3-(trimethoxysilyl)propyl] ethylene diamine reacted with 3-(triethoxysilyl)propyl isocyanate, on macroporous glass fiber and alumina membranes, followed by bromoethane treatment. The FTIR results demonstrated the successful membrane modification, and the resulted anion-exchange capacities were 6.2 and 1.5 mu equiv./cm(2), respectively, for modified glass fiber membrane and modified alumina membrane. In batch adsorption process, the corresponding adsorption capacity for plasmid DNA could be sorted by: commercial polymeric SB6407 > modified glass fiber > modified alumina membrane; while for RNA adsorption, the order became: modified glass fiber approximate to SB6407 > modified alumina membrane. The optimal elution condition found from batch desorption performance was: 2 M NaCl in 50 mM Tris-HCl, pH 8 for RNA elution, followed by 1 M NaCl and 20\% ethanol in 50 mM Tris-HCl, pH 8 for plasmid DNA elution. In membrane chromatography process, plasmid DNA and RNA could be clearly separated from the feed of 10 mu g plasmid DNA + 10 mu g RNA mixture or cell lysate by one piece of 47 mm modified glass fiber or SB6407 membrane, but not by the stacked 13 mm modified alumina membranes. The overall plasmid DNA recovery for the modified glass fiber membrane was 98-106\%, higher than that of SB6407 membrane (91-96\%). (c) 2007 Elsevier B.V. All rights reserved.}, author = {Chang, C. S. and Ni, H. S. and Suen, S. Y. and Tseng, W. C. and Chiu, H. C. and Chou, C. P.} } @article {22004, title = {Therapeutic activity of soluble CD83: Comments on Pashine et al}, journal = {Immunology Letters}, volume = {115}, number = {1}, year = {2008}, note = {Chou, C. Perry}, pages = {20-20}, author = {Chou, C. P.} } @article {21927, title = {Effect of heat-shock proteins for relieving physiological stress and enhancing the production of penicillin acylase in Escherichia coli}, journal = {Biotechnology and Bioengineering}, volume = {96}, number = {5}, year = {2007}, note = {Wu, Ming-Shen Pan, Kao-Lu Chou, C. Perry}, pages = {956-966}, abstract = {High-level expression of recombinant penicillin acylase (PAC) using the strong trc promoter system in Escherichia coli is frequently limited by the processing and folding of PAC precursors (proPAC) in the periplasm, resulting in physiological stress and inclusion body formation in this compartment. Periplasmic heat-shock proteins with protease or chaperone activity potentially offer a promise for overcoming this technical hurdle. In this study, the effect of the two genes encoding periplasmic heat-shock proteins, that is degP and fkpA, on pac overexpression was investigated and manipulation of the two genes to enhance the production of recombinant PAC was demonstrated. Both AdegP and AfkpA mutants showed defective culture performance primarily due to growth arrest. However, pac expression level was not seriously affected by the mutations, indicating that the two proteins were not directly involved in the pathway for periplasmic processing of proPAC. The growth defect caused by the two mutations (i.e., OdegP and AfkpA) was complemented by either one of the wildtype proteins, implying that the function of the two proteins could partially overlap in cells overexpressing pac. The possible role that the two heat-shock proteins played for suppression of physiological stress caused by pac overexpression is discussed.}, author = {Wu, M. S. and Pan, K. L. and Chou, C. P.} } @article {21889, title = {Engineering cell physiology to enhance recombinant protein production in Escherichia coli}, journal = {Applied Microbiology and Biotechnology}, volume = {76}, number = {3}, year = {2007}, note = {Chou, C. Perry}, pages = {521-532}, abstract = {The advent of recombinant DNA technology has revolutionized the strategies for protein production. Due to the well-characterized genome and a variety of mature tools available for genetic manipulation, Escherichia coli is still the most common workhorse for recombinant protein production. However, the culture for industrial applications often presents E. coli cells with a growth condition that is significantly different from their natural inhabiting environment in the gastrointestinal tract, resulting in deterioration in cell physiology and limitation in cell{\textquoteright}s productivity. It has been recognized that innovative design of genetically engineered strains can highly increase the bioprocess yield with minimum investment on the capital and operating costs. Nevertheless, most of these genetic manipulations, by which traits are implanted into the workhorse through recombinant DNA technology, for enhancing recombinant protein productivity often translate into the challenges that deteriorate cell physiology or even jeopardize cell survival. An in-depth understanding of these challenges and their corresponding cellular response at the molecular level becomes crucial for developing superior strains that are more physiologically adaptive to the production environment to improve culture productivity. With the accumulated knowledge in cell physiology, whose importance to gene overexpression was to some extent undervalued previously, this review is intended to focus on the recent biotechnological advancement in engineering cell physiology to enhance recombinant protein production in E. coli.}, author = {Chou, C. P.} } @article {21896, title = {Arabinose-induction of lac-derived promoter systems for penicillin acylase production in Escherichia coli}, journal = {Biotechnology Progress}, volume = {22}, number = {3}, year = {2006}, note = {Narayanan, Niju Hsieh, Ming-Yi Xu, Yali Chou, C. Perry}, pages = {617-625}, abstract = {Arabinose was shown to serve as an effective inducer for induction of the lac-derived promoters in Escherichia coli using penicillin acylase (PAC) as a model protein. Upon the induction with a conventional inducer, isopropyl-beta-D-thiogalactopyranoside (IPTG), for pac overexpression, which is regulated by the trc or (DE3)/T7 promoter, the production of PAC was limited by the accumulation of PAC precursors (proPAC) as inclusion bodies. Negative cellular responses, such as growth inhibition and cell lysis, were frequently observed, resulting in a low pac expression level and poor culture performance. Interestingly, these technical hurdles can be overcome simply through the use of arabinose as an inducer. The results indicate that arabinose not only induced the lac-derived promoter systems (i.e., trc and (DE3)/T7) for pac (or LL pac) overexpression but also facilitated the posttranslational processing of proPAC for maturation. However, the arabinose-inducibility appears to be host-dependent and becomes less observable in the strains with a mutation in the ara operon. The arabinose-inducibility was also investigated in the expression system with the coexistence of the trc promoter system regulating pac expression and another arabinose-inducible promoter system of araB regulating degP coexpression.}, author = {Narayanan, N. and Hsieh, M. Y. and Xu, Y. L. and Chou, C. P.} } @article {21931, title = {Characterization of the T7 promoter system for expressing penicillin acylase in Escherichia coli}, journal = {Applied Microbiology and Biotechnology}, volume = {72}, number = {3}, year = {2006}, note = {Xu, Yali Rosenkranz, Stefan Weng, Chiao-Ling Scharer, Jeno M. Moo-Young, Murray Chou, C. Perry}, pages = {529-536}, abstract = {The pac gene encoding penicillin acylase (PAC) was overexpressed under the regulation of the T7 promoter in Escherichia coli. PAC, with its complex formation mechanism, serves as a unique target protein for demonstration of several key strategies for enhancing recombinant protein production. The current T7 system for pac overexpression was fraught with various technical hurdles. Upon the induction with a conventional inducer of isopropyl-beta-D-thiogalactopyranoside (IPTG), the production of PAC was limited by the accumulation of PAC precursors (proPAC) as inclusion bodies and various negative cellular responses such as growth inhibition and cell lysis. The expression performance could be improved by the coexpression of degP encoding a periplasmic protein with protease and chaperone activities. In addition to IPTG, arabinose was shown to be another effective inducer. Interestingly, arabinose not only induced the current T7 promoter system for pac expression but also facilitated the posttranslational processing of proPAC for maturation, resulting in significant enhancement for the production of PAC. Glycerol appeared to have an effect similar to, but not as significant as, arabinose for enhancing the production of PAC. The study highlights the importance of developing suitable genetically engineered strains with culture conditions for enhancing recombinant protein production in E. coli.}, author = {Xu, Y. L. and Rosenkranz, S. and Weng, C. L. and Scharer, J. M. and Moo-Young, M. and Chou, C. P.} } @article {21899, title = {High-level gene expression for recombinant penicillin acylase production using the araB promoter system in Escherichia coli}, journal = {Biotechnology Progress}, volume = {22}, number = {6}, year = {2006}, note = {Narayanan, Niju Xu, Yali Chou, C. Perry}, pages = {1518-1523}, abstract = {The pac gene encoding penicillin acylase (PAC) was overexpressed under the regulation of the araB promoter (P-araB, also known as P-BAD) in Escherichia coli (E. coli). The current P-araB expression system exhibited minimum leaking pac expression in the absence of arabinose as well as fast and high-level pac expression upon induction with arabinose in a wide concentration range. The production of PAC was limited by the accumulation of PAC precursors (i.e., proPAC in both soluble and insoluble forms) and various negative cellular responses, such as growth arrest and cell lysis. The culture performance could be improved by degP coexpression and the individual contribution of DegP protease and chaperone activities to the enhancement on the production of PAC was characterized. The study highlights the importance of identifying the step(s) limiting high-level gene expression and subsequent design and construction of the host/vector system for enhancing recombinant protein production in E. coli.}, author = {Narayanan, N. and Xu, Y. L. and Chou, C. P.} } @article {21932, title = {Chaperone-mediated folding and maturation of the penicillin acylase precursor in the cytoplasm of Escherichia coli}, journal = {Applied and Environmental Microbiology}, volume = {71}, number = {10}, year = {2005}, note = {Xu, YL Weng, CL Narayanan, N Hsieh, MY Anderson, WA Scharer, JM Moo-Young, M Chou, CP}, pages = {6247-6253}, abstract = {Expression of the leaderless pac gene (LL pac), which lacks the coding region for the signal peptide of penicillin acylase (PAC), in Escherichia coli was conducted. It was demonstrated that the PAC precursor, proPAC, can be produced and even processed to form mature PAC in the cytoplasm, indicating that the posttranslational processing steps for PAC maturation can occur in both the periplasm and the cytoplasm of E. coli. The outcome of proPAC folding and PAC maturation could be affected by several factors, such as inducer type, proPAC formation rate, and chaperone availability. Misfolding of proPAC in the cytoplasm could be partially resolved through the coexpression of cytoplasmic chaperones, such as trigger factor, GroEL/ES, or DnaK/J-GrpE. The three chaperones tested showed different extents of the effect on proPAC solublization and PAC maturation, and trigger factor had the most prominent one. However, the chaperone-mediated solublization of proPAC did not guarantee its maturation, which is usually limited by the first autoproteolytic step. It was observed that arabinose could act as an effective inducer for the induction of LLpac expression regulated by the lac-derived promoter system of trc. In addition, PAC maturation could be highly facilitated by arabinose supplementation and coexpression of trigger factor, suggesting that the coordination of chaperone systems with proper culture conditions could dramatically impact recombinant protein production. This study suggests that folding/misfolding of proPAC could be a major step limiting the overproduction of PAC in E. coli and that the problem could be resolved through the search for appropriate chaperones for coexpression. It also demonstrates the analogy in the issues of proPAC misfolding as well as the expression bottleneck occurring in the cytoplasm (i.e., LL pac expression) and those occurring in the periplasm (i.e., wild-type pac expression).}, author = {Xu, Y. L. and Weng, C. L. and Narayanan, N. and Hsieh, M. Y. and Anderson, W. A. and Scharer, J. M. and Moo-Young, M. and Chou, C. P.} } @article {21929, title = {Cytoplasmic overexpression, folding, and processing of penicillin acylase precursor in Escherichia coli}, journal = {Biotechnology Progress}, volume = {21}, number = {5}, year = {2005}, note = {Xu, YL Hsieh, MY Narayanan, N Anderson, WA Scharer, JM Moo-Young, M Chou, CP}, pages = {1357-1365}, abstract = {Penicillin acylase (PAC) precursor, proPAC, was overproduced in a soluble or insoluble form in the cytoplasm of Escherichia coli through the expression of the leader-less pac gene (ll-pac) devoid of the coding region for the signal peptide of PAC. Also, a portion of the overexpressed proPAC was further processed to form mature PAC, indicating that the posttranslational processing steps for PAC maturation can occur in both the periplasm and the cytoplasm of E. coli. The cultivation performance for ll-pac expression was limited by several factors, including (1) misfolding of proPAC, resulting in the aggregation of insoluble proPAC as inclusion bodies, (2) intracellular proteolysis, leading to the degradation of the overexpressed gene products, and (3) inefficient PAC maturation, limiting the formation of active PAC. The effect of coexpression of various cytoplasmic chaperones, including trigger factor, GroEL/ES, DnaK/J-GrpE, and their combinations, on ll-pac expression was investigated. Intracellular proteolysis of the overexpressed gene products could be prevented by coexpression of GroEL/ES. On the other hand, coexpression of trigger factor appeared to be able to facilitate the folding of soluble proPAC and to improve PAC maturation. The roles of trigger factor and GroEL/ES could be coordinated to significantly improve ll-pac expression performance. DnaK/J-GrpE had an effect for solublization of proPAC and perhaps, similar to trigger factor, for improving PAC maturation. The ll-pac expression performance was also significantly improved through the simultaneous coexpression of DnaK/J-GrpE and GroEL/ES. The results of the study suggest that the folding and/or processing of proPAC could be a major issue limiting the overproduction of PAC in E. coli and the bottleneck could be eliminated through the coexpression of appropriate chaperone(s).}, author = {Xu, Y. L. and Hsieh, M. Y. and Narayanan, N. and Anderson, W. A. and Scharer, J. M. and Moo-Young, M. and Chou, C. P.} } @article {21967, title = {Fedbatch operation using Clostridium acetobutylicum suspension culture as biocatalyst for enhancing hydrogen production}, journal = {Biotechnology Progress}, volume = {19}, number = {2}, year = {2003}, note = {Chin, HL Chen, ZS Chou, CP}, pages = {383-388}, abstract = {We demonstrated the feasibility of fedbatch operation using Clostridium acetobutylicum suspension culture as a biocatalyst for the continuous production of hydrogen. The optimum operating pH and temperature of the current cultivation system for hydrogen production were pH 6.0 and 37degreesC, respectively. The volumetric loading of the bioreactor for hydrogen production can be as high as 650 mmol hydrogen/L culture with a yield at approximately 2.0 mol hydrogen/mol glucose. Acetate and butyrate made up approximately 80\% of the total metabolites. The inhibitory effect from the two metabolites on the hydrogen production process was investigated. Butyrate at a concentration higher than 13 g/L significantly inhibited not only cell growth but also hydrogen production (i.e., specific hydrogen production rate). Acetate appears to be less toxic than butyrate to the hydrogen production process. While significantly inhibiting cell growth, acetate hardly affected hydrogen production. Finally, the factors limiting cultivation performance were discussed and possible strategies for enhancing the production of hydrogen were proposed.}, author = {Chin, H. L. and Chen, Z. S. and Chou, C. P.} } @article {21983, title = {Roles of DegP in prevention of protein misfolding in the Periplasm upon overexpression of penicillin acylase in Escherichia coli}, journal = {Journal of Bacteriology}, volume = {185}, number = {10}, year = {2003}, note = {Pan, KL Hsiao, HC Weng, CL Wu, MS Chou, CP}, pages = {3020-3030}, abstract = {Enhancement of the production of soluble recombinant penicillin acylase in Escherichia coli via coexpression of a periplasmic protease/chaperone, DegP, was demonstrated. Coexpression of DegP resulted in a shift of in vivo penicillin acylase (PAC) synthesis flux from the nonproductive pathway to the productive one when pac was overexpressed. The number of inclusion bodies, which consist primarily of protein aggregates of PAC precursors in the periplasm, was highly reduced, and the specific PAC activity was highly increased. DegP was a heat shock protein induced in response to pac overexpression, suggesting that the protein could possibly suppress the physiological toxicity caused by pac overexpression. Coexpression of DegP(S210A), a DegP mutant without protease activity but retaining chaperone activity, could not suppress the physiological toxicity, suggesting that DegP protease activity was primarily responsible for the suppression, possibly by degradation of abnormal proteins when pac was overexpressed. However, a shortage of periplasmic protease activity was not the only reason for the deterioration in culture performance upon pac overexpression because coexpression of a DegP-homologous periplasmic protease, DegQ or DegS, could not suppress the physiological toxicity. The chaperone activity of DegP is proposed to be another possible factor contributing to the suppression.}, author = {Pan, K. L. and Hsiao, H. C. and Weng, C. L. and Wu, M. S. and Chou, C. P.} } @article {21977, title = {Effect of pH on high-temperature production of bacterial penicillin acylase in Escherichia coli}, journal = {Biotechnology Progress}, volume = {18}, number = {3}, year = {2002}, note = {Huang, SW Lin, YH Chin, HL Wang, WC Kuo, BY Chou, CP}, pages = {668-671}, abstract = {High-temperature-oriented production of bacterial penicillin acylase (PAC), which is usually expressed at low temperatures (less than 30 degreesC), was demonstrated in this study via heterologous expression of the Providencia rettgeri (P. rettgeri) pac gene in Escherichia coli (E. coli). While it is possible to produce PAC at a temperature as high as 37 degreesC, the environmental condition (specifically, culture pH) critically affected culture performance. Production of PAC at 37 degreesC was feasible only when culture pH was close to neutral (i.e., 6.5-7.5). Outside this pH range, cell physiology for the host/ vector system was seriously affected, resulting in poor culture performance. In acidic culture environments, temperature significantly affected the pac expression level and specific PAC activity decreased with an increase in culture temperature. In basic culture environments, cell growth was seriously inhibited though the pac expression level was minimally affected by temperature. Such unusual types of pH and temperature effects on pac expression were never reported for bacterial PACs. The results suggest that culture pH should be precisely controlled for the current host/ vector systems being applied on the overproduction of P. rettgeri PAC in E. coli at high temperatures.}, author = {Huang, S. W. and Lin, Y. H. and Chin, H. L. and Wang, W. C. and Kuo, B. Y. and Chou, C. P.} } @article {21982, title = {Strain improvement to enhance the production of recombinant penicillin acylase in high-cell-density Escherichia coli cultures}, journal = {Biotechnology Progress}, volume = {18}, number = {6}, year = {2002}, note = {Lin, YH Hsiao, HC Chou, CP}, pages = {1458-1461}, abstract = {Using fed-batch operation for high-cell-density cultivation, efforts are frequently made for optimization of culture parameters, particularly feeding strategy. The current study also emphasized the importance of selecting strains for the production of recombinant proteins in high-cell-density cultures. With Escherichia coli penicillin acylase (PAC) as a target protein, the host/vector system of MDDeltaP7 harboring pTrcKnPAC2902 and pKS12 was designed for optimization of fed-batch cultivation for recombinant protein production. The host, MDDeltaP7, potentially had a high translational and periplasmic processing efficiency for pac expression. On the other hand, the vector, pTrcKnPAC2902, was genetically constructed for pac overexpression. Coexistence of the other vector, pKS12, significantly enhanced PAC production by improving cell physiology and reducing the amount of inclusion body formation upon pac overexpression. Am extremely high volumetric PAC activity at 37,500 U/L was obtained with the use of the developed host/vector system under optimum fed-batch culture conditions.}, author = {Lin, Y. H. and Hsiao, H. C. and Chou, C. P.} } @article {21980, title = {A biochemical engineering approach for enhancing production of recombinant penicillin acylase in Escherichia coli}, journal = {Bioprocess and Biosystems Engineering}, volume = {24}, number = {4}, year = {2001}, note = {Lin, WJ Kuo, BY Chou, CP}, pages = {239-247}, abstract = {The production of recombinant penicillin acylase (PAC) in Escherichia coli was optimized in this study. The effect of using a selection of host/vector systems as well as varying culture conditions on the production of PAC was investigated. The production of PAC based on the use of the native pac promoter was inefficient and could be significantly improved by using the strong trc promoter for regulation of pac expression. A mutant strain MDDeltaP7 was shown to be a suitable host for the production of PAC since the efficiency of both pac translation and posttranslational processing for MDDeltaP7 was significantly higher than that for the parent strain HBPAC101. However, the accumulation of inclusion bodies tended to limit the production of PAC as pac transcriptional and translational efficiency was increased. It has been demonstrated that, in addition to the increase in pac transcriptional and translational efficiency, the protein synthesis flux throughout pac expression steps should be balanced for enhancing the production of PAC in E. coli. With the optimization of the host/vector system and culture conditions, culture performance for the production of recombinant PAC was greatly improved. Process bottlenecks limiting the production of PAC were also discussed.}, author = {Lin, W. J. and Kuo, B. Y. and Chou, C. P.} } @article {21978, title = {DegP-coexpression minimizes inclusion-body formation upon overproduction of recombinant penicillin acylase in Escherichia coli}, journal = {Biotechnology and Bioengineering}, volume = {73}, number = {6}, year = {2001}, note = {Lin, WJ Huang, SW Chou, CP}, pages = {484-492}, abstract = {We demonstrated the enhancement of recombinant penicillin acylase (PAC) production in Escherichia coli by increasing the intracellular concentration of the periplasmic protease DegP. Using appropriate host/vector systems (e.g., HB101 harboring pTrcKnPAC2902 or MD Delta P7 harboring pTrcKnPAC2902) in which the expression of the pac gene was regulated by the strong trc promoter, the overproduction of PAC was often limited by periplasmic processing and inclusion bodies composed of protein aggregates of PAC precursors were formed in the periplasm. The amount of these periplasmic inclusion bodies was significantly reduced and PAC activity was significantly increased upon coexpression of DegP. The specific PAC activity reached an extremely high level of 674 U/L/OD600 for MD Delta P7 harboring pTrcK-nPAC2902 and pKS12 under optimum culture conditions. However, such improvement in the production of PAC was not observed for the expression systems (e.g., MD Delta P7 harboring pCLL2902) in which the periplasmic processing was not the step limiting the production of PAC. The results suggest that DegP could in vivo assist the periplasmic processing though the enzyme is shown to be not absolutely required for the formation of active PAC in E. coli. In addition, the steps limiting the production of PAC are identified and the reasons for the formation of PAC inclusion bodies are discussed here. (C) 2001 John Wiley \& Sons, Inc.}, author = {Lin, W. J. and Huang, S. W. and Chou, C. P.} } @article {21979, title = {High-level extracellular production of penicillin acylase by genetic engineering of Escherichia coli}, journal = {Journal of Chemical Technology and Biotechnology}, volume = {76}, number = {10}, year = {2001}, note = {Lin, WJ Huang, SW Chou, CP}, pages = {1030-1037}, abstract = {The extracellular production of penicillin acylase (PAC) in genetically engineered Escherichia coli by coexpression of the brp gene encoding bacteriocin release protein (BRP) and the pac gene was demonstrated. Cell physiology was affected while PAC was released into the medium, depending on the strategy for brp expression. The performance for the production and release of PAC was optimized by taking several culture parameters, including host, inducer (mitomycin C) concentration, and induction timing for brp expression, into consideration. The effect of PAC release on inclusion body formation was also investigated. It was observed that the amount of inclusion bodies was significantly affected by brp expression. A reason for the limitation of PAC production and a strategy for resolving this problem are proposed. (C) 2001 Society of Chemical Industry.}, author = {Lin, W. J. and Huang, S. W. and Chou, C. P.} } @article {21981, title = {Improving production of penicillin acylase in Escherichia coli via efficient DegP-mediated processing of precursors in periplasm}, journal = {Process Biochemistry}, volume = {37}, number = {1}, year = {2001}, note = {Lin, YH Fang, WL Lin, WJ Huang, SW Chou, CP}, pages = {23-30}, abstract = {Penicillin acylase (PAC) from Escherichia coli has a complex enzyme formation mechanism that is unusual for prokaryotic proteins. In this study, PAC is used as a model protein to demonstrate an important concept of developing genetic strategies for improving recombinant protein production in E. coli; namely, the bottleneck gene expression step(s) limiting recombinant protein production must be precisely identified. Using the strong promoter system of trc for regulation of pac gene expression, the overproduction of PAC was often limited by translocation and/or periplasmic processing steps, resulting in intracellular accumulation of various types of PAC precursors. The over-accumulated PAC precursors formed inclusion bodies in the cytoplasm and/or periplasm. The periplasmic protease DegP could efficiently assist periplasmic processing of PAC precursors and, therefore, the amount of periplasmic inclusion bodies was significantly reduced. The PAC inclusion bodies remaining in DegP-coexpressing cells were located in the cytoplasm where the DegP function could not reach. These results indicate that the activity of DegP expression based on pKS12 was high enough for processing the over-accumulated periplasmic PAC precursors in the current expression systems. PAC activity was significantly increased due to DegP-mediated processing in the periplasm. The strategy of DegP coexpression could be further applied to estimate the amounts of various PAC precursors and pac translational efficiency for different pac expression systems could be compared. (C) 2001 Elsevier Science Ltd. All rights reserved.}, author = {Lin, Y. H. and Fang, W. L. and Lin, W. J. and Huang, S. W. and Chou, C. P.} } @article {21974, title = {An approach for enhancing heterologous production of Providencia rettgeri penicillin acylase in Escherichia coli}, journal = {Biotechnology Progress}, volume = {16}, number = {3}, year = {2000}, note = {Chou, CP Wang, WC Lin, MI}, pages = {315-318}, abstract = {Heterologous production of Providencia rettgeri penicillin acylase (PAC) was optimized in Escherichia coli. Several factors, including carbon, temperature, and host effects, were identified to be critical for the enzyme overproduction. The optimum culture conditions for the enzyme production vary for different host/vector systems. With the optimization, both volumetric and specific PAC activities could be significantly improved by more than 50-fold compared to the native expression in P. rettgeri. The heterologous production could be possibly limited by translation or posttranslational steps, depending on the culture temperature and host/vector system. To our knowledge, this is the first evidence demonstrating the limiting step for the production of P. rettgeri PAC and the existence of the P, rettgeri PAC precursor.}, author = {Chou, C. P. and Wang, W. C. and Lin, M. I.} } @article {21971, title = {Effect of carbon source on inclusion body formation upon overproduction of periplasmic penicillin acylase in Escherichia coli}, journal = {Journal of the Chinese Institute of Chemical Engineers}, volume = {31}, number = {3}, year = {2000}, note = {Chou, CP Tseng, JH}, pages = {219-224}, abstract = {The effect of carbon supplementation on penicillin acylase (PAC) production and inclusion body formation was investigated. The host effect was also examined by comparing culture performances using HB101 or its derived mutant, MD Delta P7, as the expression host. Among several carbons tested, glycerol was found to be optimum for PAC production. PAC activity was increased and the amount of inclusion bodies was reduced upon glycerol supplementation. The ef feet of carbon source on inclusion body formation appears to be strain-dependent. With the optimization on the culture parameters (i.e., host and carbon effects), volumetric PAC activity could be significantly improved at more than fourfold that of the control experiment. This information is useful for developing expression strategies for optimization of PAC production.}, author = {Chou, C. P. and Tseng, J. H.} } @article {21970, title = {Genetic strategies to enhance penicillin acylase production in Escherichia coli}, journal = {Enzyme and Microbial Technology}, volume = {27}, number = {10}, year = {2000}, note = {Chou, CP Lin, WJ Kuo, BY Yu, CC 9th European Congress on Biotechnology (ECB9) Jul 11-15, 1999 Brussels, belgium}, pages = {766-773}, abstract = {We demonstrated the improvement of penicillin acylase (PAC) production by optimization of the host/vector system using genetic engineering strategies. Several expression plasmids with improved efficiency for the transcription of the pac gene and/or translation of the pac mRNA were constructed. Mutant strains, isolated by a novel screening method, were effective for use as the expression host to produce PAC. The feasibility of using the mutant strains harboring a selection of expression plasmids for the production of PAC was evaluated. The effect of the mutation(s) resulting in the improved PAC producing ability was characterized. While the production of PAC was significantly enhanced using the optimized host/vector system, the formation of PAC inclusion bodies was shown to be another seep limiting the production of recombinant PAC. (C) 2000 Elsevier Science Inc. All rights reserved.}, author = {Chou, C. P. and Lin, W. J. and Kuo, B. Y. and Yu, C. C.} } @article {21969, title = {Production of heterologous Providencia rettgeri penicillin acylase in Escherichia coli}, journal = {Journal of the Chinese Institute of Chemical Engineers}, volume = {31}, number = {2}, year = {2000}, note = {Chou, CP Lin, MI Wang, WC}, pages = {135-144}, abstract = {In this work, we constructed several expression plasmids for the production of Providencia rettgeri penicillin acylase (EC 3.5.1.11; PAC) in Escherichia coli. DNA fragments containing the pac gene from P. rettgeri ATCC31052 were PCR-amplified and cloned in to the expression vectors so that the pac gene expression was controlled by the tac or trc promoter system. The effects of culture conditions, such as IPTG concentration, temperature, and carbon source, on the native or heterologous expression were investigated. Among a selection of expression systems, JM109 harboring pUTKnPAC2601 gave the highest PAC activity and could be of interest for industrial application. Cultivation should be performed at a temperature ranging from 28 degrees C to 33 degrees C and the medium could be supplemented with glycerol. The host/vector system offers an opportunity for high-temperature-oriented PAC production, which is usually conducted at a low temperature. Volumetric PAC activity at more than fiftyfold (similar to 820 U/L) that of the native expression in ATCC31052 (similar to 15 U/L) could be reached by optimization of the host/vector system and culture conditions.}, author = {Chou, C. P. and Lin, M. I. and Wang, W. C.} } @article {21972, title = {Effect of SecB chaperone on production of periplasmic penicillin acylase in Escherichia coli}, journal = {Biotechnology Progress}, volume = {15}, number = {3}, year = {1999}, note = {Chou, CF Tseng, JH Kuo, BY Lai, KM Lin, MI Lin, HK}, pages = {439-445}, abstract = {The effect of SecB chaperone on production of periplasmic penicillin acylase (PAC) in Escherichia coli was investigated. It appears that formation of PAC required the function of SecB chaperone and the amount of SecB required was at a basal level. The secB mutant was defective in production of PAC, and the impairment could be complemented by extrachromosomally supplementing SecB in trans. The function of SecB might be primarily stabilizing the cytoplasmic PAC precursors. Overproduction of SecB chaperone usually resulted in an increase in the amount of PAC precursors without enhancing PAC activity. In addition, most of the PAC precursors were located in the periplasm, suggesting that formation of active PAC was likely limited by periplasmic processing steps.}, author = {Chou, C. P. and Tseng, J. H. and Kuo, B. Y. and Lai, K. M. and Lin, M. I. and Lin, H. K.} } @article {21976, title = {Genetic manipulation to identify limiting steps and develop strategies for high-level expression of penicillin acylase in Escherichia coli}, journal = {Biotechnology and Bioengineering}, volume = {63}, number = {3}, year = {1999}, note = {Chou, CP Yu, CC Tseng, JH Lin, MI Lin, HK}, pages = {263-272}, abstract = {We have identified the bottleneck steps limiting expression of penicillin acylase (PAC) through comparison of the expression performance for various PAC-expression vectors constructed by genetically modulating the efficiencies of transcription and/or translation of the pac gene. To our knowledge, this is the first report demonstrating that expression of PAC could be limited by various steps, such as transcription, translation, and post-translational steps (i.e, translocation and periplasmic processing), depending on the host/vector systems. Results also indicate that the structure of the wild-type pac gene might not be optimal for direct use in production of PAC using recombinant DNA technology. To improve the gene expression, transcription was enhanced by manipulating certain DNA bases in the pac regulatory region, whereas translation was enhanced by enlarging the spacing between the ribosome binding site and the ATG initiation codon to increase the initiation efficiency. The information is useful in terms of developing genetic strategies for overproduction of recombinant PAC in Escherichia coli. (C) 1999 John Wiley \& Sons, Inc. Biotechnol Bioeng 63: 283-272, 1999.}, author = {Chou, C. P. and Yu, C. C. and Tseng, J. H. and Lin, M. I. and Lin, H. K.} } @article {21973, title = {Manipulation of carbon assimilation with respect to expression of the pac gene for improving production of penicillin acylase in Escherichia coli}, journal = {Journal of Biotechnology}, volume = {69}, number = {1}, year = {1999}, note = {Chou, CP Tseng, JH Lin, MI Lin, HK Yu, CC}, pages = {27-38}, abstract = {A strategy of genetically manipulating carbon assimilation with respect to expression of the pac gene was employed for overproduction of recombinant penicillin acylase (PAC). Two expression plasmids of pCLL2902 and pCLL3201, which contain the pac coding region but differ in the pac regulatory region, were constructed for the production experiments. Expression of the pac gene was subjected to phenyl acetic acid (PAA-) induction and glucose catabolite repression for pCLL3201, whereas it was subjected to neither of the two transcriptional regulations for pCLL2902. The specific PAC activity for strains harboring pCLL2902 was significantly higher than that for strains harboring pCLL3201 due to an improved transcription efficiency. In addition, no inclusion bodies were observed upon production of PAC using the current expression systems. The results suggest that using the native pac promoter instead of a strong promoter such as tac for regulation is a feasible approach for production of PAC. The impact of the current expression systems is also significant from a process viewpoint since, using strains harboring pCLL2902, not only could glucose replace PAA as a carbon source of Escherichia coli cultures for production of PAC but also the volumetric PAC activity was highly improved. (C) 1999 Elsevier Science B.V. All rights reserved.}, author = {Chou, C. P. and Tseng, J. H. and Lin, M. I. and Lin, H. K. and Yu, C. C.} } @article {21975, title = {Novel strategy for efficient screening and construction of host/vector systems to overproduce penicillin acylase in Escherichia coli}, journal = {Biotechnology and Bioengineering}, volume = {65}, number = {2}, year = {1999}, note = {Chou, CP Yu, CC Lin, WJ Kuo, BY Wang, WC}, pages = {219-226}, abstract = {A novel and simple method of using penicillin for screening of mutant strains with a high penicillin acylase (PAC) activity was developed. Random mutagenesis was conducted using a PAC-producing strain resistant to 6-aminopenicillanic acid (6-APA) as the parent strain and mutants were screened with penicillin at a high concentration. Results suggest that mutants with a high minimum inhibitory concentration for penicillin (MICpenG) usually overproduce PAC. Both volumetric and specific PAC activities of a mutant, MD7, were significantly higher than those of the parent strain, HBPAC101 harboring pCLL2902. The mutation(s) resulting in the enhanced expression was mapped on the host chromosome rather than the plasmid. In addition, the mutant strain of MD Delta P7, derived by elimination of the harbored plasmid in MD7, was demonstrated to be efficient in production of PAC by using the expression plasmids for which expression of the pac gene is limited by translation. An extremely high specific PAC activity of more than 350 U/L/OD600 was reached upon cultivation of MD Delta P7 harboring pTrcKnPAC2902 in a bioreactor. As such, the strategy is effective in terms of constructing PAC overproducers and improving the process yield for production of PAC. (C) 1999 John Wiley \& Sons, Inc.}, author = {Chou, C. P. and Yu, C. C. and Lin, W. J. and Kuo, B. Y. and Wang, W. C.} } @article {21968, title = {Optimization of the host/vector system and culture conditions for production of penicillin acylase in Escherichia coli}, journal = {Journal of Bioscience and Bioengineering}, volume = {88}, number = {2}, year = {1999}, note = {Chou, CF Kuo, BY Lin, WJ}, pages = {160-167}, abstract = {Culture performance for the production of penicillin acylase (PAC) in a bioreactor was investigated using HB101 or ATCC11105 as the host and pCLL2902, pCLL3201 or pTrcKnPAC2902 as the expression plasmid. We observed that the production of PAC by HB101 harboring pCLL3201 was, similar to ATCC11105, induced by phenyl acetic acid (PAA) and catabolically repressed by glucose, whereas the production of PAC by HB101 harboring pCLL2902 did not require PAA for induction and was not repressed by glucose. PAC activity of HB101 harboring pCLL2902 was significantly higher than that of HB101 harboring pCLL3201. There was no significant effect of host or carbon source on the production of PAC using pCLL2902, The production of PAC by HB101 harboring pTrcKnPAC2902, in which the pac gene expression was controlled by the trc promoter system, was about the same as that by HB101 harboring pCLL2902, when the culture was appropriately induced with isopropyl beta-D-thiogalactopyranoside (IPTG). Therefore, the use of both pCLL2902 and pTrcKnPAC2902 could be expected to be feasible for industrial applications. However, optimization of IPTG induction for HB101 harboring pTrcKnPAC2902 might be required, since formation of inclusion bodies tends to limit the production of PAC in some cases.}, author = {Chou, C. P. and Kuo, B. Y. and Lin, W. J.} }