Strain engineering of Escherichia coli for biobased production
While E. coli represents the most popular host system for biobased production, many chemicals are non-native to this microorganism. Specifically, odd-chain alcohols/acids are not natively produced by E. coli, or even most microorganisms, due to the lack of relevant C3-metabolic pathways. We have explored the production of non-native 1-propanol in E. coli by activation of the genomic Sleeping beauty mutase (Sbm) operon, which is a native gene operon in E. coli but its native expression often remains dormant. Importantly, activation of the Sbm operon has led to the implementation of a novel C3-fermentative pathway with the formation of a key metabolic intermediate, i.e. propionyl-CoA. The presence of propionyl-CoA, along with subsequently developed synthetic biology strategies, has opened a wide avenue for novel biosynthesis of many non-native valuable chemical compounds derived from propionyl-CoA using E. coli as a cell factory, including 1-propanol, propionate, butanone, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and 3-hydroxyvalerate.
Strain engineering of Bacillus subtilis for biobased production
Although B. subtilis is a popular Gram-positive bacterium designated as a Generally Recognized As Safe (GRAS) strain to generate products for human consumption, genetic tools of this microorganism are immature. We have developed a CRISPR-Cas9 tool kit by implementing CRISPR-Cas9 for genome editing and strain engineering of B. subtilis. The successful implementation of the CRISPR-Cas9 system has enabled extensive strain engineering, including gene knockout, gene knock-in, gene knock-down, site-specific mutation, and multiplexed manipulation on the genome, leading to my subsequent development of various novel engineered B. subtilis strains for biobased production of value-added products, including biopolymer (hyaluronic acid), amino acid (valine), organic acid (isobutyrate).
Strain engineering of anaerobic Clostridium for biobased production
We have been involved in engineering strict anaerobic Clostridium species, mainly Clostridium acetobutylicum and Clostridium pasteurianum, for 1-butanol production. While bacterial production of 1-butanol was widely explored, we specifically focused on an important feedstock issue by using glycerol, which is a cheap side-product associated with biodiesel production. C. pasteurianum can naturally dissimilate glycerol, but its genetic tools were unavailable. We have developed genetic tools for DNA transformation and heterologous gene expression in C. pasteurianum. Given these successful developments, genomic engineering of C. pasteurianum was still difficult. Also, C. acetobutylicum had the similar technical difficulty for genomic engineering, though several genetic tools already existed then. Hence, we have implemented the CRISPR-Cas9 system into these two microorganisms for genomic engineering, including gene knockout, gene knock-in, and gene-knock-down.
Microbial production of industrial enzymes and therapeutic proteins
We have developed various biochemical and genetic engineering strategies for industrial enzyme production in E. coli. Using penicillin G acylases (PAC) with a unique posttranslational processing mechanism, potential bottleneck steps limiting heterologous production of recombinant protein was demonstrated, leading to the construction of various host/vector systems for enhanced gene expression. An integrated approach that considers various issues in all bioprocess stages was taken to develop a bioprocess for effective PAC expression and purification. On the other hand, various genetic strategies were developed for heterologous expression of lipases in the various compartments of E. coli, including cytoplasm, periplasm, cell surface, and extracellular medium. The developed strategies can be applied to other target proteins, even with more complex gene expression steps, and offer an easy, efficient, and rational way for improving recombinant protein production. We have also been involved in developing biochemical and genetic engineering strategies for therapeutics production in E. coli. One product is the extracytoplamic region of human CD83 for potential treatment of autoimmune diseases and transplantation rejection. Another product is a therapeutic antibody fragment against human epidermal growth factor receptor 2 (HER2) for potential treatment of HER2-associated cancers.