Development of systems metabolic engineering tools and strategies and their applications on microbial production of natural compounds

Abstract

During pursuit of PhD degree, I accomplished development of novel tools and strategies for the production of various important natural compounds. As malonyl-CoA is an important intermediate for the production of various natural products including polyketides and phenylpropanoids, to accurately and easily measure precursor availability, malonyl-CoA biosensor using a polyketide synthase was developed. The biosensor is based on a type III polyketide synthase (PKS) RppA, enabling colorimetric screening. Using this biosensor, four representative natural compounds (two polyketides and two phenylpropanoids) could be overproduced. Having developed a biosensor that can screen target compound overproducers, development of an efficient tool for rapid and multiplex regulation of target gene expression was also needed. Therefore, a synthetic sRNA platform was developed to enable compatible and simultaneous knockdown of target genes in already engineered E. coli strains. An E. coli genome-scale sRNA library was constructed using this expanded sRNA expression platform, and was applied for enhanced production of two natural colorants. Next, integrated strategies encompassing systems metabolic engineering, membrane engineering, and vesicle engineering were developed to further augment the production of hydrophobic natural products, taking violacein derivatives as proof-of-concept examples. Aromatic polyketides can be produced from both type III and type II PKS systems

the type III PKS AaOKS used for construction of malonyl-CoA biosensor mentioned above and type II PKS from Photorhabdus can produce the same aromatic polyketide shunt product. Especially, employing type II PKS in heterologous model microorganisms (e.g., E. coli) – which is a relatively overlooked category of PKSs – was sought to produce valuable aromatic polyketides used as food additives, cosmetics, drugs, etc. Applying this strategy along with biochemical reaction analyses and in silico-assisted enzyme mutagenesis allowed successful production of carminic acid, which is a widely used red food colorant. Engineered E. coli strains were also developed which are capable of producing decaketides resistomycin (antibiotic) and $\varepsilon$-rhodomycinone (an aglycone of doxorubicin, an anticancer agent) for the first time. Silybin precursors (coniferyl-alcohol and dihydroxyquercetin) were successfully produced in E. coli, and the novel biosynthetic pathways towards $\beta$-lapachone analogues were also constructed.