The Metabolic Engineering of E. coli for the Enhanced Heterologous Biosynthesis and Discovery of Complex Natural Product

Microbes are the hidden treasures for various functional compounds, mainly because of its diverse metabolites.;Compared with fastidious nature of 99% of environmental microbes, E. coli possess many traits as an industrial bacterium: fast growth rate (∼20 minutes turnover rate), easy to be genetically manipulate and accessible genome sequences. These features provide both a motivation and an experimental basis for enacting heterologous biosynthesis. It has been not only engineered for over-producing its native metabolites, but also harnessed as surrogate for heterologous productions. Escherichia coli (E. coli) has the been most prevalence engineered strain used in a myriad of applications, including producing biofuels, pharmeutuical compounds, fibers, polymers etc. In this dissertation, the researches have been done on engineering E. coli strain as the mini-factory for synthesis of erythromycin, diversification of erythromycin analogues to combat emerging antibiotic resistant strains, and surrogate for discoveries of novel polyketide natural products.;In chapter 2, an improved erythromycin heterologous biosynthesis system is developed. The establishment of erythromycin production within the heterologous host E. coli marked an accomplishment in genetic transfer capacity. Namely, over 20 genes and 50 kb of DNA was introduced to E. coli for successful heterologous biosynthetic reconstitution. However, the prospect for production levels that approach those of the native host requires the application of the engineering tools associated with E. coli. In this report, metabolic and genomic engineering was implemented to improve the E. coli cellular background and the plasmid platform supporting heterologous erythromycin formation. Results include improved plasmid stability and metabolic support for biosynthetic product formation. Specifically, the new plasmid design for erythromycin formation allowed for ≥89% stability relative to current standards (20% stability). In addition, the new strain designed to improve biosynthetic carbon flow provided ∼3-fold improvements in titer levels.;In chapter 3, E. coli based erythromycin analog production platform is built by exchanging erythromycin TDP-deoxysugar with unnatural TDP-deoxysugars. As a macrolide antimicrobial agent, erythromycin acts upon binding to 50S sub unit of bacterial ribosome, inhibiting nascent peptide chain from propagating. TDP-mycarose and TDP-desosamine were demonstrated directly involved in blocking peptide chain passage in ribosome complex. Therefore, by replacing TDP-mycarose with an unnatural erythromycin sugars could incite restoration of erythromycin activities against erythromycin resistant bacterial strains. In this study, 8 unnatural TDP-sugar pathways were constructed by Gibson assembly, followed heterologous production in BL21(DE3) coupled with chaperone proteins. Totally, 6 out of 8 deoxysugars were successfully reconstituted in E. coli. Then those 6 pathways were introduced in to E. coli based erythromycin analog production platform. 5 in 6 were demonstrated successfully complete biosynthesis of E. coli-derived erythromycin deoxysugar analogs. Among them, forsosamine and noviose-erythromycin analogs were tested positively active against erythromycin resisted B. subtilis strain.;In chapter 4, an advanced E. coli strain for metagenomics discovery of novel natural products is reported. Heterologous biosynthesis of natural products is meant to enable access to the vast array of valuable properties associated with these compounds. Often motivated by limitations inherent in native production hosts, the heterologous biosynthetic process begins with a candidate host regarded as technically advanced relative to original producing organisms. Given this requirement, E. coli has been a top choice for heterologous biosynthesis attempts as associated recombinant tools emerged and continue to develop. However, success requires overcoming challenges associated with natural product formation, including complex biosynthetic pathways and the need for metabolic support. These two challenges have been heavily featured in cellular engineering efforts completed to position E. coli as a viable surrogate host. This chapter outlines steps taken to engineer E. coli with an emphasis on genetic manipulations designed to support the heterologous production of polyketide, nonribosomal peptide, and similarly complex natural products.