Engineering of microbes to produce targeted compounds often requires the engineering of multiple traits, such as pathway flux, product tolerance, and growth. For example, styrene is a high-value monomer used in the production of polymers and copolymers, most notably polystyrene, acrylonitrile-butadiene-styrene (ABS), styrene-acrylonitrile (SAN), and styrene-butadiene rubber. As one of the most important monomers in the plastics industry, its annual production exceeds 6 million metric tons per year representing a $28 billion market. However, styrene is not natively produced by E. coli, and it inhibits the growth of E. coli at concentrations below those required for economic production. To achieve economically viable styrene production targets, it is necessary to optimize the expression of enzymes in the styrene synthesis pathway, and improve styrene tolerance in E. coli. Methods for engineering such complex traits are often laborious and thus difficult to extend beyond a single trait at a time without large and sophisticated engineering teams and resources. We reported the iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method, which incorporates CRISPR editing and DNA barcoding, thereby enabling the massive and trackable editing of E. coli genomes iteratively (Fig. 1). In this study, we used 4 rounds (~1 weeks) of iCREATE to perform a comprehensive mapping of the effect of changes in the expression and specificity of the multiple proteins on styrene production. The designer libraries targeted ~120,000 mutations across 90 genes, spanning regulatory and metabolic networks, to engineer E. coli central carbon metabolism, styrene synthesis, and styrene tolerance. The combinatorial mutants were constructed and confirmed to improve styrene tolerance and production. In addition, we combined with polymerization and biosynthesis of styrene in a integration system.

Polymerization and synthesis of biostyrene by engineering E. coli using iCREATE strategy

2019 Spring National Meeting

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