(686g) Engineering Styrene Biosynthesis in Yeast Via Rational and Combinatorial Approaches

Renewable production of styrene, a versatile commodity chemical and monomer, has to date been limited by the low toxicity threshold of E. coli (~300 mg/L). To address this, multiple alternative methods have been investigated in parallel, including the development of more robust yeast platforms, as well as via multiple product removal strategies. Saccharomyces cerevisiae BY4741 as well as more robust industrial yeast strains have been investigated as initial strains for the development of novel styrene producers. As the immediate endogenous precursor to the styrene pathway is the proteinogenic amino acid phenylalanine, the first aim of this study was to engineer a feedback-resistant mutant capable of its overproduction. Multiple approaches of both rational and combinatorial nature were explored to this end.  This included engineering known feedback-resistant point mutations in the DAHP synthase (ARO4 ^K229L) and the chorismate mutase (ARO7 ^G141S) as well as the use of classical mutagenesis and anti-metabolites to increase flux through the shikimic acid pathway. Multiple rounds of mutagenesis coupled with increasing growth challenges ultimately elucidated a strain with increased flux through the phenylalanine pathway. This approach had led to the development of yeast strains capable of producing over 350 mg/L phenylalanine from glucose. To achieve styrene biosynthesis, it was found that only over-expression of phenylalanine ammonia lyase (PAL2 from Arabidopsis thaliana) to deaminate phenylalanine to trans-cinnamate was required as trans-cinnamate was then naturally decarboxylated to styrene by the native FDC1. Factors influencing FDC1 transcription and the impact of its expression level were investigated to ensure ample flux through the pathway was possible. At last, multiple approaches for in situ styrene removal from cultures including via liquid-liquid extraction and vapor stripping/collection were also investigated. These approaches led to improvements in overall productivity.