Development of microbial cell factory for enhanced production of trans-cinnamic acid based on systems level metabolic engineering

Abstract

Trans-cinnamic acid is one of phenylpropanoid having many applications as precursors for cosmetics, flavoring compounds, anti-bacterial compounds, and pharmaceuticals. Trans-cinnamic acid has conventionally produced by extraction from plant sources or chemical synthesis. However, several shortcomings have been existed in conventional methods such as low concentration in plant source or the toxic byproduct generation. Therefore, in recent decades, trans-cinnamic acid production using metabolically engineered microbes has emerged as a powerful alternative method. The main goal of this thesis was to expand the capability and improve the ability of microbial biosynthesis of trans-cinnamic acid in Escherichia coli through systems level metabolic engineering strategies. First, I developed the genetically engineered E. coli which can efficiently produce L-phenylalanine (main precursor of trans-cinnamic acid) through strain engineering and plasmid based overexpression of L-phenylalanine biosynthesis pathway genes. After that, I built a biosynthetic pathway for trans-cinnamic acid by introduction of heterologous enzyme, phenylalanine-ammonia lyase, in engineered E. coli. I also optimized the culture condition for enhanced production of L-phenylalanine and trans-cinnamic acid. Lastly, the fed-batch fermentation of trans-cinnamic acid and even L-phenylalanine also carried out to see if the production performance (titer, yield, productivity) of engineered E. coli is suitable for economically industrial production or not. As a result of native pathway engineering for L-phenylalanine target and introduction of heterologous enzyme for trans-cinnamic acid production, engineered E. coli YHP05 harboring pYHP plasmid and YHP05 harboring pYHP and pCA plasmids were developed, respectively. In flask and fed-batch cultivation of YHP05 harboring pYHP plasmid, the recombinant E. coli YHP05 harboring pYHP plasmid showed good production performance compared to parental host as well as other L-phenylalanine overproducing strains. The L-phenylalanine production titer reached up to 3.91 g/L with yield of 0.270 g/g glucose and productivity of 0.082 g/L/h in flask cultivation. And the maximum L-phenylalanine titer was 52.89 g/L with yield of 0.248 g/g glucose and productivity of 1.102g/L/h in fed-batch fermentation. In case of trans-cinnamic acid production in flask and fed-batch cultivation, the recombinant E. coli YHP05 harboring pYHP and pCA plasmids also showed good production performance compared to parental host as well as other trans-cinnamic acid overproducing strains. The trans-cinnamic acid production titer reached up to 413 mg/L with productivity of 8.6 mg/L/h in flask cultivation. And the maximum trans-cinnamic acid titer was 4.1 g/L with yield of 0.072 g/g glucose and productivity of 0.057 g/L/h in fed-batch fermentation. To the best of my knowledge, this is the first report of development the microbial cell factory for production of trans-cinnamic acid based on systems level metabolic engineering approach. Moreover, the trans-cinnamic acid titer achieved in this study is 5.13-fold higher than the reported highest titer ever in trans-cinnamic acid production. This work will significantly contribute to the field of microbial trans-cinnamic acid production and developed microbial cell factory in this study can be economically feasible production platform in the future. Moreover, the systems level metabolic engineering strategies described in this study can make the other microbes be from zero to hero strain in trans-cinnamic acid production and even more other phenylpropanoids production.