Biosynthesis of poly-γ-glutamic acid in Escherichia coli by heterologous expression of pgsBCAE operon from Bacillus.

AIMS: Poly-γ-glutamic acid (γ-PGA) is an excellent water-soluble biosynthesis material. To confirm the rate-limiting steps of γ-PGA biosynthesis pathway, we introduced a heterologous Bacillus strain pathway and employed an enzyme-modulated dismemberment strategy in Escherichia coli. METHODS AND RESULTS: In this study, we heterologously introduced the γ-PGA biosynthesis pathway of two laboratory-preserved strains-Bacillus amyloliquefaciens FZB42 and Bacillus subtilis 168 into E. coli, and compared their γ-PGA production levels. Next, by changing the plasmid copy numbers and supplying sodium glutamate, we explored the effects of gene expression levels and concentrations of the substrate l-glutamic acid on γ-PGA production. We finally employed a two-plasmid induction system using an enzyme-modulated dismemberment of pgsBCAE operon to confirm the rate-limiting genes of the γ-PGA biosynthesis pathway. CONCLUSION: Through heterologously over-expressing the genes of the γ-PGA biosynthesis pathway and exploring gene expression levels, we produced 0·77 g l-1 γ-PGA in strain RSF-EBCAE(BS). We also confirmed that the rate-limiting genes of the γ-PGA biosynthesis pathway were pgsB and pgsC. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is beneficial to increase γ-PGA production and study the mechanism of γ-PGA biosynthesis enzymes.

Multi-modular engineering for renewable production of isoprene via mevalonate pathway in Escherichia coli.

AIMS: To establish the biotechnology platforms for production of bio-based chemicals in various micro-organisms is considered as a promising target to improve renewable production of isoprene. METHODS AND RESULTS: In this study, we heterologously expressed the mevalonate (MVA) isoprene biosynthesis pathway, and explored three strategies of increasing isoprene production in Escherichia coli. We first manipulated the expression levels of the MVA pathway genes through changing the gene cassettes and promoters. To introduce cofactor engineering, we then overexpressed NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene from Clostridium acetobutylicum to supply available NADPH. To reduce the inhibitory by-product accumulation, we finally knocked out acetate-producing genes, phosphate acetyl transferase and pyruvate oxidase B in E. coliJM109 (DE3), decreasing acetate accumulation 89% and increasing isoprene production 39%. The strategies described here finally increased the isoprene titre to 92 mg l-1 in two-gene deletion strain JMAB-4T7P1Trc, increasing 2·6-fold comparing to strain JM7T7. CONCLUSION: The multimodularly engineering approaches including promoter engineering, cofactor engineering and by-product reducing could be used to improve isoprene production in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The metabolic strategies in this study show us directions for further studies to promote transformation of renewable sources to isoprene.