OPTIMIZATION OF GAMMA-PGA BIOSYNTHESIS SUPPORTED BY SYNTHETIC BIOLOGY AND METABOLIC ENGINEERING STRATEGIES

Abstract

Poly-γ-glutamate (γ-PGA) is a natural polymer made of glutamic acid residues, synthesized by the pgs operon of Bacillus subtilis. γ-PGA has a wide range of applications as food, cosmetics and pharmaceutical additive. However, to increase its industrial attractiveness, it is necessary to cut production costs utilizing cost-competitive feedstocks for fermentation. Raw glycerol is a low-cost by-product of biodiesel plants (it accounts for 10% of the final product) that can be used as feedstock. To achieve cost-competitive γ-PGA production from glycerol a multifaceted approach has been set up that includes: 1) Characterization and optimization of pgs operon regulation: the strength of the pgs operon regulatory elements has been analysed both by a synthetic biology approach, exploiting the well-characterized expression operating unit (EOU) inserted in amyE, and by a classical in-locus transcriptional fusion. Results from the two settings will be compared. These data will now be used to finely tune pgs expression through an inducible promoter to optimize γ-PGA yield. 2) Accumulation of γ-PGA precursors by metabolic engineering: a genome-scale metabolic model was used to identify suitable targets for enhancing central carbon pathway flux toward γ-PGA synthesis. The first two B. subtilis strains, engineered according to this analysis, showed enhanced polymer production. Other target genes are under investigation. 3) Enhancement of glycerol metabolism: B. subtilis tolerance to raw glycerol obtained from a biodiesel plant (from both vegetable and animal origin) was verified. Further investigations are underway to improve glycerol uptake and consumption