Poly-γ-glutamate (γ-PGA) is a natural polymer composed by glutamic acid residues, synthesized by the pgs operon of Bacillus subtilis. γ-PGA has a wide range of applications as food, cosmetic and pharmaceutical additive. However, to increase its industrial attractiveness, it is necessary to cut production costs utilizing cost-competitive feedstocks for fermentation. A low-cost by-product that can be used as feedstock is raw glycerol, that accounts for 10% (w/w) of the total biodiesel production. To achieve cost-competitive γ-PGA production from glycerol a multifaceted approach has been set up that includes: 1) improvement of pgs expression; 2) accumulation of γ-PGA precursors by metabolic engineering; 3) enhancement of glycerol metabolism. 1) 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 be then used to finely tune pgs expression and optimize γ-PGA yield. To this end, an inducible pgs operon has been constructed. 2) 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 following this analysis, showed enhanced polymer production. Other target genes are currently under investigation. 3) 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.
Optimization of γ-PGA biosynthesis supported by synthetic biology and metabolic engineering strategies
Matteo CavalettiWriting – Original Draft Preparation
;Ilaria MassaiuInvestigation
;Lorenzo PasottiConceptualization
;Paolo MagniProject Administration
;Cinzia Calvio
Writing – Review & Editing
2018-01-01
Abstract
Poly-γ-glutamate (γ-PGA) is a natural polymer composed by glutamic acid residues, synthesized by the pgs operon of Bacillus subtilis. γ-PGA has a wide range of applications as food, cosmetic and pharmaceutical additive. However, to increase its industrial attractiveness, it is necessary to cut production costs utilizing cost-competitive feedstocks for fermentation. A low-cost by-product that can be used as feedstock is raw glycerol, that accounts for 10% (w/w) of the total biodiesel production. To achieve cost-competitive γ-PGA production from glycerol a multifaceted approach has been set up that includes: 1) improvement of pgs expression; 2) accumulation of γ-PGA precursors by metabolic engineering; 3) enhancement of glycerol metabolism. 1) 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 be then used to finely tune pgs expression and optimize γ-PGA yield. To this end, an inducible pgs operon has been constructed. 2) 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 following this analysis, showed enhanced polymer production. Other target genes are currently under investigation. 3) 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
