Improving poly-3-hydroxybutyrate production in Escherichia coli by combining the increase in the NADPH pool and acetyl-CoA availability

Abstract

The biosynthesis of poly-3-hydroxybutyrate (P3HB), a biodegradable bio-plastic, requires acetyl-CoA as precursor and NADPH as cofactor. Escherichia coli has been used as a heterologous production model for P3HB, but metabolic pathway analysis shows a deficiency in maintaining high levels of NADPH and that the acetyl-CoA is mainly converted to acetic acid by native pathways. In this work the pool of NADPH was increased 1.7-fold in E. coli MG1655 through plasmid overexpression of the NADP(+)-dependent glyceraldehyde 3-phosphate dehydrogenase gene (gapN) from Streptococcus mutans (pTrcgapN). Additionally, by deleting the main acetate production pathway (ackA-pta), the acetic acid production was abolished, thus increasing the acetyl-CoA pool. The P3HB biosynthetic pathway was heterologously expressed in strain MG1655 Δack-pta/pTrcgapN, using an IPTG inducible vector with the P3HB operon from Azotobacter vinelandii (pPHB Av ). Cultures were performed in controlled fermentors using mineral medium with glucose as the carbon source. Accordingly, the mass yield of P3HB on glucose increased to 73 % of the maximum theoretical and was 30 % higher when compared to the progenitor strain (MG1655/pPHB Av ). In comparison with the wild type strain expressing pPHB Av , the specific accumulation of PHB (gPHB/gDCW) in MG1655 Δack-pta/pTrcgapN/pPHB Av increased twofold, indicating that as the availability of NADPH is raised and the production of acetate abolished, a P3HB intracellular accumulation of up to 84 % of the E. coli dry weight is attainable.

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“…Over expression of zwf gene encoding enzyme glucose-6-phosphate dehydrogenase and gnd gene encoding 6-phosphogluconate dehydrogenase in oxidative PP pathway increased the pool of NADPH that ultimately enhanced the PHB synthesis in recombinant E. coli [13]. Moreover, the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (encoded by gapN) from a mutant of Streptococcus, which catalyzes the conversion of glyceraldehyde-3-phosphate to glycerate-1, 3-bisphosphate, was over expressed in E. coli having phb genes [14]. The result showed that the PHB accumulation was increased due to the excess supply of NADPH.…”

“…The result showed that the PHB accumulation was increased due to the excess supply of NADPH. Furthermore, deletion of the main acetate pathway genes (ackA and pta) and the lactate pathway gene (ldh) in recombinant E. coli abolished acetate and lactate production leading to increased pool of acetyl-CoA, which eventually enhanced the PHB synthesis [14,15]. KDPG: 2-keto-3-deoxy-6-phospho gluconate; DHAP: dihydroxyacetone phosphate; Sedo-7-P: Sedoheptulose-7-phosphate; TCA: Tricarboxylic Acid; glk: Glucokinase; pgi: Phosphoglucose Isomerase; pfkA: Phosphofructokinse; fbaA: Fructose Bisphosphate Aldolase; zwf: Glucose-6-phosphate-1-dehydrogenase; gnd: 6-phosphogluconate Dehydrogenase; tkt: Transketolase; talA: Transaldolase; edd: Phosphogluconate Dehydratase; eda: 2-keto-3-deoxygluconate-6-phosphate Aldolase; gapA: Glyceraldehyde-3-phosphate Dehydrogenase; pgk: Phosphoglycerate Kinase; gpm: Phosphoglyceromutase; sera: D-3-phosphoglycerate Dehydrogenase; serB: Phosphoserine Phosphatase; serC: 3-phosphoserine Aminotransferase; sdaA: L-serine Deaminase; pyk: Pyruvate Kinase; ppsA: Phosphoenolpyruvate Synthase; ldh: Lactate Dehydrogenase; aceE: Pyruvate Dehydrogenase E1; aceF: Pyruvate Dehydrogenase E2; lpdA: Lipoamide Dehydrogenase; poxB: Pyruvate Oxidase; pta: Phosphate Acetyltransferase; ackA: Acetate Kinase; phaA: β-ketothiolase; phaB: NADPHdependent AcetoacetylCoA reductase; phaC: PHB Synthase; gltA: Citrate Synthase In E. coli, acetyl-CoA is mostly synthesized from pyruvate catalyzed by the pyruvate dehydrogenase (PDH) complex, which is an important intermediate metabolite in both catabolic and anabolic reactions (Figure 1).…”

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“…[317]. In addition, GAPN has been used successfully in metabolic engineering efforts intended to improve the production of metabolites whose biosynthesis depends on NADPH, such as L-lysine or poly-3-hydroxybutyrate, or to decrease the formation of undesired by-products such as glycerol in industrial ethanol production strains [318][319][320].…”

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“…Thus, a combined strategy aimed at increasing both the levels of acetyl-CoA and NADPH has been used for the production of fatty acid ethyl esters and polyhydroxybutyrate. [19][20][21] Many culture medium formulations for microbial fermentations are based on waste/low-cost sugars and it is therefore important to improve lipid accumulation from sugar-based media. 22 In this sense, the pox1D strains generated in our work were able to produce up to 9% of their CDW as lipids in rich glucose-rich medium (Fig.…”