Dataset Information

Metabolic engineering of Escherichia coli for the synthesis of polyhydroxyalkanoates using acetate as a main carbon source.

ABSTRACT:

Background

High production cost of bioplastics polyhydroxyalkanoates (PHA) is a major obstacle to replace traditional petro-based plastics. To address the challenges, strategies towards upstream metabolic engineering and downstream fermentation optimizations have been continuously pursued. Given that the feedstocks especially carbon sources account up to a large portion of the production cost, it is of great importance to explore low cost substrates to manufacture PHA economically.

Results

Escherichia coli was metabolically engineered to synthesize poly-3-hydroxybutyrate (P3HB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) using acetate as a main carbon source. Overexpression of phosphotransacetylase/acetate kinase pathway was shown to be an effective strategy for improving acetate assimilation and biopolymer production. The recombinant strain overexpressing phosphotransacetylase/acetate kinase and P3HB synthesis operon produced 1.27 g/L P3HB when grown on minimal medium supplemented with 10 g/L yeast extract and 5 g/L acetate in shake flask cultures. Further introduction succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, and CoA transferase lead to the accumulation of P3HB4HB, reaching a titer of 1.71 g/L with a 4-hydroxybutyrate monomer content of 5.79 mol%. When 1 g/L of ?-ketoglutarate or citrate was added to the medium, P3HB4HB titer increased to 1.99 and 2.15 g/L, respectively. To achieve PHBV synthesis, acetate and propionate were simultaneously supplied and propionyl-CoA transferase was overexpressed to provide 3-hydroxyvalerate precursor. The resulting strain produced 0.33 g/L PHBV with a 3-hydroxyvalerate monomer content of 6.58 mol%. Further overexpression of propionate permease improved PHBV titer and 3-hydroxyvalerate monomer content to 1.09 g/L and 10.37 mol%, respectively.

Conclusions

The application of acetate as carbon source for microbial fermentation could reduce the consumption of food and agro-based renewable bioresources for biorefineries. Our proposed metabolic engineering strategies illustrate the feasibility for producing polyhydroxyalkanoates using acetate as a main carbon source. Overall, as an abundant and renewable resource, acetate would be developed into a cost-effective feedstock to achieve low cost production of chemicals, materials, and biofuels.

SUBMITTER: Chen J

PROVIDER: S-EPMC6029019 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Metabolic engineering of Escherichia coli for the synthesis of polyhydroxyalkanoates using acetate as a main carbon source.

Chen Jing J Li Wei W Zhang Zhao-Zhou ZZ Tan Tian-Wei TW Li Zheng-Jun ZJ

Microbial cell factories 20180703 1

<h4>Background</h4>High production cost of bioplastics polyhydroxyalkanoates (PHA) is a major obstacle to replace traditional petro-based plastics. To address the challenges, strategies towards upstream metabolic engineering and downstream fermentation optimizations have been continuously pursued. Given that the feedstocks especially carbon sources account up to a large portion of the production cost, it is of great importance to explore low cost substrates to manufacture PHA economically.<h4>Re ...[more]

PMID: 29970091

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Project description:BackgroundEscherichia coli (E. coli) is a bacteria that is widely employed in many industries for the production of high interest bio-products such as recombinant proteins. Nevertheless, the use of E. coli for recombinant protein production may entail some disadvantages such as acetate overflow. Acetate is accumulated under some culture conditions, involves a decrease in biomass and recombinant protein production, and its metabolism is related to protein lysine acetylation. Thereby, the carbon and nitrogen sources employed are relevant factors in cell host metabolism, and the study of the central metabolism of E. coli and its regulation is essential for optimizing the production of biomass and recombinant proteins. In this study, our aim was to find the most favourable conditions for carrying out recombinant protein production in E. coli BL21 using two different approaches, namely, manipulation of the culture media composition and the deletion of genes involved in acetate metabolism and Nε-lysine acetylation.ResultsWe evaluated protein overexpression in E. coli BL21 wt and five mutant strains involved in acetate metabolism (Δacs, ΔackA and Δpta) and lysine acetylation (ΔpatZ and ΔcobB) grown in minimal medium M9 (inorganic ammonium nitrogen source) and in complex TB7 medium (peptide-based nitrogen source) supplemented with glucose (PTS carbon source) or glycerol (non-PTS carbon source). We observed a dependence of recombinant protein production on acetate metabolism and the carbon and nitrogen source employed. The use of complex medium supplemented with glycerol as a carbon source entails an increase in protein production and an efficient use of resources, since is a sub-product of biodiesel synthesis. Furthermore, the deletion of the ackA gene results in a fivefold increase in protein production with respect to the wt strain and a reduction in acetate accumulation.ConclusionThe results showed that the use of diverse carbon and nitrogen sources and acetate metabolism knockout strains can redirect E. coli carbon fluxes to different pathways and affect the final yield of the recombinant protein bioprocess. Thereby, we obtained a fivefold increase in protein production and an efficient use of the resources employing the most suitable strain and culture conditions.

Project description:BACKGROUND:Acetyl-CoA is an important metabolic intermediate and serves as an acetylation precursor for the biosynthesis of various value-added acetyl-chemicals. Acetyl-CoA can be produced from glucose, acetate, or fatty acids via metabolic pathways in Escherichia coli. Although glucose is an efficient carbon source for acetyl-CoA production, the pathway from acetate to acetyl-CoA is the shortest and fatty acids can produce acetyl-CoA through fatty acid oxidation along with abundant NADH and FADH2. In this study, metabolically engineered E. coli strains for efficiently supplying acetyl-CoA from glucose, acetate, and fatty acid were constructed and applied in one-step biosynthesis of N-acetylglutamate (NAG) from glutamate and acetyl-CoA. RESULTS:A metabolically engineered E. coli strain for NAG production was constructed by overexpressing N-acetylglutamate synthase from Kitasatospora setae in E. coli BW25113 with argB and argA knockout. The strain was further engineered to utilize glucose, acetate, and fatty acid to produce acetyl-CoA. When glucose was used as a carbon source, the combined mutants of ?ptsG::glk, ?galR::zglf, ?poxB::acs, ?ldhA, and ?pta were more efficient for supplying acetyl-CoA. The acetyl-CoA synthetase (ACS) pathway and acetate kinase-phosphate acetyltransferase (ACK-PTA) pathway from acetate to acetyl-CoA were investigated, and the ACK-PTA pathway showed to be more efficient for supplying acetyl-CoA. When fatty acid was used as a carbon source, acetyl-CoA supply was improved by deletion of fadR and constitutive expression of fadD under the strong promoter CPA1. Comparison of acetyl-CoA supply from glucose, acetate and palmitic acid revealed that a higher conversion rate of glutamate (98.2%) and productivity (an average of 6.25 mmol/L/h) were obtained when using glucose as a carbon source. The results also demonstrated the great potential of acetate and fatty acid to supply acetyl-CoA, as the molar conversion rate of glutamate was more than 80%. CONCLUSIONS:Metabolically engineered E. coli strains were developed for NAG production. The metabolic pathways of acetyl-CoA from glucose, acetate, or fatty acid were optimized for efficient acetyl-CoA supply to enhance NAG production. The metabolic strategies for efficient acetyl-CoA supply used in this study can be exploited for other chemicals that use acetyl-CoA as a precursor or when acetylation is involved.

Dataset Information

Metabolic engineering of Escherichia coli for the synthesis of polyhydroxyalkanoates using acetate as a main carbon source.

Background

Results

Conclusions

Publications

Metabolic engineering of Escherichia coli for the synthesis of polyhydroxyalkanoates using acetate as a main carbon source.

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