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

Abstract

BackgroundHigh 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.ResultsEscherichia 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 α-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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0949-0) contains supplementary material, which is available to authorized users.

Metabolic engineering of Escherichia coli carrying the hybrid acetone-biosynthesis pathway for efficient acetone biosynthesis from acetate

“…On the other hand, acetate utilization and acetyl-CoA metabolism in E. coli have been thoroughly studied, which makes acetic acid more feasible to be used as an alternate carbon. In recent years, acetate has been used to synthesize a series of value-added products, such as medium chain fatty acids [18], lipids [15], ethanol [19], itaconic acid [20], polyhydroxyalkanoates [21], mevalonate [22] and other acetyl-CoA derivatives. The CoA related acetate transportation in the acetone synthetic pathway of C. acetobutylicum made it a better substitution than other bio-pathways from acetate to acetone.…”

Metabolic engineering for efficient supply of acetyl-CoA from different carbon sources in Escherichia coli

“…Acetate has been used as a carbon source in the production of polyhydroxyalkanoates, itaconic acid, and other chemicals. Chen et al used acetate as a main carbon source to synthesize polyhydroxyalkanoates, and strains overexpressing the ACK-PTA pathway produced nearly fourfold levels of poly-3-hydroxybutyrate, twofold levels of poly(3-hydroxybutyrate- co -4-hydroxybutyrate), and twofold levels of poly(3-hydroxybutyrate- co -3-hydroxyvalerate) compared to control strains [14]. By overexpressing acs and inactivating iclR , the conversion rate of acetate to itaconic acid was improved to 16.7% of the theoretical maximum yield [12].…”

“…The studies are still in the stage of proof of concept and more works are needed to make it possible for industrial application. Recently, acetate was used as an alternative carbon source and to produce chemicals such as succinate [10, 11], itaconic acid [12], isobutanol [13], and polyhydroxyalkanoates [14]. Two pathways can convert acetate to acetyl-CoA: (1) the reversible acetate kinase-phosphate acetyltransferase (ACK-PTA) pathway and (2) the irreversible ACS pathway catalyzed by acetyl-CoA synthetase (ACS).…”

Microbial Upgrading of Acetate into Value-Added Products—Examining Microbial Diversity, Bioenergetic Constraints and Metabolic Engineering Approaches

“…In the case of tyrosine, the pathway is natural to the E. coli host strain, but the expression levels of the involved enzymes were optimized to favor tyrosine production [ 128 , 144 ]. For the production of acetone, N-acetylglutamate, PHB, 3-hydroxypropionic acid, itaconic acid, mevalonate, and phloroglucinol the pathways contained one gene derived from a different organism than E. coli , resulting in the construction of expression cassettes containing extra copies of the E. coli genes and the heterologous ones [ 60 , 113 , 116 , 118 , 119 , 121 , 123 ]. Truly synthetic pathways incorporating genes from several organisms were employed for the production of β-caryophyllene, isobutanol, isopropanol, and succinate [ 33 , 114 , 120 , 125 , 145 ].…”