Dataset Information

Comparison of Glucose, Acetate and Ethanol as Carbon Resource for Production of Poly(3-Hydroxybutyrate) and Other Acetyl-CoA Derivatives.

ABSTRACT: Poly(3-hydroxybutyrate) (PHB) is a biodegradable and biocompatible thermoplastic, and synthesized from the central metabolite acetyl-CoA. The acetyl-CoA synthesis from glucose presents low atomic economy due to the release of CO₂ in pyruvate decarboxylation. As ethanol and acetate can be converted into acetyl-CoA directly, they were used as carbon source for PHB production in this study. The reductase mutant AdhE A267T/E568K was introduced into Escherichia coli to enable growth on ethanol, and acetate utilization was improved by overexpression of acetyl-CoA synthetase ACS. Comparison of the PHB production using glucose, ethanol or acetate as sole carbon source showed that the production and yield from ethanol was much higher than those from glucose and acetate, and metabolome analysis revealed the differences in metabolism of glucose, ethanol and acetate. Furthermore, other acetyl-CoA derived chemicals including 3-hydroxypropionate and phloroglucinol were produced from those three feedstocks, and similar results were achieved, suggesting that ethanol could be a suitable carbon source for the production of acetyl-CoA derivatives.

SUBMITTER: Sun S

PROVIDER: S-EPMC7396591 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Comparison of Glucose, Acetate and Ethanol as Carbon Resource for Production of Poly(3-Hydroxybutyrate) and Other Acetyl-CoA Derivatives.

Sun Shenmei S Ding Yamei Y Liu Min M Xian Mo M Zhao Guang G

Frontiers in bioengineering and biotechnology 20200723

Poly(3-hydroxybutyrate) (PHB) is a biodegradable and biocompatible thermoplastic, and synthesized from the central metabolite acetyl-CoA. The acetyl-CoA synthesis from glucose presents low atomic economy due to the release of CO<sub>2</sub> in pyruvate decarboxylation. As ethanol and acetate can be converted into acetyl-CoA directly, they were used as carbon source for PHB production in this study. The reductase mutant AdhE A267T/E568K was introduced into <i>Escherichia coli</i> to enable growth ...[more]

PMID: 32850713

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Project description:Acetyl-CoA synthetase (ACS) and acetate ligase (ACD) are widespread among microorganisms, including archaea, and play an important role in their carbon metabolism, although only a few of these enzymes have been characterized. Anaerobic methanotrophs (ANMEs) have been reported to convert methane anaerobically into CO2, polyhydroxyalkanoate, and acetate. Furthermore, it has been suggested that they might be able to use acetate for anabolism or aceticlastic methanogenesis. To better understand the potential acetate metabolism of ANMEs, we characterized an ACS from ANME-2a as well as an ACS and an ACD from ANME-2d. The conversion of acetate into acetyl-CoA (Vmax of 8.4 μmol mg-1 min-1 and Km of 0.7 mM acetate) by the monomeric 73.8-kDa ACS enzyme from ANME-2a was more favorable than the formation of acetate from acetyl-CoA (Vmax of 0.4 μmol mg-1 min-1 and Km of 0.2 mM acetyl-CoA). The monomeric 73.4-kDa ACS enzyme from ANME-2d had similar Vmax values for both directions (Vmax,acetate of 0.9 μmol mg-1 min-1 versus Vmax,acetyl-CoA of 0.3 μmol mg-1 min-1). The heterotetrameric ACD enzyme from ANME-2d was active solely in the acetate-producing direction. Batch incubations of an enrichment culture dominated by ANME-2d fed with 13C2-labeled acetate produced 3 μmol of [13C]methane in 7 days, suggesting that this anaerobic methanotroph might have the potential to reverse its metabolism and perform aceticlastic methanogenesis using ACS to activate acetate albeit at low rates (2 nmol g [dry weight]-1 min-1). Together, these results show that ANMEs may have the potential to use acetate for assimilation as well as to use part of the surplus acetate for methane production. IMPORTANCE Acetyl-CoA plays a key role in carbon metabolism and is found at the junction of many anabolic and catabolic reactions. This work describes the biochemical properties of ACS and ACD enzymes from ANME-2 archaea. This adds to our knowledge of archaeal ACS and ACD enzymes, only a few of which have been characterized to date. Furthermore, we validated the in situ activity of ACS in ANME-2d, showing the conversion of acetate into methane by an enrichment culture dominated by ANME-2d.

Project description:BACKGROUND: The biotechnology industry has extensively exploited Escherichia coli for producing recombinant proteins, biofuels etc. However, high growth rate aerobic E. coli cultivations are accompanied by acetate excretion i.e. overflow metabolism which is harmful as it inhibits growth, diverts valuable carbon from biomass formation and is detrimental for target product synthesis. Although overflow metabolism has been studied for decades, its regulation mechanisms still remain unclear. RESULTS: In the current work, growth rate dependent acetate overflow metabolism of E. coli was continuously monitored using advanced continuous cultivation methods (A-stat and D-stat). The first step in acetate overflow switch (at ? = 0.27 ± 0.02 h(-1)) is the repression of acetyl-CoA synthetase (Acs) activity triggered by carbon catabolite repression resulting in decreased assimilation of acetate produced by phosphotransacetylase (Pta), and disruption of the PTA-ACS node. This was indicated by acetate synthesis pathways PTA-ACKA and POXB component expression down-regulation before the overflow switch at ? = 0.27 ± 0.02 h(-1) with concurrent 5-fold stronger repression of acetate-consuming Acs. This in turn suggests insufficient Acs activity for consuming all the acetate produced by Pta, leading to disruption of the acetate cycling process in PTA-ACS node where constant acetyl phosphate or acetate regeneration is essential for E. coli chemotaxis, proteolysis, pathogenesis etc. regulation. In addition, two-substrate A-stat and D-stat experiments showed that acetate consumption capability of E. coli decreased drastically, just as Acs expression, before the start of overflow metabolism. The second step in overflow switch is the sharp decline in cAMP production at ? = 0.45 h(-1) leading to total Acs inhibition and fast accumulation of acetate. CONCLUSION: This study is an example of how a systems biology approach allowed to propose a new regulation mechanism for overflow metabolism in E. coli shown by proteomic, transcriptomic and metabolomic levels coupled to two-phase acetate accumulation: acetate overflow metabolism in E. coli is triggered by Acs down-regulation resulting in decreased assimilation of acetic acid produced by Pta, and disruption of the PTA-ACS node.

Dataset Information

Comparison of Glucose, Acetate and Ethanol as Carbon Resource for Production of Poly(3-Hydroxybutyrate) and Other Acetyl-CoA Derivatives.

Publications

Comparison of Glucose, Acetate and Ethanol as Carbon Resource for Production of Poly(3-Hydroxybutyrate) and Other Acetyl-CoA Derivatives.

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