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A synthetic biosensor to detect peroxisomal acetyl-CoA concentration for compartmentalized metabolic engineering.

ABSTRACT: Background:Sub-cellular compartmentalization is used by cells to create favorable microenvironments for various metabolic reactions. These compartments concentrate enzymes, separate competing metabolic reactions, and isolate toxic intermediates. Such advantages have been recently harnessed by metabolic engineers to improve the production of various high-value chemicals via compartmentalized metabolic engineering. However, measuring sub-cellular concentrations of key metabolites represents a grand challenge for compartmentalized metabolic engineering. Methods:To this end, we developed a synthetic biosensor to measure a key metabolite, acetyl-CoA, in a representative compartment of yeast, the peroxisome. This synthetic biosensor uses enzyme re-localization via PTS1 signal peptides to construct a metabolic pathway in the peroxisome which converts acetyl-CoA to polyhydroxybutyrate (PHB) via three enzymes. The PHB is then quantified by HPLC. Results:The biosensor demonstrated the difference in relative peroxisomal acetyl-CoA availability under various culture conditions and was also applied to screening a library of single knockout yeast mutants. The screening identified several mutants with drastically reduced peroxisomal acetyl-CoA and one with potentially increased levels. We expect our synthetic biosensors can be widely used to investigate sub-cellular metabolism and facilitate the "design-build-test" cycle of compartmentalized metabolic engineering.

SUBMITTER: Huttanus HM

PROVIDER: S-EPMC7485502 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A synthetic biosensor to detect peroxisomal acetyl-CoA concentration for compartmentalized metabolic engineering.

Huttanus Herbert M HM Senger Ryan S RS

PeerJ 20200908

<h4>Background</h4>Sub-cellular compartmentalization is used by cells to create favorable microenvironments for various metabolic reactions. These compartments concentrate enzymes, separate competing metabolic reactions, and isolate toxic intermediates. Such advantages have been recently harnessed by metabolic engineers to improve the production of various high-value chemicals via compartmentalized metabolic engineering. However, measuring sub-cellular concentrations of key metabolites represent ...[more]

PMID: 33194349

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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.

Project description:Cytosolic acetyl-coenzyme A is a precursor for many biotechnologically relevant compounds produced by Saccharomyces cerevisiae. In this yeast, cytosolic acetyl-CoA synthesis and growth strictly depend on expression of either the Acs1 or Acs2 isoenzyme of acetyl-CoA synthetase (ACS). Since hydrolysis of ATP to AMP and pyrophosphate in the ACS reaction constrains maximum yields of acetyl-CoA-derived products, this study explores replacement of ACS by two ATP-independent pathways for acetyl-CoA synthesis. After evaluating expression of different bacterial genes encoding acetylating acetaldehyde dehydrogenase (A-ALD) and pyruvate-formate lyase (PFL), acs1M-NM-^T acs2M-NM-^T S. cerevisiae strains were constructed in which A-ALD or PFL successfully replaced ACS. In A-ALD-dependent strains, aerobic growth rates of up to 0.27 h-1 were observed, while anaerobic growth rates of PFL-dependent S. cerevisiae (0.21 h-1) were stoichiometrically coupled to formate production. In glucose-limited chemostat cultures, intracellular metabolite analysis did not reveal major differences between A-ALD-dependent and reference strains. However, biomass yields on glucose of A-ALD- and PFL-dependent strains were lower than those of the reference strain. Transcriptome analysis suggested that reduced biomass yields were caused by acetaldehyde and formate in A-ALD- and PFL-dependent strains, respectively. Transcript profiles also indicated that a previously proposed role of Acs2 in histone acetylation is probably linked to cytosolic acetyl-CoA levels rather than to direct involvement of Acs2 in histone acetylation. While, for the first time, demonstrating that yeast ACS can be fully replaced by alternative reactions, this study demonstrates that further modifications are needed to achieve optimal in vivo efficiencies of the supply of acetyl-CoA as product precursor. To investigate the impact of introduced changes in native pathway of cytosolic acetyl-CoA formation in S. cerevisiae, a DNA microarray-based transcriptome analysis was performed on aerobic or anaerobic, glucose-limited chemostat cultures.

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A synthetic biosensor to detect peroxisomal acetyl-CoA concentration for compartmentalized metabolic engineering.

Publications

A synthetic biosensor to detect peroxisomal acetyl-CoA concentration for compartmentalized metabolic engineering.

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