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Transcriptome analysis of C. glutamicum ΔaceE Δpyc versus ΔaceE

ABSTRACT: The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative decarboxylation of pyruvate yielding acetyl-CoA and CO2. The PDHC-deficient Corynebacterium glutamicum strain ΔaceE is therefore lacking an important decarboxylation step in central metabolism. Additional inactivation of pyc, encoding pyruvate carboxylase, resulted in a >15 hour lag phase in the presence of glucose, while no growth defect was observed on gluconeogenetic substrates like acetate. Transcriptome analysis as well as an adaptive laboratory evolution experiment of strain ΔaceE Δpyc revealed the reduction of glucose uptake as a key adaptive measure to enhance growth on glucose/acetate mixtures.

ORGANISM(S): Escherichia coli Pseudomonas putida KT2440 Bacillus subtilis subsp. subtilis str. 168 Corynebacterium glutamicum Gluconobacter oxydans Corynebacterium glutamicum ATCC 13032

PROVIDER: GSE134218 | GEO | 2019/07/31

REPOSITORIES: GEO

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Project description:Previous findings have demonstrated that the NADH/NAD+ ratio has a strong impact on the glycolytic flux in C. glutamicum under anaerobic conditions in the absence of external electron acceptors. During an attempt to rewire anaerobic metabolism to achieve high yield formation of ethanol, we inactivated the malate dehydrogenase and lactate dehydrogenase in a C. glutamicum strain expressing pyruvate decarboxylase and alcohol dehydrogenase, to eliminate formation of the by-products succinate and lactate, respectively. This modification increased the yield of ethanol but had a negative effect on glycolysis, which we found to correlate with an elevated NADH/NAD+ ratio. The pyruvate dehydrogenase (PDH) of C. glutamicum is active under anaerobic conditions, and can potentially exacerbate the negative effect on glycolysis, due to NADH formation. To reduce PDH activity under anaerobic conditions, we decided to replace the gene encoding the E3 subunit of PDH with its Escherichia coli counterpart, as E. coli PDH has been reported to be functional under aerobic conditions only. The resultant strain JS133 produced far less acetate with a further increased ethanol production, however, the glycolytic flux was still low. After observing differences in glycolytic flux for JS133 on glucose and fructose, we speculated that the pentose phosphate pathway (PPP) might be involved in the reduced flux on glucose. To prove this, we deleted the zwf gene, encoding glucose-6-phosphate dehydrogenase, which is the entry point into PPP, and immediately observed a stimulating effect on glycolysis. Subsequent characterization revealed a direct correlation between the intracellular NADH/NAD+ and NADPH/NADP+ ratios under anoxic conditions. Based on these findings we managed to re-channel the metabolism of C. glutamicum successfully towards either to ethanol or D-lactate with 92% and 98% of the theoretical yield respectively, which is the highest yields for D-lactate production thus far reported in the literature.

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Transcriptome analysis of C. glutamicum ΔaceE Δpyc versus ΔaceE

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