Modular control of multiple pathways of Corynebacterium glutamicum for 5-aminolevulinic acid production

Abstract

Abstract5-aminolevulinic acid (ALA) has broad potential applications in the medical, agricultural and food industries. Several strategies have been implemented successfully to try to improve ALA synthesis. Nonetheless, the low yield has got in the way of large-scale bio-manufacture of 5-ALA. In this study, we explored strain engineering strategies for high‐level 5‐ALA production in Corynebacterium glutamicum F343 using the C4 pathway. Initially, the glutamate dehydrogenase-encoding gene gdhA was deleted to reduce glutamate yield. Then the C4 pathway was introduced in the gdhA mutant strain F2-A (∆gdhA + hemA), resulting in a 5-ALA yield of up to 3.2 g/L. Furthermore, the accumulations of downstream metabolites such as heme, porphobilinogen, and protoporphyrin IX, were decreased. After evaluating the mechanisms of this synthetic pathway by RNA-Seq, the results showed that genes involved in both the C5 pathway and heme pathways were down-regulated in strain F2-A (∆gdhA + hemA). Interestingly, upstream genes of succinyl-CoA in the tricarboxylic acid (TCA) cycle, such as icd, lpdA, were up-regulated, while its downstream genes, including sucC, sucD, sdhB, sdhA, sdhCD, were down-regulated. These changes amplify the sources of succinyl-CoA and reduce its expenditure, before pulling the carbon flux to produce 5-ALA. Furthermore, the down-regulation of most genes of the heme pathway could reduce the drainage of 5‐ALA, which further enhance its accumulation. To alleviate competition between glyoxylate and the TCA cycle, the isocitrate dehydrogenase-encoding gene aceA was also knocked out, resulting in 3.86 g/L of 5‐ALA. Finally, the fermentation conditions were optimized, resulting in a maximum 5-ALA yield of 5.6 g/L. Overall, the blocking of the glutamate synthesis pathway could be a powerful strategy to re-allocate the carbon flux to produce 5-ALA. It could also enable the efficient synthesis of other TCA derivatives in C. glutamicum.

Transcriptomic and enzymatic analysis reveals the roles of glutamate dehydrogenase in Corynebacterium glutamicum

“…There is a possibility that gdh double deficiency would be beneficial to the accumulation of pyruvic acid and its various fermentation products such as butanol, malic acid, lactic acid, propionic acid. In the TCA, the genes including acnA , idh upstream of 2-OG were strongly downregulated, suggesting gdh deletion blocked the synthesis of glutamic acid and caused reduction of the carbon flux to 2-OG by re-allocating the TCA carbon flux (Ge et al 2021 ).…”

“…Corynebacterium glutamicum , as a non-pathogenic, soil-derived gram-positive actinobacterium, is used as a safe industrial producer of various amino acids (especially L-glutamate, L-lysine and L-arginine), nucleotides, and organic acids (Tsuge et al 2021 ; Sheng et al 2021 ; Ge et al 2021 ). Furthermore, the availability of genetic engineering methods and easy cultivation has helped to make C. glutamicum as a model organism and a cell factory of choice in industrial biotechnology (Wang et al 2021 ; Mei et al 2016 ).…”

Transcriptomic and enzymatic analysis reveals the roles of glutamate dehydrogenase in Corynebacterium glutamicum

“…These results con rmed that gdh double de ciency initiated the enhancement of the absorption and utilization of carbon sources. In the TCA, the genes including acnA, idh upstream of 2-OG were strongly downregulated, suggesting gdh deletion blocked the synthesis of glutamic acid and caused reduction of the carbon ux to 2-OG by re-allocating the TCA carbon ux (Ge et al, 2021).…”

“…Corynebacterium glutamicum, as a non-pathogenic, soil-derived gram-positive actinobacterium, is used as a safe industrial producer of various amino acids (especially L-glutamate, L-lysine and L-arginine), nucleotides, and organic acids (Tsuge et al, 2021;Sheng et al, 2021;Ge et al, 2021). Furthermore, the availability of genetic engineering methods and easy cultivation has helped to make C. glutamicum as a model organism and a cell factory of choice in white biotechnology (Wang et al, 2021;Mei et al, 2016).…”

Application of cofactors in the regulation of microbial metabolism: A state of the art review

“…precursor (Liu et al, 2017). Ge et al (2021) obtained 3.86 g/L of 5-aminolevulinic acid in Corynebacterium glutamicum (C. glutamicum) by alleviating the competition between glyoxylate and TCA cycle and knocking out the gene encoding isocitrate dehydrogenase, with a maximum yield of 5.6 g/L of 5-aminolevulinic acid after optimization of fermentation conditions.…”