Glutaric acid production by systems metabolic engineering of an l-lysine-overproducing Corynebacterium glutamicum

Abstract

There is increasing industrial demand for five-carbon platform chemicals, particularly glutaric acid, a widely used building block chemical for the synthesis of polyesters and polyamides. Here we report the development of an efficient glutaric acid microbial producer by systems metabolic engineering of an l-lysine-overproducing Corynebacterium glutamicum BE strain. Based on our previous study, an optimal synthetic metabolic pathway comprising Pseudomonas putida l-lysine monooxygenase (davB) and 5-aminovaleramide amidohydrolase (davA) genes and C. glutamicum 4-aminobutyrate aminotransferase (gabT) and succinate-semialdehyde dehydrogenase (gabD) genes, was introduced into the C. glutamicum BE strain. Through system-wide analyses including genome-scale metabolic simulation, comparative transcriptome analysis, and flux response analysis, 11 target genes to be manipulated were identified and expressed at desired levels to increase the supply of direct precursor l-lysine and reduce precursor loss. A glutaric acid exporter encoded by ynfM was discovered and overexpressed to further enhance glutaric acid production. Fermentation conditions, including oxygen transfer rate, batch-phase glucose level, and nutrient feeding strategy, were optimized for the efficient production of glutaric acid. Fed-batch culture of the final engineered strain produced 105.3 g/L of glutaric acid in 69 h without any byproduct. The strategies of metabolic engineering and fermentation optimization described here will be useful for developing engineered microorganisms for the high-level bio-based production of other chemicals of interest to industry.

Keywords: Corynebacterium glutamicum; glutaric acid; metabolic engineering; multiomics.

Fig. 1.

Overview for construction of the glutaric acid biosynthesis pathway in the C. glutamicum BE strain. (A) Schematic of the glutaric acid biosynthesis pathway from glucose. The genes in light green are obtained from P. putida, and the genes in blue are obtained from C. glutamicum. Each gene encodes the following: davA, 5-aminovaleramide amidohydrolase; davB, l-lysine 2-monooxygenase; davT, 5-aminovalerate aminotransferase; davD, glutarate semialdehyde dehydrogenase; gabT, 4-aminobutyrate aminotransferase; and gabD, succinate-semialdehyde dehydrogenase. (B) Schematic of plasmids used for glutaric acid biosynthesis. (C) Flask cultivation results of glutaric acid-producing strains. Error bars represent SD, and the white circles represent individual data points. Experiments were performed in triplicate. Cells were cultured for 48 h. (D) Fed-batch fermentation profile of the BE strain harboring pGA4. ASP, l-aspartate; AVA, 5-aminovaleric acid; GLC, glucose; GLT, glutaric acid; LYS, l-lysine; OAA, oxaloacetate; PYR, pyruvate.

Fig. 2.

Flux response analysis results for glutaric acid production. Each box represents the response of glutaric acid production flux to varying flux of reactions of genes in glycolysis, PPP, the TCA cycle, and the glutaric acid biosynthetic pathway. Each reaction of the genes in glycolysis, PPP, the TCA cycle, and the glutaric acid biosynthetic pathway was gradually increased from the minimum flux to the maximum flux while maximizing glutaric acid production flux. The negative flux of each reaction indicates the reverse reaction. Genes shown here are listed in SI Appendix, Note S1.

Fig. 3.

Comparative transcriptome analysis between the BE and WT strains. The fold change value (BE/WT) of each gene indicating the comparative gene expression level between the BE and WT strains is represented in color. ACT, acetate; AKB, α-ketobutyrate; ASP, l-aspartate; FA, fatty acid; GLC, glucose; GLU, l-glutamate; KIV, ketoisovalerate; LA, lactate; LEU, l-leucine; LYS, l-lysine; MET, l-methionine; THR, l-threonine; OAA, oxaloacetate; PAN, pantoate; PRM, pyrimidine; PYR, pyruvate; VAL, l-valine. The genes shown here are listed in SI Appendix, Note S2.

Fig. 4.

Construction of the glutaric acid-producing platform strain. (A) Overview of the engineering strategies to enhance glutaric acid flux. The blue and dotted arrows represent up-regulated and down-regulated flux, respectively. The strategies that brought no significant increase in glutaric acid production are shown in gray boxes. (B) Comparison of glutaric acid titers, cell growth, and yields obtained by flask cultivation of engineered C. glutamicum strains. Each strain harboring pGA4 was tested. The P values of the data showing increases compared with BE (pGA4) are *P < 0.05, **P < 0.01, and ***P < 0.001. (C) The effect of putative glutaric acid exporter gene amplification on glutaric acid production. Each plasmid was transformed to the GA16 strain together with pGA4, and the generated strains were cultured in flasks. The P values of the data showing increases compared with GA16 (pGA4, pEKEx1) are *P < 0.05, **P < 0.01, and ***P < 0.001. The blue and gray bars indicate glutaric acid titer (g/L) and OD₆₀₀, respectively, while the white circles indicate yield. The error bars represent SD. Experiments were performed in triplicate. Cells were cultured for 48 h. AKG, α-ketoglutarate; ASA, l-aspartyl-semialdehyde; ASP, l-aspartate; ASPP, l-aspartate-phosphate; DAP, diaminopimelate; DHDP, l-2,3-dihydropicolinate; GLC, glucose; GLT, glutaric acid; ICT, isocitrate; LYS, l-lysine; OAA, oxaloacetate; PDC, l-piperidine-2,6-discarboxylate; PEP, phosphoenolpyruvate; PYR, pyruvate.

Fig. 5.

Fed-batch fermentation profile of the GA17 strain harboring pGA4. The black triangle represents OD₆₀₀; the white diamond, glucose (g/L); blue circles, glutaric acid (g/L). For reproducibility, the results of another fed-batch culture performed independently are shown in SI Appendix, Fig. S13.