Metabolic engineering of Corynebacterium glutamicum for enhanced production of 5-aminovaleric acid

Abstract

Background5-Aminovaleric acid (5AVA) is an important five-carbon platform chemical that can be used for the synthesis of polymers and other chemicals of industrial interest. Enzymatic conversion of l-lysine to 5AVA has been achieved by employing lysine 2-monooxygenase encoded by the davB gene and 5-aminovaleramidase encoded by the davA gene. Additionally, a recombinant Escherichia coli strain expressing the davB and davA genes has been developed for bioconversion of l-lysine to 5AVA. To use glucose and xylose derived from lignocellulosic biomass as substrates, rather than l-lysine as a substrate, we previously examined direct fermentative production of 5AVA from glucose by metabolically engineered E. coli strains. However, the yield and productivity of 5AVA achieved by recombinant E. coli strains remain very low. Thus, Corynebacterium glutamicum, a highly efficient l-lysine producing microorganism, should be useful in the development of direct fermentative production of 5AVA using l-lysine as a precursor for 5AVA. Here, we report the development of metabolically engineered C. glutamicum strains for enhanced fermentative production of 5AVA from glucose.ResultsVarious expression vectors containing different promoters and origins of replication were examined for optimal expression of Pseudomonas putida davB and davA genes encoding lysine 2-monooxygenase and delta-aminovaleramidase, respectively. Among them, expression of the C. glutamicum codon-optimized davA gene fused with His6-Tag at its N-Terminal and the davB gene as an operon under a strong synthetic H36 promoter (plasmid p36davAB3) in C. glutamicum enabled the most efficient production of 5AVA. Flask culture and fed-batch culture of this strain produced 6.9 and 19.7 g/L (together with 11.9 g/L glutaric acid as major byproduct) of 5AVA, respectively. Homology modeling suggested that endogenous gamma-aminobutyrate aminotransferase encoded by the gabT gene might be responsible for the conversion of 5AVA to glutaric acid in recombinant C. glutamicum. Fed-batch culture of a C. glutamicum gabT mutant-harboring p36davAB3 produced 33.1 g/L 5AVA with much reduced (2.0 g/L) production of glutaric acid.Conclusions Corynebacterium glutamicum was successfully engineered to produce 5AVA from glucose by optimizing the expression of two key enzymes, lysine 2-monooxygenase and delta-aminovaleramidase. In addition, production of glutaric acid, a major byproduct, was significantly reduced by employing C. glutamicum gabT mutant as a host strain. The metabolically engineered C. glutamicum strains developed in this study should be useful for enhanced fermentative production of the novel C5 platform chemical 5AVA from renewable resources.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0566-8) contains supplementary material, which is available to authorized users.

Advances in bio‐based production of dicarboxylic acids longer than C4

“…However, the currently achieved titers from these pathways were much lower than those with the AMV pathway for glutarate production [28]. However, the currently achieved titers from these pathways were much lower than those with the AMV pathway for glutarate production [28].…”

“…Various P450 monoxygenases, oxidoreductases and esterases have been identified responsible for conversion of the terminal methyl to carboxyl group in carboxylic acids, and have been engineered with superior catalytic activity with an expanding substrate spectrum [18][19][20]. (A) ω-Oxidation, the ω-oxidation reactions converting the methyl group to a carboxylic group at the ω-position of carboxylic acids [17,19,20], FAS, fatty acid synthase; (B) AMV pathway, aminovalerate pathway started from L-lysine to yield glutarate [25][26][27][28], 2-KG, α-ketoglutarate, L-Glu, L-glutamate; (C) C+1 elongation, the C1 elongation pathway started from α-ketoglutarate (AKG) to yield α-ketoadipate (AKA) or α-ketopimelate (AKP) [29,30]; α-Keto decarboxylation and oxidation, the α-ketoacid decarboxylation pathway started from AKA or AKP to yield glutarate or adipate [31,32]; (D), α-Keto reduction, the α-ketoacid reduction pathway started from AKG to yield glutarate [34]; (E) Reversal β-oxidation, the reversal β-oxidation pathway started from the condensation of acetyl-CoA and acyl-CoA (C = n) to yield free fatty acid (C = n+2) [40][41][42][44][45][46]; (F) PKSs, engineered polyketide synthase (PKS)-based pathway started from the condensation of succinyl-CoA and malonyl-CoA to yield adipate [49]; (G) Biotin-fatty acid pathway, the engineered biotin synthetic pathway by removing the activity for biotin synthesis (BioH) and overexpression of specific thioesterase to yield odd-chain DCAs [51], SAM, S-adenosyl-L-methionine, SAH, S-adenosylhomocysteine; Muconic acid pathway, a extended muconic acid biosynthetic pathway converting cis, cis-muconic acid into adipate [119,120] . For example, an engineered pathway for the synthesis of medium chain carboxylic acids (C6-C10) was recently established ( Fig.…”

“…More recently, overexpression of davB and davA genes of Pseudomonas in lysine overproducers of C. glutamicum resulted in simultaneous production of AMV and glutarate as a major byproduct [27,28]. In these engineered strains, the unexpected, partial conversion of AMV to glutarate was related to the presence of endogenous genes coding for 5-aminovalerate aminotransferase (gabT) and glutarate semialdehyde dehydrogenase.…”

“…The primary consideration is to tune the expression levels of multiple enzymes of the synthetic pathway to achieve a good balance. Besides, various expression vectors with differing copy numbers and promoters were tested to construct a functional AMV pathway, and an optimal cassette for overexpression of the davBA genes was obtained to permit more efficient production of glutarate [28]. Therefore, fine-tuning expression with a combination of suitable promoters and ribosome binding sites, and dynamic regulation of key enzymes are useful for enhancing the production from synthetic pathways [57][58][59][60][61][62].…”

“…AMV pathway [25][26][27][28] 4. Repression of the pathway from AKG to succinyl-CoA to enrich AKG in E. coli.…”