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

Abstract

Background: 5-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.

Results: Various 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.

Keywords: 5-Aminovaleric acid; Corynebacterium glutamicum; Glutaric acid; L-Lysine; Metabolic engineering.

Fig. 1

Metabolic engineering strategies for 5AVA production using C. glutamicum. Heterologous expression of the P. putida davB gene (encoding l-lysine 2-monooxygenase) and the davA gene (encoding delta-aminovaleramidase) results in conversion of l-lysine into 5AVA. 5AVA5, 5-aminovalerate; ASP5 l-aspartate; ASP-P aspartyl phosphate; ASP-SA aspartate semialdehyde; LYS l-lysine

Fig. 2

Growth and production characteristics of the C. glutamicum BE strain harboring the pKCA212davAB, pJS30, or pJS38 vectors after 44 h of shake-flask cultivation (n = 3, error bars = SD). The C. glutamicum BE strain containing no plasmids was used as a non-engineered control. a The final OD₆₀₀ at the end of cultivation is shown for all strains tested, and an experimentally determined correlation factor (0.28) was used to determine the biomass yield (Y _X/S). b The production characteristics include the final titers for l-lysine (light-grey bars), 5AVA (dark-grey bars), and glutaric acid (black bars). c The molar yields from glucose for l-lysine (white bars), 5AVA (grey bars), and glutaric acid (black bars) are also shown. When appropriate, gene expression was induced by addition of IPTG at a final concentration of 0.5 mM when the growth reached an OD₆₀₀ of 0.5–0.6

Fig. 3

Production of 5AVA from glucose by fed-batch cultures of (a, c) C. glutamicum BE (pJS38) and (b, d) C. glutamicum BE (p36davAB3). Characteristics of the fed-batch cultivation profile, including growth (filled circles, OD₆₀₀), residual sugar (empty circles; g/L), l-lysine (filled diamonds), 5AVA (magenta diamonds), and glutaric acid (green triangles) production titers, are plotted against the cultivation time

Fig. 4

Growth and production characteristics of the C. glutamicum BE strain harboring the pJS59, pJS60, or p36davAB2 vector after 44 h of shake-flask cultivation (n = 3, error bars = SD). The C. glutamicum BE strain containing no plasmids was used as a non-engineered control. a The final OD₆₀₀ at the end of cultivation is shown for all strains tested, and an experimentally determined correlation factor (0.28) was used to determine the biomass yield (Y _X/S). b The production characteristics include the final titers for l-lysine (light-grey bars), 5AVA (dark-grey bars), and glutaric acid (black bars)

Fig. 5

Growth and production characteristics of the C. glutamicum BE strain harboring p36davAB1 or p36davAB3 after 44 h of shake-flask cultivation (n = 3, error bars = SD). a The final OD₆₀₀ at the end of cultivation is shown for the strains tested. b The production characteristics include the final titers for l-lysine (light-grey bars), 5AVA (dark-grey bars), and glutaric acid (black bars)

Fig. 6

Growth and production characteristics of the AVA2 strain for 44 h of shake-flask cultivation (n = 3, error bars = SD). OD₆₀₀ (filled circle), glucose (empty circle) and l-lysine (filled diamond) are shown

Fig. 7

Fed-batch cultivation of C. glutamicum AVA2 harboring p36davAB3 for the production of 5AVA in laboratory-scale bioreactor from glucose. a Characteristics of the fed-batch cultivation profile including growth (filled circles, OD₆₀₀), residual sugar (empty circles; g/L) and b production titers of products including l-lysine (dark diamonds), 5AVA (magenta diamonds), and glutaric acid (green triangles) are plotted against the cultivation time