L-citrulline production by metabolically engineered Corynebacterium glutamicum from glucose and alternative carbon sources

Abstract

L-citrulline plays an important role in human health and nutrition and is an intermediate of the L-arginine biosynthetic pathway. L-citrulline is a by-product of L-arginine production by Corynebacterium glutamicum. In this study, C. glutamicum was engineered for overproduction of L-citrulline as major product without L-arginine being produced as by-product. To this end, L-arginine biosynthesis was derepressed by deletion of the arginine repressor gene argR and conversion of L-citrulline towards L-arginine was avoided by deletion of the argininosuccinate synthetase gene argG. Moreover, to facilitate L-citrulline production the gene encoding a feedback resistant N-acetyl L-glutamate kinase argB (fbr) as well as the gene encoding L-ornithine carbamoylphosphate transferase argF were overexpressed. The resulting strain accumulated 44.1 ± 0.5 mM L-citrulline from glucose minimal medium with a yield of 0.38 ± 0.01 g[Symbol: see text]g(-1) and a volumetric productivity of 0.32 ± 0.01 g[Symbol: see text]l(-1)[Symbol: see text]h(-1). In addition, production of L-citrulline from the alternative carbon sources starch, xylose, and glucosamine could be demonstrated.

Figure 1

L-arginine pathway in C. glutamicum (modified from (Wendisch et al.[2014])). gdh: L-glutamate dehydrogenase, cg3035: anaplerotic N-acetylL-glutamate synthase, argJ: L-ornithine N-acetyltransferase, argB: N-acetylL-glutamate kinase; argC: N-acetyl-gamma-glutamyl-phosphate reductase; argD: acetylL-ornithine aminotransferase; argE: acetylL-ornithine deacetylase; argF: L-ornithine carbamoyltransferase; argG: argininosuccinate synthetase; argH: argininosuccinate lyase. Oxoglutarate is an intermediate of the central carbon metabolism.

Figure 2

Biomass formation by various C. glutamicum strains. The cultivation was performed in CGXII minimal medium containing 20 g L-1 glucose, 1 mM IPTG, 750 μM L-arginine and 25 μg L-1 kanamycin. OD₆₀₀ was determined of CIT0(pVWEx1) (open squares), CIT0(pVWEx1-argF) (gray circles) and CIT0(pVWEx1-argFB^fbr) (black diamonds). Values and error bars represent the mean and the standard error of triplicates.

Figure 3

Biomass formation and production of ornithine and citrulline on glucose by various C. glutamicum strains: cell dry weight (hatched bars), L-ornithine concentration (open bars) and L-citrulline concentration (filled bars). The cultivation was performed in CGXII minimal medium containing 20 g/L glucose, 1 mM IPTG, 750 μM L-arginine and 25 μg/L kanamycin. The amino acid concentrations in the supernatant were determined after the consumption of glucose. Values and error bars represent the mean and the standard error of triplicates.

Figure 4

Amino acid production by various C. glutamicum strains. L-ornithine production by C. glutamicum CIT0(pVWEx1) (filled squares) (A) and L-citrulline accumulation (filled squares) and glucose consumption (open triangles) by strain CIT0(pVWEx1-argFB^fbr) (B). The experiments were performed in CGXII minimal medium with 20 g/L glucose, 1 mM IPTG, 25 μg/L kanamycin and supplemented with 750 μM L-arginine. Values and error bars represent the mean and the standard error of triplicates.

Figure 5

L-citrulline concentration in the engineered strains after the consumption of the respective carbon source. CIT1(pEC-XT99A), CIT1(pAmy) with 10 g/L soluble starch, 2,5 g/L glucose after 31 h. CIT1(pEKEx2-xylAB) with 15 g/L xylose after xylose consumption. CIT1(pEKEx3-nagB) with 10 g/L glucosamine after glucosamine consumption. Values and error bars represent the mean and the standard error of triplicates.