A New Strategy for Production of 5-Aminolevulinic Acid in Recombinant Corynebacterium glutamicum with High Yield

Abstract

5-Aminolevulinic acid (ALA), a nonprotein amino acid involved in tetrapyrrole synthesis, has been widely applied in agriculture, medicine, and food production. Many engineered metabolic pathways have been constructed; however, the production yields are still low. In this study, several 5-aminolevulinic acid synthases (ALASs) from different sources were evaluated and compared with respect to their ALA production capacities in an engineered Corynebacterium glutamicum CgS1 strain that can accumulate succinyl-coenzyme A (CoA). A codon-optimized ALAS from Rhodobacter capsulatus SB1003 displayed the best potential. Recombinant strain CgS1/pEC-SB produced 7.6 g/liter ALA using a mineral salt medium in a fed-batch fermentation mode. Employing two-stage fermentation, 12.46 g/liter ALA was produced within 17 h, with a productivity of 0.73 g/liter/h, in recombinant C. glutamicum Through overexpression of the heterologous nonspecific ALA exporter RhtA from Escherichia coli, the titer was further increased to 14.7 g/liter. This indicated that strain CgS1/pEC-SB-rhtA holds attractive industrial application potential for the future.

Importance: In this study, a two-stage fermentation strategy was used for production of the value-added nonprotein amino acid 5-aminolevulinic acid from glucose and glycine in a generally recognized as safe (GRAS) host,Corynebacterium glutamicum The ALA titer represented the highest in the literature, to our knowledge. This high production capacity, combined with the potential easy downstream processes, made the recombinant strain an attractive candidate for industrial use in the future.

FIG 1

ALA biosynthesis pathway in C. glutamicum. The enzymes encoded by the corresponding genes are as follows: GdhA, glutamate dehydrogenase; GltX, glutamyl-tRNA synthetase; HemA, glutamyl-tRNA reductase; HemL, glutamate-1-semialdehyde aminotransferase (in the C₅ pathway); HemA, 5-aminolevulinate synthase (in the C₄ pathway). Cross in pathway, sucC and sucD, encoding succinyl-CoA synthetase, were deleted; circled gene, HemA in the C₄ pathway was exogenously expressed.

FIG 2

Time course of ALA production by recombinant C. glutamicum strains. Wild-type C. glutamicum and CgS1 harboring the ALAS of R. sphaeroides 2.4.1 were cultured in a 300-ml baffled shake flask containing 50 ml modified CGXII medium, at 30°C and 180 rpm, and the pH was maintained at approximately 6.5. The initial glucose (Glc) concentration was 40 g/liter. The precursor glycine (Gly) was added at 2 g/liter every 12 h, and 10 g/liter succinate (Suc) was added when needed. Results are averages from three independent experiments, with standard deviations indicated by error bars.

FIG 3

Comparison of ALASs from different sources. CgS1 strains harboring different ALASs were compared for ALA production in batch fermentation performed under the same conditions as in Fig. 2. Cell extracts of the five strains were used to determine ALAS activity as described previously (15). Results are averages from three independent experiments, with standard deviations indicated by error bars.

FIG 4

Fed-batch fermentation profile of CgS1/pEC-SB for ALA production. Fermentation was performed in a 1-liter baffled shake flask containing 100 ml modified CGXII medium, at 30°C and 180 rpm, and the pH was maintained at approximately 6.5. The initial glucose concentration was about 40 g/liter. The precursor glycine was added at 2 g/liter every 12 h. For clarity, a representative curve from three independent experiments is shown, with standard differences (relative standard deviations [RSDs]) of less than 10%.

FIG 5

Two-stage fermentation profile of CgS1/pEC-SB for ALA production under optimized conditions. Fermentation was performed in a 300-ml baffled shake flask containing 50 ml 2 mM MgSO₄ in 250 mM potassium phosphate buffer, at 30°C and 180 rpm, and the pH was maintained at approximately 6.5. The initial glucose and glycine concentrations were ∼20 g/liter. For clarity, a representative curve from three independent experiments is shown, with standard differences (RSDs) of less than 10%.

FIG 6

ALA production by two-stage fermentation in various recombinant C. glutamicum strains. Fermentation was performed under the same conditions as in Fig. 5. Results are averages from three independent experiments, with standard deviations indicated by error bars.