Engineering Corynebacterium glutamicum to produce 5-aminolevulinic acid from glucose

Abstract

Background: Corynebacterium glutamicum is generally regarded as a safe microorganism and is used to produce many biochemicals, including L-glutamate. 5-Aminolevulinic acid (ALA) is an L-glutamate derived non-protein amino acid, and is widely applied in fields such as medicine and agriculture.

Results: The products of the gltX, hemA, and hemL genes participate in the synthesis of ALA from L-glutamate. Their annotated C. glutamicum homologs were shown to be functional using heterologous complementation and overexpression techniques. Coexpression of hemA and hemL in native host led to the accumulation of ALA, suggesting the potential of C. glutamicum to produce ALA for research and commercial purposes. To improve ALA production, we constructed recombinant C. glutamicum strains expressing hemA and hemL derived from different organisms. Transcriptome analysis indicated that the dissolved oxygen level and Fe(2+) concentration had major effects on ALA synthesis. The downstream pathway of heme biosynthesis was inhibited using small molecules or introducing genetic modifications. Small-scale flask cultures of engineered C. glutamicum produced 1.79 g/L of ALA.

Conclusion: Functional characterization of the key enzymes indicated complex regulation of the heme biosynthetic pathway in C. glutamicum. Systematic analysis and molecular genetic engineering of C. glutamicum may facilitate its development as a system for large-scale synthesis of ALA.

Fig. 1

The ALA biosynthesis pathway in C. glutamicum. The enzymes encoded by the corresponding genes are: gdh glutamate dehydrogenase; gltX glutamyl-tRNA synthetase; hemA glutamyl-tRNA reductase; hemL glutamate-1-semialdehyde aminotransferase; hemB 5-aminolevulinic acid dehydratase. Glu glutamate; Gln ^tRNA Glutamyl-tRNA; GSA glutamate 1-semialdehyde aminotransferase; ALA 5-aminolevulinic acid; PBG porphobilinogen

Fig. 2

The complementation and ALA accumulation experiments in recombinant E. coli. a The ALA auxotrophic E. coli JP1449 containing pGX. b The ALA auxotrophic E. coli SASX41B containing pHA. c The ALA auxotrophic E. coli GE1377containing pHL. d The ALA accumulation in recombinant E. coli DH5α expressing gltX, hemA and hemL from C. glutamicum. Complementation experiments using the ALA auxotrophic strains were grown in LB medium supplemented with 50 μg/mL ALA (ALA+) or without ALA (ALA−). The wild type E. coli DH5α was respectively transformed with pUC19, pGX, pHA and pHL, generating the strain PD19, PDGX, PDHA and PDHL. Samples were taken and measured at 24 h with an interval of 4 h. The results were the average of three individual experiments

Fig. 3

Effects on the growth, ALA and PBG accumulation in C. glutamicum SEAL with addition of LA, MA and PA. 40 g/L glucose was added initially as the sole carbon source. The results are the average of three individual experiments

Fig. 4

The growth, ALA and PBG accumulation in C. glutamicum SEAL and SEAL1. SEAL contains the plasmid pSEAL, and SEAL1 contains the plasmid pSEAL and the ASV tag in the C-terminus of ALAD. 40 g/L glucose was added initially as the sole carbon source. The results are the average of three independent experiments