Engineering the C4 pathway of Corynebacterium glutamicum for efficient production of 5-aminolevulinic acid / 生物工程学报

5-aminolevulinic acid (5-ALA) plays an important role in the fields of medicine and agriculture. 5-ALA can be produced by engineered Escherichia coli and Corynebacterium glutamicum. We systematically engineered the C4 metabolic pathway of C. glutamicum to further improve its ability to produce 5-ALA. Firstly, the hemA gene encoding 5-ALA synthase (ALAS) from Rhodobacter capsulatus and Rhodopseudomonas palustris were heterologously expressed in C. glutamicum, respectively. The RphemA gene of R. palustris which showed relatively high enzyme activity was selected. Screening of the optimal ribosome binding site sequence RBS5 significantly increased the activity of RphemA. The ALAS activity of the recombinant strain reached (221.87±3.10) U/mg and 5-ALA production increased by 14.3%. Subsequently, knocking out genes encoding α-ketoglutarate dehydrogenase inhibitor protein (odhI) and succinate dehydrogenase (sdhA) increased the flux of succinyl CoA towards the production of 5-ALA. Moreover, inhibiting the expression of hemB by means of sRNA reduced the degradation of 5-ALA, while overexpressing the cysteine/O-acetylserine transporter eamA increased the output efficiency of intracellular 5-ALA. Shake flask fermentation using the engineered strain C. glutamicum 13032/∆odhI/∆sdhA-sRNAhemB- RBS5RphemA-eamA resulted in a yield of 11.90 g/L, which was 57% higher than that of the original strain. Fed-batch fermentation using the engineered strain in a 5 L fermenter produced 25.05 g/L of 5-ALA within 48 h, which is the highest reported-to-date yield of 5-ALA from glucose.

Bacteriocin from Purple Nonsulfur Phototrophic Bacteria, Rhodobacter capsulatus

To find whether productivity of bacteriocin is controlled between different species under unusual cultural conditions, we used Rhodobacter capsulatus ATCC 17016 as a producer and Rhodopseudomonas palustris ATCC 17003 as an indicator. Rhodobacter capsulatus was cultured under aerobic conditions in the dark in Lascelles medium containing 0.3% Triton X-100. As a result, bacteriocin productivity increased enormously. The optimal pH range of bacteriocin production was 6~7.8. Through partial purification of bacteriocin, the molecular weight was roughly estimated at 14 kDa. Plasmid had no influence on bacteriocin production by Rhodobacter capsulatus. Our findings indicate that culture conditions affect bacteriocin productivity between more distantly related species, and bacteriocin of Rhodobacter capsulatus is not encoded by a plasmid.