5-Aminolevulinic Acid Production By Recombinant E. Coli Via Host Selection And Process Improvement

5-aminolevulinic acid (ALA) is a common precursor in the biosynthesis of heme, porphyrin, chlorophyll and vitamin B12 and other tetrapyrrole compounds, widely exists in microorganisms, plants and animal cells. ALA which has no toxicity to humans and animals, can be easily degraded in the environment. Therefore it is a pollution-free green pesticide.In 1999, the FDA formally approved ALA as a photodynamic drug for the treatment of skin cancer. ALA shows a broad market prospect in the field of agriculture and medicine.ALA production by using the wild type E. coli MG1655 was also optimized. The use of different nitrogen sources, different carbon sources were investigated and compared in shake flasks. Then the results of shake flasks were amplified in a 15-1 fermenter. After cultivation for 28 hours, ALA yield reached 4.63 g/1. In the meanwhile, the ALA synthase gene was synthesized after the rare codons were optimized. Then we successfully constructed a recombinant strain of E. coli MG1655 and (DE3)/pET28a (+)-OP. hemA. It was found that the new strain showed high synthetic potential of ALA. These results provide some new ideas and directions in further increasing the ALA yield, reducing the cost of raw materials of fermentation.The fermentation process of recombinant Escherichia coli Rosetta (DE3)/pET28a (+)-hemA was optimized. A short-term anaerobic fermentation strategy was applied during fermentation in shake flask, and the ALA production was improved. This protocol was then confirmed with the fed-batch fermentation in a 15-1 fermenter and an ALA concentration of 8.6 g/1 was achieved. Moreover, up to 9.4 g/1 (72 mM) of ALA was accumulated in the 151 fermenter when D-glucose and precursors was continuously fed into the fermenter 6 hours after inoculation. This is the highest reported concentration of ALA in the fermentation broth, which was nearly 29% higher than the highest level obtained by recombinant E. coli reported to date.Finally, this work successfully constructed two homologous recombination fragments in the ALA metabolic pathway. This lays a certain foundation for the further integration of the ALA synthase gene to E.coli chromosome and the replacement of ALA dehydratase gene.