Study On Corynebacterium Glutamicum Producing 5-aminolevulinic Acid Via C4 Pathway

5-aminolevulinic acid(ALA),a non-protein amino acid with high added-value,has been applied not only in pharmaceutical field for photodynamic therapy and diagnosis of cancer,but also in agriculture field as a plant growth accelerator and an important component of herbicides and pesticides.Currently major industrial method for producing ALA is chemical synthesis with disadvantages of low efficiency,high cost and high pollution,which caused high price and limited application of ALA.Microbial fermentation method for ALA production has been developed recently using Escherichia coli as a common host strain,which has an apparent drawback because of endotoxin synthesis in E.coli.Corynebacterium glutamicum,a GRAS strain,has been widely used in biological fermentation.Our laboratory has constructed a recombinant ALA produing strain by rebuiding foreign C4 pathway in C.glutamicum ATCC13032.The present study is carried out on this basis,and the main results are shown as follows:First of all,a complex medium for shake flask culture using yeast extract as organic nitrogen source was established.By adjusting inducer concentration,precursor concentration and inoculum amount,a stable shake flask fermentation system was eventually set up.The accumulation of ALA using this system increased by 45.54%.Secondly,biotin and nitrogen source,together with fermentation process parameters,were further optimized in a 5 L bioreactor.Under the optimum condition,the amount of ALA reached 28 g/L,which is to our best knowledge the highest level using C.glutamicum as a host strain.Thirdly,expression of ALAS encoding genes from different organisms was compared,in C.glutamicum ATCC 13032,and results showed that the hemA gene from Rhodopseudomonas palustris ATCC 17001 was more beneficial to ALA production.In combination of RBS sequence optimization,the ALA production was enhanced by 16.67%compared with the control strain.Fourthly,expression of ppc gene,encoding phosphoenolpyruvate carboxylase PPC,was improved by RBS sequence optimization.The results showed that the maximum amount of ALA reached 5.52 g/L at the flask level,33.01%the control strain.In summary,this work established a flask and bioreactor fermentation system for C4 pathway-based recombinant C.glutamicum strain.Together with fine-tuning the expression of the key enzmes ALAS and PPC,ALA production was significantly improved.This work offers an important foundation for the industrial production and application of ALA.