Modification Of The Metabolic Pathway Of Corynebacterium Glutamate For Improting The Yield Of5-aminolevulinic Acid

5-aminolevulinic acid（ALA）is a non-protein amino acid widely existing in microorganisms,plants and animal cells.It provides key precursors for the biosynthesis of tetrapyrrol compounds such as chlorophyll,heme,porphyrin and vitamin B₁₂,and has broad application prospects in agriculture,medicine and food.The traditional ALA chemical synthesis method is difficult to put into industrial production due to the disadvantages such as high raw materials,many by-products,and difficulty in separation and purification.In recent years,the biosynthesis of ALA has attracted more and more attention of researchers,and a lot of research has been carried out around the metabolic pathway and gene modification to increase the production of ALA.Nowdays,the production of biosynthetic ALA is still at a low level,and the production cost is high,which greatly limits the application of ALA in practical production.In this study,Corynebacterium glutamicum ATCC 13032（abbreviated as W）was used as the starting strain,and the metabolic pathway of ALA synthesis was modified as follows:（1）Firstly,the expression plasmid of 5-aminolevulinic acid synthase（ALAS）coding gene hemA was constructed.The ALAS coding gene hemA（abbreviated as A）from Rhodobacter capsulatus ATCC 11166 was cloned into the expression vector p XMJ19 and the recombinant expression plasmid p XMJ19-hemA was obtained.（2）Secondly,mutant strains with deletion of glutamate dehydrogenase gene（gdh A）or succinyl-Co A synthase gene（suc CD）were constructed by suicide knockout plasmid p K18mob Sac B.The first step is to construct a mutant strain with deletion of gdh A gene to block the pathway of transformation from alpha-ketoglutarate to glutamic acid,so as to increase the flow of metabolic pathway to succinyl-Co A,and obtain the recombinant strain Corynebacterium glutamicum△gdh A,or G1 for short;the second step is to knock out suc CD gene of W and G1 strains to block the metabolic pathway of succinyl-Co A to succinic acid,respectively.Corynebacterium glutamicum△suc CD,abbreviated as S1,and Corynebacterium glutamicum△gdh A△suc CD,abbreviated as GS1,were obtained.In the third step,in order to test the effect of gene knockout on ALA metabolic pathway of Corynebacterium glutamicum,strains W,G1,S1 and GS1 were fermented for production of glutamic acid.The results showed that the glutamic acid produced by strain W was 2.12 g/L,while the glutamic acid content of G1 and GS1 was very low,which were 0.22 g/L and 0.31g/L,respectively.It indicates that the inactivation of the genes did affect the synthesis of glutamic acid,which resultly changed the metabolic pathway of Corynebacterium glutamicum.（3）Finally,the conditions of ALA production by shaking flask fermentation of the modified recombinant strain were optimized.The expression plasmids p XMJ19-hemA were transformed into strains W,G1,S1 and GS1,respectively,and the recombinant strains W-A,G1-A,S1-A and GS1-A were obtained.The four recombinant strains were fermented in shaking flask.The results showed that the maximum ALA yield of GS1-A strain was 2.06 g/L,which was 2.26 times higher than that of the control strain W-A.In order to further improve the production of ALA,recombinant strain GS1-A was selected to optimize the shaking flask fermentation conditions.The results showed that when the concentration of glucose was 45 g/L,the concentration of Fe²⁺was 0.2 mg/L,the concentration of glycine was 7 g/L,the concentration of inducer（IPTG）was 0.4mmol/L,the induction temperature was 30℃,the p H of fermentation broth was maintained at 6.5 and the initial inoculation volume was 5%,the recombinant strain GS1-A was incubated for 40 hours,the accumulation of ALA reached the highest level of 4.06 g/L,which was 95.2%higher than that before optimization,and reached the highest level of shaking flask fermentation reported in the literature.