| 5-Aminolevulinic acid（5-ALA）is an important functional non-proteinogenic amino acid,which is widely used in the fields of medicine and agriculture.Recently,it is mainly synthesized by biological fermentation from Escherichia coli and Corynebacterium glutamicum.In this study,we constructed and optimized the C4 metabolic pathway to increase the production of 5-ALA in C.glutamicum ATCC 13032.The main contents are summarized as follows:（1）Since the 5-aminolevulinic acid synthase ALAS from Rhodobacter capsulatus and Rhodopseudomonas palustris had certain advantages over the enzyme activity,they were selected to express in C.glutamicum ATCC 13032 heterologously.The RphemA gene from R.palustris was used as the key synthetase gene,and the ribosome binding site RBS5 was screened which can significantly enhance the enzyme activity of RphemA.The ALAS activity of the recombinant strain CA5 reached（221.87±3.10）U·mg-1,which was 39.70%higher than the original specific enzyme activity of strain CA2(158.80±4.08 U·mg-1).The highest yield of 5-ALA accumulated by the strain CA5 in shake flask fermentation reached up to 9.47g·L-1.（2）By increasing the metabolic flux of glucose to the direction of 5-ALA precursor succinyl CoA,it promoted the accumulation of 5-ALA.The succinyl CoA synthase sucD gene was knocked out in C.glutamicum ATCC 13032,and the sucD deletion strain was used as the starting engineering strain.After overexpressing the RphemA gene,the recombinant strain CA10 accumulated 7.80 g·L-15-ALA.In addition,the RphemA gene was overexpressed in the double deletion mutant strain that knocking out theα-ketoglutarate dehydrogenase inhibitor gene（odhI）and succinate dehydrogenase gene（sdhA）.The 5-ALA synthesis ability of strain CA12 under shake flask fermentation was greatly enhanced,reaching the level of 9.94 g·L-1,which was 31.13%higher than the control strain CA2(7.58 g·L-1).（3）Explored the effect ofα-ketoglutarate dehydrogenase complex enzyme genes（odhA-sucB-lpd）on the synthesis of 5-ALA.The study found that the highest yield of the recombinant strain CA16 expressing RphemA and odhA-sucB-lpd in tandem was increased to 8.20 g·L-1,which was 8.18%higher than that of CA2.The highest yield of the recombinant strain CA14 expressing RphemA and sucB in tandem was slightly higher than that of the original strain CA2,while expressing RphemA in tandem with odhA and lpd,the yield of the recombinant strain was decreased compared with the control.The results indicated that the gene sucB may be the key gene of theα-ketoglutarate dehydrogenase complex enzyme system.（4）Overexpression of the threonine and homoserine efflux protein RhtA and the cysteine/O-acetylserine transporter EamA from E.coli in the strain CA2,the extracellular secretion capacity of 5-ALA can be enhanced.The overexpression of EamA can promote 5-ALA exocytosis efficiency more than RhtA.The 5-ALA synthesis of the strain CA18 overexpressing the eam A gene was increased to 9.56 g·L-1,while RhtA also transported 5-ALA eventually reaching 8.10 g·L-1.（5）Suppress hem B expression by sRNA to reduce 5-ALA downstream degradation.The recombinant strain CA21 containing sRNA sequence was constructed based on the strain CA2.The 5-ALA content was increased to 7.82 g·L-1 by sRNA inhibition through shaking flask fermentation,which was slightly higher than the control strain CA2.（6）Finally,the recombinant strain CA22 was constructed by combining the efficient RBS5 sequence to express RphemA,knocking out the odhI and sdhA genes,overexpressing the transporter EamA,and inhibiting hemB activity with sRNA.The strains CA22 was carried out shake flask fermentation under the optimal conditions of the substrate glycine and the maximum output of 5-ALA reached 11.90 g·L-1.Applying fed-batch fermentation in a 5 L fermenter,the recombinant strain synthesized 25.05 g·L-1 of 5-ALA within 48 h and this research had high prospects for industrial application. |
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