| Adipic acid,a key precursor for the synthesis of nylon 6,6.At present,due to the long production cycle and low production efficiency of adipic acid produced by biological method,it is difficult for microbial synthesis of adipic acid to meet the needs of industrial production.Therefore,a strain of Escherichia coli suitable for adipic acid production was developed in this study as a chassis strain,which significantly improved the titer and yield of adipic acid by removing the path rate-limiting steps,metabolite spectrum analysis and regulating precursor balance.The main research results are as follows:（1）Construction of adipic acid production strain:Firstly,based on the literature,this study reconstructed an adipate synthesis pathway with glucose as substrate,acetyl-co A and succinyl-Co A as precursors,and five pathway enzymes involved.Secondly,E.coli DH5α,E.coli JM109（DE3）,E.coli BL21（DE3）,E.coli MG1655,E.coli FMME N-2,E.coli W3110were selected as the optimal chassis strain based on the concentration of intra-cell adipic acid synthetic precursor.Finally,the recombinant plasmids p Trc His A-0875-2399-0067 and p Tet-1647-2576-7 were used to construct the adipic acid synthesis pathway,and then introduced into the chassis strain to obtain E.coli JL00.After 72 h shaking fermentation,adipic acid production was up to 0.34 g·L-1 and 0.08 g·g-1 glucose in strain E.coli JL00.（2）Comparative Genomic Analysis of adipic acid producing strains:First,E.coli FMME N-2 and E.coli BL21（DE3）were compared by genome resequencing.The differential genes were mainly distributed in the glucose transport system,central metabolic pathway,amino acid metabolic pathway and transcriptional regulatory metabolic pathway,and 40%of the differential genes were concentrated in the central metabolic pathway.Secondly,among the differential genes related to the central metabolic pathway,E.coli FMME N-2 had higher transcription levels of acs,pank,and bts T genes than the control strains,and the acs and pank genes were closely related to the synthesis of the precursor acetyl-co A.Finally,the activity of acetyl-co A synthetase and pantothenic acid kinase in E.coli FMME N-2 was higher than that in E.coli BL21（DE3）.Therefore,it is possible to enhance the expression of acs and pank genes to increase the supply of precursors,so as to improve the production of adipic acid.（3）Optimization of adipic acid synthesis path:Firstly,the key rate-limiting enzyme in the synthesis path was identified through in vitro experiments,and Tfu＿1647 was identified as the key rate-limiting enzyme in the pathway.Secondly,strain E.coli JL01 was constructed by regulating the expression of rate-limiting enzyme Tfu＿1647 with high intensity of RBS03.The titer of adipic acid was 0.87 g·L-1,and the yield of glucose was 0.12 g·g-1,which was2.56 and 1.5 times higher than that of control strain E.coli JL00,respectively.Finally,E.coli JL12 strain was obtained by a combination strategy of overexpression of acetyl-Co A synthetase acs gene,mutation of glutamate at lpd gene 354 to lysine,and knockout ofβ-subunit suc D gene encoding succinyl-Co A to balance the precursor supply of adipic acid.At the shaking flask level,E.coli JL12 obtained 1.51 g·L-1 adipic acid and 0.25 g·g-1 glucose.（4）Fermentation optimization and test of adipic acid producing strain:Firstly,the induction temperature and induction time in shaking flask fermentation system were optimized,and the optimum fermentation conditions were determined as follows:induction temperature 37℃and concentration of inducer 3.0 mmol·L-1,the titer of adipic acid was 1.69g·L-1,and the yield of glucose was 0.15 g·g-1.Secondly,the fermentation process（inoculation amount,residual sugar concentration,rotational speed,ventilation and dissolved oxygen）was optimized in a 2.4-L bioreactor.When inoculation amount was 10%,residual sugar concentration was 2.5-5 g·L-1,rotational speed was 600 r·min-1,ventilation was 0.5 vvm and dissolved oxygen was not controlled,the highest yield of adipatic acid was 22.31 g·L-1,and the yield was 0.21 g·g-1.Finally,by enlarging the fermentation system to a 5-L fermenter,the yield of adiponic acid reached 25.50 g·L-1,the yield was 0.27 g·g-1,and the production intensity was 0.85 g·L-1·h-1. |
