Engineering The Reductive TCA Pathway To Dynamically Regulate The Biosynthesis Of Adipic Acid In Escherichia Coli

Adipic acid is a versatile aliphatic dicarboxylic acid,it is applied mainly in the polymerization of nylon 6,6.In order to realize the green production and industrial upgrading of adipic acid,microbial production of adipic acid had gradually become a current research hotspot.A competitive reverse adipate-degradation pathway（RADP）was obtained to synthesize adipic acid in our previous research.Although previous studies had obtained a high yield of adipic acid,they faced a common problem,that was,there were two steps of decarboxylation in the process of synthesis of succinyl-CoA through the oxidative TCA（Tricarboxylic acid）pathway,which resulted in a theoretical yield of only 54.07%and made it difficult to further improve the output of adipic acid.In order to solve the above-mentioned new challenge,based on the adipic acid synthetic engineering strain Escherichia coli Mad1415,our research contents are as follows:（1）Proposal and verification of the strategy for the synthesis of adipic acid based on the reductive TCA pathway.In order to maximize the utilization of carbon atoms,we proposed a reductive TCA pathway,which can bypass the decarboxylation step to synthesize succinyl-CoA,the precursor substance of adipic acid.The theoretical yield of adipic acid was calculated as 81.11%based on the stoichiometric formula,which was nearly 50%higher than before.By adding organic acid of the reductive TCA pathway,the reductive TCA pathway was verified to be feasible to synthesize adipic acid.At the same time,we analyzed and obtained that strengthening these key enzymes of succinyl-CoA synthase alpha subunit（sucD）,pyruvate carboxylase（pyc）,malate dehydrogenase（mdh）,and fumarate reductase（frdABCD）were beneficial to increase the synthesis of succinyl-CoA.（2）Dynamic regulation to enhance the synthesis of adipic acid by the reductive TCA pathway.Since the reductive TCA pathway is usually activated under anaerobic conditions,the production of adipic acid depends on the anaerobic fermentation environment in our study.To achieve dynamic regulation of the reductive TCA pathway under different oxygen environments,we introduced an oxygen-responsive dynamic regulation system,which based on the transcription factor fumarate nitrate reductase（FNR）.Under the control of the promoter P_{fnr F8},the expression of green fluorescent protein under anaerobic conditions was6.14 times that under aerobic conditions.On this basis,several key potential genes that might strengthen the reductive TCA pathway were overexpressed individually or in combination.It showed that the reaction catalyzed by fumarate reductase（frdABCD）was the rate-limiting step in the production of adipic acid in the metabolic pathway.At the same time,when the gene sucD,pyc,and frdABCD were co-expressed in a monocistronic structure,it was most conducive to the accumulation of adipic acid.The recombinant strain E.coli Mad1415-F8NAspf produced 0.30 g·L^-1 adipic acid,which was 23.56-fold that of the control strain E.coli Mad1415.（3）Expression optimization of reductive TCA pathway.The metabolic flux of the reductive TCA pathway is the key to determining the accumulation of succinyl-CoA under anaerobic conditions.Therefore,optimizing the expression levels of sucD,pyc,and frdABCD would help to further increase the production of adipic acid.By changing the distance between the transcription factor FNR binding site and the-35 region of the P_{fnr F8} promoter,we screened and obtained anaerobic inducible promoters with different response strengths.Based on the above research,by maintaining the high-level expression of the rate-limiting step（frdABCD）and optimizing the expression of sucD and pyc with high,medium,and low strength anaerobic inducible promoters,we concluded that when the gene frdABCD,sucD,and pyc were expressed at high,medium,and low levels,respectively,the accumulation of adipic acid was increased by 76.67%compared to before optimization,and the titer was 0.53g·L^-1,which was 41.62-fold that of the control strain E.coli Mad1415.