Intermediates Transport Regulation And Co-substrate Regeneration To Promote Production Of Glutarate

Glutarate is an important C5 dicarboxylic acid building block of nylon-4,5 and nylon-5,5 and other important polymers,which has important application in the field of pharmaceutical and chemical synthesis.The synthesis of glutarate by traditional chemical methods has high cost and serious pollution.Therefore,the synthesis of glutarate by biological methods is of great significance for the synthesis of polymers.In this study,a new pathway of biosynthesis of glutarate,which consists entirely of endogenous enzymes was designed using synthetic biology techniques and strategies.Through the in vitro screening of the key enzymes in the pathway,the results showed that patA can effectively catalyze the transamidation of cadaverine,with an optimal pH of 8.5 and a conversion rate of up to 10 mM·h-1.In addition,patD catalyzes the initial rate of 5-aminovaleric acid transamination up to 1.67 mM·h-1.The accumulation of glutarate was achieved by in vivo transformation experiments by constructing pathway in the host E.coli.In view of the unstable production of the original strain,we divided the metabolic pathway into three modules:cadA,patAD,and gabTD.Then the modular optimization was performed by linking the three modular genes to plasmids with different copy numbers pZE12-luc,pCS27,and pSA74.The results showed that pSA-cadA and pCS-patAD-gabTD were the best combination of modules.The initial yield of glutarate in the shake flask test reached 1.36 g·L-1.During the modular optimization,we found that the intermediates 5AVA and cadaverine were accumulated in the fermentation broth due to the limited transport.In order to solve the 5AVA transport problem,this study first tried to express the 4-aminobutyric acid transporter(gabP).The results showed that gabP can also effectively transport 5AVA,no 5AVA was detected in the fermentation broth and the yield of glutarate reached 2.34 g·L-1,with an increase of 72.06%.Similarly,this study attempted to knock out cadB,sapB,potE or overexpression of potE,puuP to reduce cadaverine accumulation,but did not achieve the desired results.The reason may be that neutralization of cadaverine with glutarate is more conducive to cell growth.This study also achieved the recycling of the co-substrate a-ketoglutarate by knocking out gdhA,which further increased the yield of glutarate to 2.57 g·L-1.Finally,the target product yield was further reached to 3.09 g·L-1 by adding the appropriate amount of co-substrate.