Design And Verification Of Synthetic Pathways In Escherichia Coli For De Novo Biosynthesis Of Dicarboxylic Acids

Dicarboxylic acids are bulk chemicals widely used in many areas such as food,pharmaceuticals,materials and textiles.In particular,they can be polymerized into polyamides to produce engineering plastics and synthetic fibers,which are well-known as nylons with a large market and an important application value.The traditional production of dicarboxylic acids with C5 or longer carbon chains mainly depends on petroleum resources.Due to the limited fossil resources and growing concerns of environmental pollution,bio-based "green" production of chemicals has received more and more attention around the world in recent decade.For example,researches on bio-based adipate and glutarate are emerging in recent years.The development of synthetic biology enables rational design of synthetic pathways,assembling functional elements and modules and demonstration for de novo biosynthesis of chemicals.Escherichia coli is looked at as a chasis for synthetic biology because it has advantages of clear genetic background,matured methods for genetic manipulation and fast growth.Therefore,this thesis aims to design and demonstrate novel biosynthetic pathways for de novo biosynthesis of dicarboxylic acids-adipate and glutarate in E.coli.Firstly,a reversal beta-oxidation pathway was designed for the biosynthesis of dicarboxylic acids.The biosynthetic pathway comprises condensation of acetyl-CoA and succinyl-CoA to form the C6 backbone and subsequent reduction,dehydration,hydrogenation,and release of adipate from its thioester.The pathway was first tested in vitro with reconstituted pathway enzymes and then functionally introduced into Escherichia coli for the biosynthesis and excretion of adipate into the culture medium.The production titer was increased by approximately 20-folds through the combination of recruiting enzymes that were more suitable to catalyze the synthetic reactions,increasing availability of the condensation substrates and modifying the synthetic pathway with more thermodynamicly-favorable reactions.As the titer still remains very low,the production of adipate may be further elevated by exploring the enzymes with better compatibility that allows superior catalytic activities toward the C6 substrates of each synthetic step.I also designed an alternative pathway for dicarboxylic acids biosynthesis based on a-keto acid reduction pathway,which was previously reported for glutaconate production.The artificial pathway comprises reduction of the central carbon metabolite a-ketoglutarate to 2-hydroxyglutarate,activation to 2-hydroxyglutaryl-CoA,dehydration to trans-glutaconyl-CoA and remains a gap from trans-glutaconyl-CoA to saturated dicarboxylic acids.Two enzymes,trans-enoyl-CoA reductase(Ter)and thioesterase(TesB),which were proved to be able to catalyze the reduction of 2,3-dehyroadipyl-CoA and the hydrolysis of adipyl-CoA,were employed to catalyze the conversion from trans-glutaconyl-CoA to glutarate.The entire pathway introduced into E.coli made the strain a glutarate producer,which demonstrated the feasibility of producing saturated dicarboxylic acids from a-keto acid reduction pathway.Besides,a site mutangensis of traps-enoyl-CoA reductase from Treponema denticola led to a 50%higher production of glutarate.Owing to the oxygen-sensitive nature of 2-hydroxyglutaryl-CoA dehydratase(HgdABC)while the anaerobic cell metabolism is not so active,an aerobic-anaerobic shift strategy was developed by fast growing cells aerobically,followed by a shift to anaerobic cultivation to ensure the activity of HgdABC for glutarate biosynthesis to achieve higher production of glutarate.The two-stage cultivation strategy resulted in approximately 30%higher production titer than the original anaerobic cultivation.Furthermore,an anaerobically-inducible nar promoter was employed to control the hgdABC expression,and the newly engineered strain showed efficient expression of hgdABC responding to aerobic-anaerobic shift during 5 h shift two-stage cultivation.Finally,the glutarate production titer was doubled to 11.6 mg/L,while the glutaconate and 2-hydroxyglutarate titers were increased by approximately 5 and 3 times,reaching 108.8 mg/L and 0.4 g/L respectively.These results suggested the combined strategy was useful to the dicarboxylic acid production based on a-keto acid reduction pathway by the combined strategy.In summary,two artificial biosynthetic pathways were designed for dicarboxylic acid production based on reversal ?-oxidation and a-keto acid reduction pathway,respectively.The functional artificial pathways in E.coli were successfully demonstrated by de novo synthesizing adipate and glutarate.They were subsequently reformed in a synthetic biology manner including pathway designing,recruiting suitable enzymes,metabolic engineering and process engineering.These results may be helpful in the development of biobased routes for renewable production of dicarboxylic acids,as well as other non-native platform chemicals.