Multi-modular Engineering Of 1,3-propanediol Biosynthesis System

1,3-Propanediol(1,3-PDO)is a monomer for the synthesis of various polyesters,particularly used for polytrimethylene terephthalate(PTT)synthesis.It is widely used in industries including cosmetics,solvents and lubricants.The production of 1,3-PDO by biotechnological process showed advantages over the conventional chemical one.The natural producing strain,such as Klebsiella pneuwmoniae(K.pneumoniae),has advantages of good tolerability,high conversion rate and so on.However,there are serious problems,such as high concentration of byproduct,insufficient supply of NADH and inefficient genetic modification.In this work,the multi-modular engineering was applied to regulate the l,3-PDO bio-synthesis system in K.pneumoniae.Firstly,the glycerol pathway of K.pneumoniae has been optimized.Afterwards,the relationship between the regulation of NADH and carbon metabolism has been studied,and a variety of regulatory modules and strategies have been constructed to drive the synthesis of 1,3-PDO.In addition,the effects of intracellular concentration of NADH on 1,3-PDO synthesis and cell growth have been studied and suggests that the coupling strategies of carbon metabolism and energy metabolism maximizes and speeds the 1,3-PDO metabolic flux and can fundamentally enhance the ability to produce l,3-PDO.The design and reconstruction of non-natural metabolic pathways have important implications for expanding the production capacity of cell factories for producing natural and non-natural products.At present,there are many successful cases.Here,an entirely new non-natural synthetic pathway for 1,3-PDO was designed and constructed.Compared with the Dupont glucose-transformed 1,3-PDO via the glycerol pathway,VB12 is not required.Compared with the synthesis of 1,3-PDO from glucose by homoserine pathway,this pathway contains shorter and fewer unknown enzymes and broadens the route of microbial synthesis of 1,3-PDO.Therefore,based on the above two research directions,the main research results of this paper are as follows.1.Optimization of the glycerol pathway.The endogenous pathway of K.pne2umoniae was optimized.The tolerance and concentration of 1,3-PDO were increased by 36.0%and 62.5%,respectively.The concentrations of 2,3-butanediol(2,3-BDO)and lactic acid were decreased by 40.0%and 98.0%,respectively.The err was deleted and the recombinant strain could use the glycerol and glucose simultaneously.The concentration of l,3-PDO was increased by 25.0%and the productivity and yield were increased to 2.26 g/L h and 0.52 mol/mol,respectively.2.Effect of glycerol transport system and NADH regeneration system on the synthesis of l,3-propanediol.The glycerol transport system and NADH regeneration system were successfully constructed for the synthesis of 1,3-PDO,which increased the concentration,productivity and conversion of 1,3-PDO by 26.4%,19.0%,and 13.5%,respectively.The glycerol consumption rate,concentration of 1,3-PDO and intracellular NADH/NAD+ ratio was increased by the construction of the glycerol transport system.Intracellular NADH/NAD+ ratio was increased by 27.4%when NADH regeneration system was construction.The concentration of 1,3-PDO,the productivity and the yield was increased to 86.1 g/L,2.69 g/L h and 0.59 mol/mol,respectively.The results show that the regulation of carbon metabolism not only changes the distribution of carbon metabolism,but also affects the presence of intracellular cofactors and their concentrations.At the same time,the regulation of energy metabolism also redirects the distribution of intracellular metabolic flux.3.Coupling regulation strategy for the optimization of 1,3-propanediol synthesis system.The construction of Entner-Doudoroff(ED)pathway,overexpression of transhydrogenase and 1,3-PDO oxidoreductase(PDOR),and asRNA regulation strategies were couplied to increase the concentration of NADH and the ratio of NADH/NAD+.The NADH can directionally drive the synthesis of 1,3-PDO and the concentration and conversion of 1,3-PDO were increased to 83.3 g/L and 0.62 mol/mol,respectively.The construction of ED pathway and overexpression of PDOR not only accelerated the synthesis of NADH and NADPH,but also promoted the utilization of glucose and glycerol.The concentration of intracellular coenzyme ? increased by 59.9%and the concentration of coenzyme ? increased by 66.8%.The ratio of NADH/NAD+ and NADPH/NADP+ were increased by 58.9%and 30.3%,respectively.The overexpression of transhydrogenase could chang the existing forms of intracellular coenzymel and coenzymell,and the NADH/NAD+ ratio was further increased by 21.3%.The asRNA strategy successfully weakened the 2,3-BDO pathway and the weakening efficiency reached over 50%.These results suggest that the fluctuation of NADH/NAD+ not only redirects the distribution of intracellular metabolic flux,but also affects the transcription of genes involved in the central carbon metabolism.4.Effect of material and cofactor balance on 1,3-PDO synthesis system.In order to consider both the growth of strains and the efficient synthesis of products and avoid the waste of raw materials and energy,the CRISPRi,cofactor preferences modification and riboswitch-coupled regulatory strategies were used to maintain the relative balance of intracellular substances and energy.Using CRISPRi technology to weaken intracellular competition for product pathways that consume NADH,the concentrations of NADH and 1,3-PDO were increased by 63.3%and 34.6%,respectively.The concentrations of 2,3-BDO,ethanol and acetic acid were decreased by 12.3%.55.6%and 51.9%,respectively.The cofactor preference of PDOR was successfully changed and the multiple-site mutants made the concentration of 1,3-PDO increased 58.2%.The ATP-responsive riboswitch ydaO regulates the concentration of intracellular ATP and acetic acid to further increase the concentration of 1,3-PDO and the biomass.Then these strategies were coupled.The biomass and the concentration of 1,3-PDO were increased by 19.3%and 56.5%,respectively compared with the cofactor-preferred strategy.When compared to CRISPRi strategy regulation,the biomass and the concentration of 1,3-PDO were increased by 6.9%and 14.0%,respectively.5.Exploration of a new pathway for 1,3-PDO synthesis from glucose.Using the reverse synthesis method combined with on-line database analysis,the feasible biosynthetic pathway of 1,3-PDO was explored and analyzed.The viable non-natural 1,3-PDO synthesis pathway was successfully obtained.Through the screening of key enzyme sources,a novel 1,3-PDO pathway based on succinyl-CoA was successfully constructed in K.pneumoniae and E.coli.The shake flask fermentation of the best recombinant strain was 244 mg/L.