Research And Application Of 2,3-Butanediol Forming Operon In Enterobacter Cloacae

At present,the lack of the oil resource and the rising price of non-renewable resources make the environmental-friendly biorefinery technology be the strategic demands to guarantee transforming economic increasing mode,achieving the virtuous circle of ecological chain and realizing the sustainable development of the world.Systematic metabolic engineering can realize the selectivity modification of microorganisms.It can enhance the pathway of target compound,block the pathway of by-products,regulate the reducing power in vivo and expand the scope of substrates.Thus,systematic metabolic engineering is regarded as the key technology to realize the biotechnological synthesis of compounds.Acetoin and diacetyl are precursors of 2,3-butanediol,and both the compounds have widespread application value.Acetoin is used in foods as a common additive.In addition,acetoin has a moderate boiling point and this makes it advantageous among cigarette additives.The acetoin molecule has two functional groups-"hydroxy" and"ketone",and this makes acetoin the precursors of various compounds.Because of its versatile usages and great potential of bulk ndustrial production from renewable biomass,acetoin is classified as one of the 30 platform chemicals that are given the priority to their development and utilization by the US Department of Energy.The other precursor of 2,3-butanediol is diacetyl that is another important food additive.Thus,bio-production of acetoin and diacetyl based on the biorefinery?using biomass renewable resources as ras materials?and systematic metabolic engineering technology are the first research center in this manuscript.Various microorganisms,for example,Bacillus,Serratia,Klebsiella and Enterobacter,contains 2,3-butanediol forming operon,and these microorganisms accumulate 2,3-butanediol to be the vital fermentative products.Based on the fermentative characteristics in different strains,researchers have gained good progress in the applied research on 2,3-butanediol and its derivatives.However,the other research focus on fundamental research related in 2,3-butanediol,especially the physiological meaning of 2,3-butanediol accumulation in microorganisms.Thus,exploring the physiological meaning of 2,3-butanediol production is the other research center in this manuscript.Enterobacter cloacae subsp.dissolvens SDM can grow rapidly in nutrient-poor medium.This strain also can utilize various substrate directly.In view of these characteristics,strain SDM was selected as the host to produce acetoin in chapter two of this manuscript.This chapter describes three strategies to enhance the accumulation of acetoin,which involves redirecting the metabolic flux,fine tuning reducing power,and eliminating carbon catabolite repression.Firstly,after the knock-out of the acetoin reductase encoding gene budC,acetoin production arised significantly.Secondly,to further improve the acetoin production,we choose the NADH oxidase NOX from the three candidate enzyme,which involves NADH dehydrogenase NDH,Vitreoscilla hemoglobin VHb and NOX to regenerate NAD⁺.Thirdly,NOX encoding gene nox was subsequently differently expressed using the promoter library.And after that,NADH/NAD+ in vivo was fine tuned and yield of acetoin was further improved.Fourthly,the pathways of by-products were blocked.Moreover,after analyzing the mechanism of glucose effect,we deleted the EIIBGlc encoding gene ptsG,and over-expressed the galactose permease encoding gene gale.In the end,a strain that could utilize glucose and xylose efficiently and simultaneously was constructed.Using fed-batch fermentation of this engineered strain,45.6 g L^-1 acetoin was produced at a rate of 1.52 g L'-h^-1 using the lignocellulosic hydrolysate as substrate.This is the first report of acetoin production using lignocellulosic hydrolysate as substrate.In chapter three of this manuscript,we realize the high production of diacetyl?the other derivative of 2,3-butanediol?using the strategies of metabolic engineering and chemical conversion in E.cloacae SDM.Firstly,for realizing the accumulation of precursor of diacetyl-a-acetolactate,we blocked the enzymatic transformation from a-acetolactate to acetoin by inactivation of a-acetolactate decarboxylase.Secondly,for reducing the reduction of diacetyl to acetoin and 2,3-butanediol,we made the 2,3-butanediol dehydrogenase inactivation.Then,a fermentation validation for diacetyl production was conducted.After 12 h of bioconversion,59.8 mg L^-1 diacetyl was obtained from 15 g L^-1 glucose.The yield of diacetyl was only at 0.83%of the theoretical value.However,a-acetolactate?the precursor of diacetyl?was accumulated 2.94 g L^-1.Thus,diacetyl production could be further enhanced by the transformation of a-acetolactate accumulated in medium.It was reported that a-acetolactate could be further converted to diacetyl by addition of Fe³⁺.And the addition time of Fe³⁺ was optimized subsequently.In the end,diacetyl at a concentration of 1.45 g L^-1 was obtained with a high productivity(0.13 g L^-1 h^-1).This is the highest yield and productivity report about diacetyl production until now.In chapter four of this manuscript,the physiological meaning of 2,3-butanediol accumulation in E.cloacae was explored.We firstly verified and partly refuted the three hypothesis proposed by former researchers.We found that E.cloacae SDM has no the phenotype of "acetate overflow metabolism" in bacteria.In other words,strain SDM has the phenomenon of "acetate overflow elimination".According to the means of gene knock-outs and fermentative analysis,we found the production of 2,3-butanediol and ethanol participating in the process of "acetate overflow elimination" in strain SDM.Based on the analysis of reaction equation of 2,3-butanediol production and acetate degradation?resulting ethanol?,this chapter proposed a coordination mechanism that 2,3-butanediol synthesis might cooperate with alcohol production to eliminate the acetate overflow.The universality and extensiveness of the acetate overflow elimination metabolism were also be verified in microorganisms.In addition,a putative "regulation model of acetate overflow elimination" was proposed by isotopic tracer experiments,?-galactosidase analysis,RT-PCR and other technologies.This work proposed and verified a new hypothesis of physiological meaning of 2,3-butanediol forming operon,giving advice of elimination of "acetate overflow" in engineered strain.