Construction Of Microbial Cell Factories For Efficient Synthesis Of 2,3-butanediol

2,3-Butanediol(2,3-BD)is an important bio-based C4 platform compound,which has great application prospects in the fields of food,chemical,pharmaceutical and fuel,etc.Due to meeting the concept of sustainable development,the production of 2,3-BD by microbial fermentation has attracted incresing attention.However,the industrial fermentation of 2,3-BD has long faced three bottlenecks:(i)The cost of fermentation substrates is relatively high;(ii)heavy consumption of precious fresh water resources;and(iii)the fermentation process is susceptible to microbial contamination.In order to solve the above problems,microbial cell factories with excellent performance were constructed based on Bacillus licheniformis and Klebsiella pneumoniae,two 2,3-BD producers with industrial potential.The purpose of relevant research is to improve the competitiveness of 2,3-BD biosynthesis and promote its industrialization process.The main results of this dissertation are listed as following:1.Modular metabolic engineering of Bacillus licheniformis MW3 was implemented to efficiently produce 2,3-BD with cheap and non-food inulin as feedstock.The modified modules included inulin hydrolysis module,sporulation elimination module,carbon flux redistribution module and enantiopure meso-2,3-BD synthesis module.Firstly,the inulin saccharification rate and 2,3-BD synthesis efficiency were enhanced by introducing endo-inulinase.Secondly,sporulation was blocked by spoIIE gene knockout,thus strengthen the control of engineered strain in the fermentation process.Thirdly,more carbon flux was shunted into the 2,3-BD synthesis pathway via exchanging the start codon of alsS gene,knocking out acoR gene and blocking by-products synthesis pathways.Finally,the glycerol dehydrogenase gene gdh was knocked out for producing the chiral pure meso-2,3-BD.Encouragingly,82.5 g/L meso-2,3-BD with 99.3%purity was obtained by the final engineered strain MWO-8 in the fed-batch fermentation,with a productivity of 1.38 g/L/h and a yield of 0.439 g/g.To our knowledge,this is the highest titer of meso-2,3-BD so far produced by simultaneous saccharification and fermentation of inulin in the condition of without exogenously adding inulinase.2.The ability of engineered strain MWO-8 to tolerate salt-stress was investigated,and its salt-tolerance mechanism was studied.The results showed that the low salinity(1%～5%NaCl)had little effect,while the high salinity(6%～7%NaCl)exhibited significant inhibition effect on the cell growth of MWO-8.Subsequently,through transcriptomic analysis,it was found that osmotic regulation and stress response were the two main strategies of MWO-8 to tolerate saltstress.In terms of osmotic regulation,MWO-8 regulated osmotic pressure by absorbing K+and excreting Na+,and accumulating compatible solute proline in vivo to quickly adapt salt-stress.Additionally,the synthesis of proline under salt-stress was mediated by proHJAA operon.In terms of stress response,the σB-mediated general stress response played a major role,while the ECF σ factors-mediated specific stress response played a relatively minor role.3.Based on the excellent salt-tolerance property of MWO-8,the seawater-based 2,3-BD fermentation was performed.The results showed that in seawater medium,the titer of 2,3-BD was 27.5 g/L,the fermentation time was 26 h,and the maximum biomass OD600 was 15.58.In contrast,in the conventional fermentation medium,the corresponding values were 48.5 g/L,16 h and 21.72,respectively,which indicating that the fermentation performance of MWO-8 decreased significantly in seawater medium.In order to enhance the fermentation performance of MWO-8 in seawater medium,MWO-8 was adaptively precultured by gradually increasing the salt content of seed medium,thus the 2,3-BD titer was increased to 31.7 g/L,the fermentation time was shortened to 22 h,and the maximum biomass was increased to 18.24.Additionally,by adding betaine to seawater medium,the 2,3-BD titer and maximum biomass were further increased to 34.6 g/L and 19.37,and the fermentation time was shortened to 20 h.Finally,using the improved fermentation strategy,27.8 g/L 2,3-BD was obtained in seawater medium by simultaneous saccharification and fermentation of inulin.4.From the perspective of nutrient assimilation,non-sterilized fermentation of 2,3-BD was achieved by an engineered Klebsiella pneumoniae KP-OU7 with an anti-microbial contamination system.Firstly,it was found that KP-OU7 has phosphite and urea utilization gene cluster,thus KP-OU7 can rapidly grow in the auxotrophic MOPS medium,which phosphite and urea are used as phosphorus and nitrogen sources,while other microorganisms starve to death due to lack of these two metabolic pathways,making it can greatly reduce the risk of microbial contamination.Secondly,the by-products synthesis pathways of KP-OU7 were blocked to enhance the 2,3-BD fermentation performance.Additionally,wabG gene was knocked out and adaptive laboratory evolution was performed to eliminate the pathogenicity of fermentation strain.Finally,under the non-sterilized auxotrophic MOPS medium,84.53 g/L of 2,3-BD was obtained by engineered strain KP-OU7-ME5 in the fed-batch fermentation,with a productivity of 1.17 g/L/h and a yield of 0.38 g/g.