Analysis Of The Role Of 2,3-butanediol Synthesis In Acetate Overflow Elimination And Low-cost Production Of 2,3-butanediol

With the depletion of petroleum resources and the enhancement of human environmental awareness,the biorefinery technology which uses renewable biomass resources to produce platform compounds has attracted increasing attention from researchers.2,3-Butanediol is a valuable C4 platform compound,which is widely used in food,chemical,agricultural,cosmetics and other industries.At present,the general method for the production of 2,3-butanediol requires butene in the cracked gas as a raw material.This process is carried out under high temperature and high pressure conditions,which has disadvantages such as high production cost,complex process,and environmental pollution.Therefore,the production of 2,3-butanediol by microbial fermentation with mild reaction conditions and simple operation has been favored by researchers2,3-Butanediol biosynthesis pathway exists in a variety of microorganisms,and these microorganisms can produce 2,3-butanediol with different carbon sources as substrate.The synthesis of 2,3-butanediol in bacteria starts from the condensation of pyruvate to form α-acetolactate by α-acetolactate synthase.Then,acetoin is produced from α-acetolactate by α-acetolactate decarboxylase.Fianally,acetoin is reduced to 2,3-butanediol through acetoin reductase/2,3-butanediol dehydrogenase.Although 2,3-butanediol synthesis pathways are widely distributed in different microorganisms,the physiological meaning of 2,3-butanediol synthesis for microorganisms is still unclear Under aerobic,high glucose consumption and rapid growth condition,microorganisms will behave like under anaerobic state.This phenotype is called acetate overflow.During acetate overflow,glucose is partially oxidized to accumulate acetate for ATP supply instead of completely oxidation through TCA.Aceate overflow will inhibit the growth of microorganisms,reduce the yield of target products and increase the cost of downstream purification processes.The previous research of our group found that acetate is accumulated at the beginning of growth and then eliminated in the 2,3-butanediol production strain Enterobacter cloacae SDM.It is speculated that the synthesis of 2,3-butanediol may be involved in the elimination of acetate overflow.The research in this paper confirms that the elimination of acetate overflow is accompanied by the synthesis of 2,3-butanediol;subsequently,the mechanism of acetate overflow elimination in E.cloacae SDM was revealed through gene knockout,gene overexpression,gene transcription level analysis,isotopic tracing,reducing power perturbation,etc.The role of the 2,3-butanediol synthesis was identified to be promoting the elimination of acetate by providing NADH to support the reductive conversion of acetate to ethanol.Chitin is the most abundant polysaccharide in nature only second to cellulose.It is a structural homopolysaccharide formed by the polymerization of N-acetylglucosamine through β-1,4-glucosidic bond.Acetate will be inevitably produced during the bioavailability of chitin monomer N-acetylglucosamine.In this study,the acetate overflow elimination mechanism is applied to the N-acetylglucosamine resource recycling process and the NADH produced by the 2,3-butanediol synthesis process is used to promote the reduction of acetate to ethanol After knocking out the by-product synthesis pathways consuming reducing power,blocking the reductive conversion of acetoin to 2,3-butanediol,and combining anaerobic fermentation technology,the recombinant strain E.cloacae SDM(ΔldhAΔfrdAΔbudCΔgdh)can use N-acetylglucosamine as substrate for the co-production of acetoin and ethanol.In addition,E.cloacae SDM(ΔldhAΔfrdAΔbudCΔgdh)can also metabolize chitin hydrolysate and produce 14.39 g/L acetoin and 8.10 g/L ethanol,with the yields reaching 92%and 89%of the maximum theoretical yields,respectively.The above studies confirmed the practical application value of the acetate overflow elimination mechanism,and provided theoretical guidance for the effective elimination of toxic compound acetate in other renewable resources.The 2,3-butanediol fermentation technologies reported now mainly use monosaccharides and starches as carbon sources.Finding new cheap raw materials has become a key point of breaking in the production of 2,3-butanediol.This study attempts to select whey,the pollutant of the dairy industry,as a carbon source to explore the feasibility to produce 2,3-butanediol by microbial metabolism.Whey is a by-product of the dairy industry.Considering its high biochemical oxygen demand and chemical oxygen demand,whey is regarded as the most important pollutant.The main ingredient in whey is lactose,which accounts for about 70%-75%of the dry weight of whey.This study compares the lactose metabolism of several microbial strains with the ability to produce 2,3-butanediol,and selects Klebsiella oxytoca PDL-0 as the initial strain that can efficiently metabolize lactose to produce 2,3-butanediol.Then,metabolic engineering methods were used to block the synthesis pathway of by-products such as acetate,succinate,lactate and formate in K.oxytoca PDL-0.Using lactose as a substrate,the constructed recombinant strain K.oxytoca PDL-K5 can metabolize 173.2 g/L lactose and accumulate 74.9 g/L 2,3-butanediol within 33 h through fed-batch fermentation.The yield of 2,3-butanediol is 0.43 g/g,and the productivity reaches 2.27 g/L/h.When whey powder is used as substrate,the recombinant strain K.oxytoca PDL-K5 can consume 148.3 g/L lactose within 24 h,and the yield and productivity of 2,3-butanediol are 0.44 g/g and 2.73 g/L/h,respectivelyThe fermentation process of 2,3-butanediol also has disadvantages such as energy consumption in sterilization and fresh water resources consumption in fermentation This study explored the feasibility of using the marine bacteria Vibrio natriegens ATCC 14048 as the host for 2,3-butanediol production.It was firstly proved that the strain can be used for non-sterile open fermentation by seawater medium.Subsequently,the optimal conditions for 2,3-butanediol fermentation were determined through optimization of fermentation pH,sugar concentration,and concentration of externally added acetate.The genes related to succinate and lactate synthesis in this strain were successfully knocked out.The recombinant strain V.natriegensΔfrdAΔldhA-pETRABC could metabolize 105 g/L glucose in 12 h and accumulate 41.27 g/L 2,3-butanediol in seawater medium through non-sterile open fermentation.The yield of 2,3-butanediol is 0.39 g/g,and the productivity reaches 3.44 g/L/h.