Multigene Overexpression And Knockout To Improve 2,3-butanediol Production By Enterobacter Aerogenes

2,3-Butanediol is an important platform compound,which is widely used in the fields of medicine,cosmetics,food and aerospace fuel.The methods for producing2,3-butanediol include chemical cleavage and biological fermentation.The former is to produce 2,3-butanediol by hydrolysis of 2,3-butene oxide,which would lead to obvious disadvantages such as high cost,complicated process,high risk and environmental pollution.While the latter is the process in which microorganism finally produce 2,3-butanediol through a series of metabolic reactions in the cell with pyruvate as precursor.This method has the advantages of mild reaction conditions,non-pollution,environmental friendliness and wide substrate range,so it is an ideal choice for industrial production.Enterobacter aerogenes is a 2,3-butanediol-producing strain which has been studied in recent years.Its wide substrate utilization range,fast growth rate and high substrate conversion rate make it an optimal strain for biological production of 2,3-butanediol.However,the fermentation of E.aerogenes is not a completely metabolized process,with lactic acid,formic acid,acetic acid and so on as by-products,which could affect the utilization rate of carbon source and the yield of 2,3-butanediol.Therefore,reducing by-products production and improving substrate utilization rate by metabolic regulation are the important way to facilitate2,3-butanediol biosynthesis.In this paper,E.aerogenes IAM1183(E.a)was used as the starting strain.budB and budC were overexpressed to facilitate biosynthesis of precursor of 2,3-butanediol.To redistribute metabolic flux toward 2,3-butanediol pathway,pyruvate formate lyase(pfl)and lactate dehydrogenase(ldh)were knocked out.The main results of this research are as following:1.E.aerogenes pET-budB(E.a-1),E.aerogenes pET-budC(E.a-2)and E.aerogenes pET-budB/budC(E.a-3)were obtained by overexpression of?-acetolactate synthase and acetoin reductase in E.aerogenes IAM1183.After 24 h aerobic shake flask fermentation,the pH value of the fermentation broth of each strain are as follows:E.a-3=E.a>E.a-2=E.a-1.After entering the stable growth period,the density of E.a-1 and E.a-2 was lower than that of wild type,while the density of E.a-3 was similar to that of E.a.The results of shake flask fermentation with different carbon sources showed that overexpression of budB and budC genes could promote the synthesis of 2,3-butanediol by E.aerogenes and improve the utilization rate of carbon sources.The yield of 2,3-butanediol of E.a-3 was the highest and reached 51.11 m M in sucrose-based medium.2.CRISPR/Cas9 gene editing method was used to knock out pyruvate formate lyase gene(pfl),and?-Red homologous recombination method was used to knock out lactate dehydrogenase gene(ldh).Then,we obtained three gene deletion mutant E.a-4(E.aerogenes?pfl),E.a-5(E.aerogenes?ldh)and E.a-6(E.aerogenes?pfl?ldh).Mutant strains were cultured in aerobic shake flask for 24 h,and the pH value of the fermentation broth of each strain are as follows:E.a-6>E.a-5>E.a-4>E.a.After entering the stable growth period,the cell density of the mutant was significantly higher than that of the original strain,and the highest was E.a-6.Compared with the original strain,the ratio of NADH/NAD~+and the yield of 2,3-butanediol in the mutant increased significantly,while the acidic by-products decreased.The highest yield of2,3-butanediol was 43.8 m M,producing by E.a-6 in sucrose-based medium.3.The best combined expression strain E.a-3 and the best combined knockout strain E.a-6 were selected to scaled up fermentation with sucrose as carbon source,keeping pH at 6.8.The results showed that the production rate of 2,3-butanediol was directly proportional to the carbon consumption rate of the three strains during the fermentation process.The maximal production yield of 2,3-butanediol of E.a-3 was83.25 m M at 28 h,and the highest production rate of 2,3-butanediol of E.a-6 was193.47 m M at 16 h.Other products increased with the extension of fermentation time,wihle 2,3-butanediol yield increased first and then decreased.The yield of acidic by-products of E.a-6 was lower than that of E.a-3,and E.a-6 produces almost no lactic acid.However,the yield of 2,3-butanediol of E.a-6 was higher than that of E.a-3.So compared with E.a-3,E.a-6 is the promising strain for industrial production of2,3-butanediol.