Lactate Dehydrogenase Disrupted And Small RNA RyhB Expression Effect Of Metabolite 2,3-butanediol In Enterobacter Aerogenes

2,3-Butanediol has received extensive attention because of its good application prospects in many fields such as fuel,chemical industry,and medical equipment.At present,the production methods of 2,3-butanediol mainly include chemical synthesis method and biological fermentation method.The chemical synthesis method is complicated in operation,high in risk,and serious in pollution.It is difficult to achieve large-scale industrial production.The biological fermentation method can only use microorganisms Renewable resources are converted to 2,3-butanediol and the whole process is simple to operate,high safety factor,and low pollution,so the biological fermentation method is the preferred way to produce 2,3-butanediol.Enterobacter aerogenes,as the dominant strain of 2,3-butanediol,can use a variety of carbon sources for fermentation to produce 2,3-butanediol,but during the fermentation process will produce a variety of by-products.Lactic acid,as one of the main by-products,has attracted attention because it needs to consume pyruvate during the production process,and NADH will compete with 2,3-butanediol to form a substrate.And the continuous production of lactic acid will lower the pH of the fermentation broth and inhibit the normal growth of E.aerogenes,further affecting the production of 2,3-butanediol.Non-coding small RNA RyhB participates in the regulation of cellular iron homeostasis,inhibits NADH dehydrogenase(NDH-1)gene transcription,regulates the cell's overall metabolism,and promotes 2,3-butanediol production under anaerobic conditions.The research content of this paper is to use genetic engineering to knock out the key gene ldh,which plays an important role in the synthesis of lactic acid,and overexpress the small RNA RyhB to regulate the metabolic process of E.aerogenes and produce 2,3-butanediol industry provides new ideas and efficient strains,mainly including the following two aspects:1.Using the genetically engineered bacterium E.aerogenes ?nuoCD(Ea-1)with the NADH dehydrogenase hydrophilic subunit nuoCD knocked out,knock out the key gene ldh of lactate dehydrogenase through the ?-Red recombination system to obtain E.aerogenes ?nuoCD ?ldh(Ea-2)mutant strain.During the 20-hour aerobic culture,the pH value is Ea-2>Ea-1>wild-type strain,the Ea-2 exponential growth phase is significantly extended and the cell density is higher than that of Ea-1 and the original strain when entering the stationary phase.It shows that the deletion of nuoCD or(and)ldh can reduce the acid accumulation in the fermentation environment,to a certain extent,it can alleviate the inhibitory effect of acid on the growth of the strain and is conducive to cell proliferation.In an aerobic environment,the deletion of nuoCD or(and)ldh can increase the overall NADH/NAD+ratio of E.aerogenes.With aerobic fermentation,the yield of Ea-2 2,3-BD was increased by 48.27%and 6.49%compared to the original strain and Ea-1,and the lactic acid content was reduced by 77.93%and 76.12%,indicating that the knockout of the nuoCD gene can increase 2,3-BD production and the loss of lactate production from the loss of the ldh significantly reduced pyruvate for the synthesis of 2,3-BD.2.Under aerobic conditions,the growth curve,NADH/NAD+,and metabolic products of the mutant E.aerogenes ?nuoCD-RyhB(Ea-3)and E.aerogenes ?nuoCD?ldh-RyhB(Ea-4)containing small RNA RyhB Determination and analysis.The cell density and NADH/NAD+of all mutant strains containing RyhB were found to be smaller than that of the original mutant strains,indicating that RyhB has a certain inhibitory effect on cell proliferation and reduces the overall cell NADH/NAD+in aerobic environment.The RyhB-containing mutant strain also had a smaller 2,3-BD yield than the original mutant strain,which was different from RyhB promotion of 2,3-BD production by E.aerogenes during anaerobic fermentation.It can be seen that RyhB has an inhibitory effect on the growth of E.aerogenes.Under aerobic conditions,it will reduce the overall cell NADH/NAD+inhibition of 2,3-BD production,and anaerobic conditions will promote 2,3-BD production.Comprehensively considering factors such as economy and 2,3-BD production capacity,the best carbon source and the best strain from four carbon source substrates and wild-type,Ea-1,Ea-2,Ea-3,and Ea-4 strains were screened.Stable fermentation pH value is 6.8 for expanded culture.During the fermentation process,the production rate of 2,3-BD is proportional to the rate of carbon source consumption.The output of 2,3-BD in all the metabolites tested is much larger than other by-products,-The BD content reached a maximum of 283.96 mM at 24 h.