A Preliminary Study On Responses Of Actinobacillus Succinogenes To Succinic Acid Stress

Actinobacillus succinogenes is a natural succinate-producing strain with no pathogenicity.It has a wide spectrum of carbon source utilization,a high production of succinate and low production of other acids by anaerobic fermentation.However,during the fermentation of Actinobacillus succinogenes,the increasing concentration of succinate will inhibit the growth of cells,which limits the further increase of the succinate.In this thesis,the original Actinobacillus succinogenes named SW and high-yield strain ZK preserved by laboratory were used as research objects.In order to seek ways for increasing the resistance of Actinobacillus succinogenes to succinic acid,their differences in physiological responses and differentially expressed genes?DEGs?under succinic acid stress was compared.The details were as follows:?1?After comparising the tolerance of Actinobacillus succinogenes SW,SF,YQ,QZ,F₃-21,XM,ZK,and F₃F₅ preserved in laboratory to succinic acid and sodium succinate,we chose the original strain SW and the high-producing strain ZK as the main research objects in this thesis.After studying the natural growth conditions of the strain,it was determined that the acid stress condition was at pH 4.7;?2?The response characteristics of intracellular pH,H⁺-ATPase,the fatty acid content of cell membrane,cell membrane permeability,and cell morphology were examined using Actinobacillus succinogenes SW and ZK.The results showed that:strain ZK had a better ability in maintaining intracellular pH than strain SW,and the intracellular pH of both strains increased significantly after sodium succinate stress.The activity of H⁺-ATPase of both strains decreased in response to acid stress and increased in response to sodium succinate stress,indicating that H⁺-ATPase was not helpful to acid resistance,but could play a role under salt stress.An appropriate concentration of magnesium sulfate could increase H⁺-ATPase activity.After acid stress,the cell membrane fatty acid unsaturation of SW increased,while ZK's decreased.The cell membranes of both strains lacked cyclopropane fatty acids.After acid stress,the cell membranes of both strains were damaged to a certain extent,and the membrane permeability of SW increased more than that of ZK.?3?The protection of glutamic acid on strains under acid stress was studied.It was found that the protection of glutamate on Actinobacillus succinogenes may be achieved by glutamate dehydrogenase catabolizing glutamate to increase the intracellular and extracellular pH.?4?RNA-seq technology was used for the transcriptomic analysis of Actinobacillus succinogenes SW and ZK.It was found that there were 39 genes differentially expressed in strain SW after acid stress,and 49%of them were stress proteins and transporters.A total of 80 genes were differentially expressed in strain ZK,and 20%of up-regulated genes were associated with cysteine and methionine transport and metabolism.Before acid stress,SWO-ZKO had a total of 113 DEGs,and 36.3%of up-regulated genes were related to the transport and metabolism of carbohydrates,acids,alcohols and other carbon sources.After acid stress,SWX-ZKX had a total of 198 DEGs,and 27.5%of up-regulated genes were related to the transport and metabolism of carbon sources such as sugar,acid,and alcohol,and C₄ transporters.The asnB,dass,cdaR,and htpG genes encoding aconitate hydratase,anion permease,transcriptional regulators,and heat stress protein were chosen to construct overexpression vectors to study the growth of overexpression strains of Actinobacillus succinogenes under low pH conditions.Results showed that the overexpression of asnB and dass had no effect on the strains.The overexpression of htpG promoted strain growth,while the overexpression of cdaR had an adverse effect.?5?The fermentation results showed that with certain concentration of magnesium carbonate in flask,the succinate-producing capacity of five strains was in accordance with its growth ability at low pH.In order to study the effect of glutamate decarboxylase exert on the growth of Actinobacillus succinogenes,the glutamate decarboxylase gene gadB from Lactobacillus buchneri was heterologously expressed in Actinobacillus succinogenes.It was found that the growth ability of heterologously expressed strain decreased with the addition of glutamate,which was probably due to the introduction of glutamate decarboxylase that broke the original metabolic balance and affected the growth of Actinobacillus succinogenes.The physiological and transcriptional response characteristics of Actinobacillus succinogenes to succinate stress were explored in this thesis,which could provide reference for seeking acid resistance strategies of Actinobacillus succinogenes.