Mining And Application Of Energy Utilizing Related Genes In Favour Of Heterologous Expression System Of Corynebacterium Glutamicum

Corynebacterium glutamicum(C.glutamicum)is wildly used in amino acid industry due to its remarkable advantage.With the development of the research on C.glutamicum expression system,an increasing amount of heterologous proteins was expressed using C.glutamicum as the host.However,the disadvantages of C.glutamicum limit its application,such as lacking of optional expression vectors and inefficient expression.Our research used Corynebacterium glutamicum CGMCC 1.15647(named as C.glutamicum BZH001)as the research model.Key genes involved in the process of heterologous protein expression were further researched and explored.The in-parallel fermentation was carried out under the condition of 0%,30% and 50% DO(Dissolved Oxygen)by C.glutamicum BZH001 and C.glutamicum eGFP(an eGFP expressing strain constructed by C.glutamicum BZH001)strain.The transcriptome and metabolism analysis were used to deeply explore the effect of different DO conditions on transcription and metabolism when C.glutamicum BZH001 expressing eGFP.The Multivariate Data Analysis(MVDA)was also used to analysis the transcriptome and metabolism data,and three key genes about energy metabolism were determined,providing potiential gene modification targets for improving heterologous protein expression ability of C.glutamicum.These three genes' function and their regulation mechanism on heterologous protein expression were revealed by knock-out and overexpression experiments,determination of the important metabolic physiological parameters and the relative gene expression pattern analysis.Not only our research provide a kind of host cell for C.glutamicum expression system,but also gave a huge basis knowledge for establishing the gene regulatory network which affect heterologous protein expression.The major conclusions are as follows:1.The effect of different DO on metabolism and transcriptome of C.glutamicum BZH001.The transcriptome and metabolism analysis of C.glutamicum BZH001 were carried out using the data of fermentation under 0%,30% and 50% DO condition.The results showed that 30% and 50% DO had less effect on metabolism of C.glutamicum BZH001 whereas 0% DO inhibit the growth of bacteria.The concentration of ATP was lowest under 0% DO;under this condition the electron transport chain(ETC)was suppressed,then in order to balance the redox state and product more ATP,substrate level phosphorylation and glucose comsumption increased,TCA shunt: firstly,the activity of pyruvate dehydrogenase reduced,and malate synthase activity was up-regulated;secondly the product of Cys,Arg and Glu increased activating the TCA shunt;finally the up-regulation of the glyoxylate pathway increased the TCA shunt under low DO condition.The glyoxylate pathway offered more malic acid for TCA,increased the concentration of oxaloacetic acid,decreased the carbon flux of redox arm of TCA,decreased the accumulation of NADH,and further promoted the balance of redox level.The transcriptome data showed that ribosome and phosphorylation pathway were the two pathways that had the most enrichment of the differentially expressed genes.2.The transcriptome and metabolism analysis of C.glutamicum under the stress of DO and heterologous protein expression.We described RNA-seq for C.glutamicum eGFP under different DO levels(50%,30% and 0%),and the results revealed that 0% DO condition have the maximum effect on C.glutamicum eGFP.In order to get an enough energy for living from metabolism and maintain the redox balance under low DO,the metabolism shifted.For carbon metabolism,the glycolytic pathway was down-regulated,glycolytic pathway and TCA showed a carbon overflow: the amount of lactic acid,glyoxylic acid and acetic acid increased,and the pathway of Val,Ile,Iso synthesized was down-regulated.For energy metabolism,it was redox state under low DO.In order to maintain the intracellular balance of redox,TCA was downregulated by less NADH product,the glyoxylic acid pathway increased,and the genes of ETC were up-regulated.The concentration of ATP and eGFP were both lower than that under high DO concentration.For substrate transport,the PTS system was down-regulated under low DO condition.The ABC transporter like GluB,GluA was down-regulated.The DEGs was enriched on amino acid transportation and metabolism,carbohydrate transportation and metabolism,energy metabolism,signal transduction and cytothesis of cell function.3.The mining of energy-utilization related genes of C.glutamicum.The transcriptome and metabolism analysis of C.glutamicum eGFP and C.glutamicum BZH001 were done under 30% DO concentration to explore the alteration of metabolism and gene expression level when expressing heterologous protein.Extracellular and intracellular metabolites and key enzymes' activity of both strains were investigated.The metabolism-like anaerobic conditions was discovered.The ribosomal protein gene related to protein synthesis,secretion and regulation was down-regulated when expressing eGFP,whereas the Sec pathway and some genes regulating glyco-metabolism was up-regulated.The MVDA analysis was carried out on metabolism and transcriptome data.The RPKM data of C.glutamicum eGFP was firstly analysis by MVDA.Fifty nine genes related to heterologous protein expression was determined by OPLS-DA.In comparison with DEGs,3 critical genes were obtained,which,provide new targets for improving heterologous protein expression.Meanwhile,the amount of intracellular and extracellular metabolites of different timing were analized using MVDA method and we found that ATP,Glu,acetic acid,Glu-in,Met,lactic acid was the critical metabolites during the process of heterologous protein expression.4.The functional identification of these three critical genes related to heterologous protein expression and energy regulation.Three important genes(NCgl0909,NCgl2632 and NCgl1522)were obtained by RNA-seq and MVDA data analysis.The knockout and overexpressing strains of these three genes were constructed respectively to identify their functions on heterologous protein expression and regulation mechanism.The result showed that overexpressing NCgl0909 and NCgl1522 could increase the expression of eGFP,as well as NCgl2632 knockout.The ?-amylase and ScFv were also expressed in the above overexpressiong or knockout strains separately to verified the role of these three genes on heterologous protein expression.