Effects Of Cofactor And Global Transcription Factor Metabolic Engineering Modified Corynebacterium Crenatum On L-arginine Production

L-arginine and its metabolites,NO,polyamine,ornithine and proline,play an important role in regulating body metabolism,promoting hormone secretion,improving immune function,preventing cardiovascular disease and endothelial cell dysfunction,maintaining skeletal muscle and brain function,repairing damaged tissues and other aspects.Because of its great value in medicine and food,how to produce L-arginine on a large scale has attracted extensive attention of researchers at home and abroad.At present,the main production mode of L-arginine is microbial fermentation.The key of this method is to breed an engineering strain with high yield and stable heredity.Corynebacterium crenatum was isolated from soil by Chinese scientific researchers.Because of its characteristics of no endotoxin,clear genetic background and low culture cost,it is often used as chassis bacterium to construct high-yielding arginine strain.Therefore,in this study,C.crenatum was used as host strain for genetic modification to enhance the supply of cofactor NADPH by optimizing the expression of pentose phosphate pathway,TCA cycling pathway and NAD kinase gene.Remove the restriction of global transcription factors on the absorption pathway of carbon and nitrogen sources;An engineering strain of C.crenatum with high yield of L-arginine was constructed by increasing the supply of precursor glutamic acid.（1）According to the principle of gene homologous recombination,the directed metabolic modification of C.crenatum was carried out.Firstly,degradation tag was added to the end of the gene encoding 6-glucophosphate isomerase pgi,the RBS binding site of the first gene zwf in the pentose phosphate pathway was replaced,strong promoter P_tac was inserted into the tkt operon,and tandem combination modification was performed.It regulates the carbon flux of pentose phosphate pathway,promotes the regeneration of cofactor NADPH,and discusses the effect on arginine synthesis.Compared with the original strain,the tkt operon optimized strain CCM04 had the best fermentation effect,and the arginine yield reached 17.00 g/L after 120 h shaking flask fermentation,which increased by 10.9%.（2）In order to further enhance the supply of cofactor NADPH required for L-arginine synthesis,P_eftu and NAD kinase ppnk genes were inserted into P_sod upstream of icd gene encoding isocitrate dehydrogenase respectively by promoter engineering to improve their expression levels.The engineered bacteria CCM07,which overexpressed ppnk,was fermentated for 120 h.The accumulated arginine reached18.38 g/L,which was 11.6%higher than the control strain.（3）In order to effectively improve the absorption and conversion efficiency of carbon and nitrogen sources in fermentation medium and enhance the metabolic flux of arginine synthesis,a series of global transcriptional regulatory factors such as ramA,ramB and amtR were knocked out.The arginine yield of ramB-knockout strain CCM09 and amtR knockout strain CCM10 reached 17.66 g/L and 17.98 g/L,respectively.This indicates that by removing the inhibition effect of global regulatory factors,it can help to reduce the unnecessary formation of precursor substances,accumulate common intermediates,and promote the further flow of carbon and nitrogen sources to the synthesis of L-arginine.（4）In order to improve the supply of precursor glutamic acid,the genes encoding phosphorylase pknG and pknB were knocked out to deactivate ODHC by OdhI,and the metabolic flux ofα-ketoglutaric acid was transferred to the L-arginine synthesis pathway.The results showed that pknG knockout strain CCM11 yielded 20.25 g/L by shaking flask fermentation,which effectively optimized the intracellular glutamic acid supply and enhanced L-arginine synthesis.Finally,the recombinant strain CCM13 was subjected to shaking fermentation,and its arginine yield was 50.35%higher than that of the original strain CCM01,which effectively optimized the fermentation performance of the strain and provided a reference for industrial arginine production.