Physiological Mechanism Of Cofactor NADPH Regulating Intracellular Microenvironment Of Corynebacterium Glutamicum When Synthesizing L-lysine

Nicotinamide adenine dinucleotide phosphate?NADPH?plays an important role in the regulation of cell growth and products biosynthesis in Corynebacterium glutamicum.In this paper,a recombinant strain with blocking the biosynthesis of NADPH was constructed,and the cell growth and products biosynthesis were investigated.To do this,the glucose-6-phosphate dehydrogenase-coding gene zwf and malic enzyme-coding gene malE were deleted,and the native NADP⁺-dependent isocitrate dehydrogenase gene icd_Cg was replaced by Streptococcus mutans NAD⁺-dependent isocitrate dehydrogenase gene icd_Sm that resulted in the final engineered strain C.glutamicum LYS?zwf?malE?icd_Cg::icd_Sm.The effects of blocking intracellular NADPH synthesis pathway on cell growth,glucose metabolism and L-lysine synthesis were analyzed.In addition,the optimal NADPH threshold range for maintaining the L-lysine synthesis ability of the cell was investigated by adding different concentrations of NADPH solution,and then analyzed the change rule of intracellular micro-environment,thus primarily expounding the physiological mechanism for regulating intra-cellular microenvironment by the intracellular cofactor NADPH level.The main results are as follows:?1?The recombinant strain showed the higher intracellular NADH(1.97?mol·?g DCW?^-1 vs.2.73?mol·?g DCW?^-1),but it showed the lower intracellular NADPH(1.43?mol·?g DCW?^-1 vs.0.15?mol·?g DCW?^-1).In addition,ATP levels decreased by 13.10%,while ADP and AMP levels increased.The results of shake flask fermentation showed that the glucose metabolism of the recombinant strain was significantly weakened,the growth was relatively slow,and the biomass of the cells decreased by 4.36%.At the same time,L-lysine production decreased by 87.69%,and a large amount of by-products such as pyruvic acid and lactic acid accumulated in the cells.?2?Different final concentrations of NADPH solution were added to the medium to investigate the effect of NADPH on the L-lysine production.Compared with the non-added NADPH,When the final concentration of NADPH was 0.2,0.5,1,2,4 mmol·L^-1,the bacterial biomass was increased by 7.98%,11.77%,22.28%,15.86%,6.52%,and the L-lysine production was increased by 1.16,4.87,9.59,11.54,7.61 times,while the L-lysine production intensity increased by 0.08,3.36,6.84,8.96,and 6.14 times,respectively,compared with that without adding NADPH.The experimental results showed that the growth rate,L-lysine and bacterial biomass of recombinant strain were significantly improved during adding a certain amount of cofactor NADPH in the culture medium.However,addition of the excess NADPH is bad for cell growth and L-lysine synthesis.More significant,with the increase of NADPH content,the L-lysine production intensity also showed a tendency to increase and then gradually weaken.?3?Intracellular energy cofactors?ATP,ADP,AMP?,redox cofactors?NAD⁺,NADH,NADP⁺,NADPH?,acetyl-CoA,and ROS were quantitatively analyzed and compared at different NADPH concentrations of 0,0.5,2,and 4 mmol·L^-1.When the NADPH con-centration was 0,0.5,2,4 mmol·L^-1,the intracellular NADPH levels were 0.15,0.24,1.83,2.65 mol?g DCW?^-1,the ATP levels were 4.21,4.32,4.48,4.67 mol?g DCW?^-1,and the intracellular ROS levels were increased by 1.35%,1.79%and 15.21%,respectively.The experimental results showed that the intracellular NADPH,ATP level and L-lysine production were effectively increased during growed on media with?2 mmol·L^-1,the supply of acetyl-CoA were enhanced and the central carbon metabolism pathway were strengthen.However,when the concentration of NADPH exceeds 2 mmol·L^-1,the increase of con-centration gradually reduces the production intensity of l-lysine.Meanwhile,the intracellular NADH level increases with the decrease of acetyl-CoA and the central carbon metabolism weakens.In addition,reactive oxygen species?ROS?gradually increased at high NADPH concentrations,while higher ROS levels caused cellular oxidative stress,which ultimately led to apoptosis and decreased bacterial biomass.