Metabolism Related To Ros Changes Of SigE In Corynebacterium Crenatum In Response To High Dissolved Oxygen Fermentation

In industrial fermentation,microbial strains often face environmental pressures such as oxidative stress,temperature,pH,osmotic pressure,and nutrient starvation.Especially in the middle and late stages of fermentation,reactive oxygen species are very unfavorable for strains to maintain good physiological traits and industrial production performance.The production of L-arginine by Corynebacterium crenatum SYPA5-5 under high dissolved oxygen fermentation conditions produces a large amount of ROS,which significantly affects its cell growth and the metabolic synthesis efficiency of target products.In this study,SigE（RNA polymerase sigma factor,SigE）of Corynebacterium crenatum was used as the research object to explore the role of SigE in resisting intracellular oxidative stress,explore the target genes regulated by SigE,analyze the mutual regulation mechanism of SigE and CseE（Anti-sigma factor,CseE）.Finally,the key binding sites of interacting proteins were analyzed by structural simulations to influence their mechanism of binding SigE.The main research contents are as follows:（1）Comparative transcriptomic analysis found that changes in dissolved oxygen levels led to increased transcription levels of genes involved in multiple metabolic pathways in Corynebacterium glutamicum and Corynebacterium crenatum.The transcriptional levels of oxidative stress,such as DNA protective protein Dps,chaperone protein ClpB,thioredoxin disulfide reductase TrxB,catalase CAT,superoxide dismutase SOD and stress H₂O₂ transcriptional regulator OxyR,were significantly increased.The sig E encoded by C629＿06440 was up-regulated 18.37-fold at the transcriptional level in Corynebacterium crenatum.（2）First,the overexpression strain Cc-SigE,the knockout strain Cc-ΔSigE and the CseE overexpression strain Cc-CseE were constructed.The change trend of intracellular ROS content,SOD and CAT enzyme activity in recombinant strains showed that the increase of dissolved oxygen level would lead to the increase of intracellular oxidation level.Secondly,when the high dissolved oxygen fermentation was carried out for 96 h,the intracellular ROS content,SOD enzyme and CAT enzyme activities of Cc-ΔSigE were increased by 35.32%,7.79%and 13.61%,respectively,compared with the original strain.When the high dissolved oxygen fermentation reached 96 h,the intracellular NAD⁺/NADH ratio of Cc-ΔSigE was 2.26,which was 7.61%higher than that of the original strain.The intracellular NADP⁺/NADPH ratio of the original strain was 0.75,which was 18.38%lower than that of Cc-ΔSigE.The results confirmed that SigE plays an important role in maintaining cellular redox homeostasis and enhancing the ability of cells to respond to oxidative stress.（3）Transcriptomics combined with Chip-Seq analysis found that SigE was involved in many important metabolic pathways such as cell growth,aerobic respiration,cell wall formation,tricarboxylic acid cycle,electron transport chain and environmental stress in Corynebacterium crenatum.SigE may regulate transcription levels such as thiol peroxidase Tpx,molecular chaperone DnaJ,RNA polymerase sigma factor SigE,whiB family transcription regulatory factors WhcB,RNA polymerase sigma factor SigB,and NAD（P）/FAD-dependent oxidoreductase NoxA,directly or indirectly participate in ROS stress process.（4）Through RT-qPCR and His Pull down experiments,it was found that SigE in Corynebacterium sp.can regulate the expression of sigE and cseE at the transcriptional level and its activity is controlled by CseE protein.ITC experiments found that the enthalpy of CseE protein binding to Zn²⁺becomesΔH=-21.9±0.6 kcal·mol^-1,the entropy becomes TΔS=-14.3 kcal·mol^-1,and the separation constant Kd=2.7±0.5μmol·L^-1.The results of site-directed mutagenesis showed that the binding ability of CseE_C87A-C90A and CseE_{His83-C87A-C90A} mutants to SigE was slightly decreased.（5）Molecular dynamics simulation analysis showed that the binding energy between SigE and CseE protein was-23.17 kcal·mol^-1,while the binding energy between mutant SigE-CseE_C87A-C90Aand SigE-CseE_{His83-C87A-C90A} was-17.23 kcal·mol^-1 and-14.06 kcal·mol^-1,which were 22.8%and36.9%lower than that of the unmutated system,respectively.Binding indicated that His83,Cys87and Cys90 in the ZAS domain were the key amino acid sites affecting the binding of CseE to SigE.