The Relationship Between The Expression Of IrrE From Deinococcus Radiodurans And The Organic Solvents Tolerance Of Arthrobacter Simplex

Arthrobacter simplex is a commonly used C1,2 dehydrogenation strain for the production of steroid industry.The bioavailability of these substrates in the bioconversion process is low because of the remarkably high hydrophobic nature of steroids.Therefore,the organic solvent has been widely applied to improve the solubility of steroids accessibility.However,the addition amounts of organic solvents are commonly restricted to low concentrations due to their toxicity towards whole-cell biocatalysts,which in turn cripples the solubility of hydrophobic substrates and consequently decreases the biotransformation productivity.Therefore,the development of microorganisms with higher organic solvent resistance by genetic engineering methods would be an effective way to solving the urgent solubility problem in steroid bioconversion.IrrE was a crucial transcriptional regulator from Deinococcus radiodurans R1.It has been shown that the irrE gene when transferred into non-native hosts could enhance certain stress tolerances of the recombinant hosts,such as,organic solvent tolerance,ionizing radiation,oxidative stress tolerance,salt tolerence and so.on.But the hosts manily focus on some model microorganisms and gram-negative bacteria.In this paper,IrrE-expressing A..simplex strains which has been constructed in our previous work was studied.The effects of IrrE on the cell growth,physiological metabolism and activity of catalytic enzyme of A.simplex were investigated.The results showed that IrrE had no significant effect on the cell growth,glucose metabolism and Cl,2 dehydrogenase activity under no pressure condition.The results also suggested that there was no obvious effect of IrrE on the efficiency of A.simplex Cl,2 dehydrogenation reaction under non stress condition as well as low stess condition with low concentration of ethanol.With the increasing ethanol and substrate concentration,the IrrE-expressing strain showed higher PA production capacity.In the transformation system containing with 10%(v/v)ethanol and 8 g/L CA,no obvious PA production was observed in the control strain whereas the maximum PA production of IrrE-expressing strain reached 6.95 g/L after transformation for 36 h.Further analysis found that IrrE-expressing strain exhibited higher Cl,2 dehydrogenase activity,cell vability and physiological metabolism ability than the control strain in the above transformation system.Enhancement of stress tolerance of IrrE-expressing strain would be the main reason for its better production capacity.The effects of IrrE on the stress tolerance of A.simplex were investigated.The results showed that IrrE expression could not only improve the cell growth character under appropriate methanol and ethanol pressure,but also enhance the cells survival after high concentration pressure shock(including 16%ethanol,20%methanol,2 mol/L NaCl,0.1%H2O2).A similar result was also observed when IrrE was heterogeneously expressed in A.globiformis.These results indicated that IrrE was able to horizontal transfer between different microorganisms and it showed good application prospect in improving microbial stress tolerance.The molecular mechanism of IrrE related to the organic solvent tolerance enhancement of A.simplex was studied by analyzing key genes expression levels,key enzyme activities and key metabolites concentration.The results showed that IrrE-expressing strain enhanced ROS elimination,accumulated higher intracellular compatibility solute,increased energy level of cells,enhance the ability of DNA damage repair,up-regulated the expression levels of heat shock protein and affects the expression levels ofother global transcription factors(such as the up-regulation of rpoD involved in stress tolerance).Finally,the maximal production capacity of IrrE-expressiong strain was evaluated by using the growing cells as biocatalyst in transformation experiments.The results showed that the conversion ratio and PA production of IrrE-expressing strain in the transformation system were higher than those of the control strains.In the system containing 10%ethanol and 35 g/L CA,the PA production of IrrE-expression strain reached 21.1 g/L,while no obvious PA production was observed in the control strain.The results of this study provide new strategies and methods for the construction of high efficient C1,2 dehydrogenation reaction strains with good industrial application properties.