High-throughput Screening For High-yielding Sophorolipids Strain And Preliminary Exploration Of The Mechanism

Sophorolipids(SLs)is a green biosurfactant with great market potential,which has been widely applied in cosmetics,medicine,detergents and other fields.In the actual SLs production process,the SLs titer is not very high.Excellent strains in industrial production can increase the fermentation yield and enhance enterprise competitiveness.In this paper,a rational high-throughput screening model was optimized for high-yielding SLs strains,and then a mutant with high yield of SLs was successfully screenedout.Finally,the high yield mechanism was preliminarily explored from the levels of key gene transcription,key enzyme activity and intracellular reducing power pool.Firstly,the combination of atmospheric room temperature plasma and sodium nitrite mutagenesis,the relaxation culture and the combination of rational screening pressures of iodoacetic acid,malonic acid and chlorpromazine were introduced into the high-throughput screening model.And the mutation rate and positive mutation rate were used as indicators to evaluate the effects of high-throughput screening before and after optimization.The results showed that combined mutagenesis,relaxation culture and combined screening pressures could significantly increase the mutation rate by more than 20%and the positive mutation rate by more than 55%.Secondly,by applying the optimized high-throughput screening model,a mutant library with 6240 mutants was constructed.After a primary screening with 24-well plates as well as the rescreening and secondary rescreening with shake flasks,a high-yielding mutant named Candimid bombicola H1.5 was successfully obtained.After 168 h fermentation in a 5 L fermenter,the H1.5 was able to produce 292.9 g/L of SLs,which was 26.9%higher than the parent strain.Moreover,the SLs productiviey and yield of H1.5 also were increased by 27.0%and 35.0%,respectively,compared with the parent strain.After 5 subcultures,the SLs production was fluctuated within 5%,and the verification of the fifth generation strain indicated that,and the fluctuation range of yield,yield and conversion rate were within 5%,indicating that H1.5 has a good genetic stability.Finally,through comparing the targeted sites of the screening pressures from the levels of key gene trascription,key enzyme activity and intracellular reducing power NADPH pool,it was found that the P450 monooxygenase enzyme activity and transcriptional level,which was acted by chlorpromazine,as well as intracellular NADPH pool in H1.5 were obviously higher than the parent strain.In constract,the glyceraldehyde phosphate dehydrogenase enzyme activity and transcriptional level,which was acted by iodoacetic acid,and cis aconitase enzyme activity and transcriptional level,which was acted by malonate presented lower trends in H1.5.Based on these results,it was preliminarily speculated that the increases of P450 monooxygenase activity and the reducing power of NADPH could contribute to the enhancement of SLs productivity in H1.5.The reduction of glyceraldehyde phosphate dehydrogenase activity intensified the pentose phosphate pathway,thereby promoting the cell growth and the supply of NADPH.On the other hand,the reduction of cis aconitase enzyme activity weakened the TCA cycle,and further increased the supply level of acetyl-CoA,which could reduce the inflow of rapeseed oil into the β-oxidation pathway,so as to improve the utilization of substrate and SLs yield.In summary,the mechanism for the high-yielding strain to efficiently synthesize SLs could be ascribed to:(1)the accelerated cell growth is beneficial for the cell to enter the SLs production period earlier,thereby prolonging the production time,(2)both the key enzyme acitivity of P450 and the reducing power pool of NADPH were increased to enhance the SLs productivity,(3)the improved utilization of oil promotes the SLs production efficiency.