Effects Of Transcription Factor Cody Of Bacillus Licheniformis On The Lichenysin Biosynthesis

Lichenysin is a lipopeptide biosurfactant and has broad application prospects in oil exploitation,washing products,environmental remediation,agriculture,and so on.However,the large-scale industrial production and commercialization of lichenysin are limited due to high cost and low yield.Bacillus subtilis CodY can inhibit the synthesis of branched chain amino acids that are the precursor amino acids of lichenysin biosynthesis under amino acid-rich growth conditions,and be likely to regulate the biosynthesis of lichenysin.This study examined effects of precursor amino acids amended on lichenysin fermentation in a starting strain,Bacillus licheniformis WX02-Psrflch.Also,an engineered strain(a knockout of codY gene)was constructed from the starting strain WX02-Psrflch for increasing lichenysin production,and then the effect of codY knockout on lichenysin biosynthesis was analyzed.The concentration changes of free precursor amino acids during lichenysin fermentation in the starting strain,B.licheniformis WX02-Psrflch were detected,and the maximum concentrations of lichenysin composition amino acids,aspartic acid,glutamine isoleucine,leucine and valine were only 0.4 mg/L,6.0 mg/L,0.3 mg/L,0.3 mg/L and 1.6 mg/L,respectively.However,the yield of lichenysin decreased to various degrees when the precursor amino acids were added to the culture medium of B.licheniformis WX02-Psrflch.It is expected that there was a close relationship between CodY protein content and concnetrations of precursor amino acids for lichenysin biosynthesis.In order to explore this relationship,we constructed a series of codY differential expression strains based on B.licheniformis WX02-Psrflch,including codY knockout strain(WX02-Psrflch?codY)and different levels of codY overexpression strains(WX02-Psrflch/pHY-P43codY?WX02-Psrflch/pHY-PsrfcodY).For the codY knockout strain,we optimized the strategy for precursor amino acid amendment,and our results showed that an addition of 0.5 g/L equally mixed precursor amino acids achieved the best effect,resulting in an increase by 25.7%compared to the starting strain,and a significant decrease in codY overexpression strains.Batch fermentation of lichenysin was studied with both starting and codY knockout strains.Compared with the starting strain,the lichenysin production of codY knockout strain increased by 31%,and the fermentation period was shortened from 36 h to 24 h.Also,the concentrations of main overflow metabolites:acetate,acetoin and 2,3-butanediol were decreased by 50.9%,41.4%and 25.9%,respectively in codY knockout strain.In addition,the concentrations of intracellular free precursor amino acids were tested.During the rapid biosynthesis period(6-12 h)of lichenysin,the intracellular concentrations of precursor amino acids(including Val,Leu,Ile and Gln)were higher in the codY knockout than in the starting strain,except that both strains had almost the same concentration of Asp.The results showed that the codY knockout decreased synthesis of overflow metabolites and enhanced concentrations of intracellular Gln,Ile,Leu and Val.We further analyzed the transcriptional levels of key genes in lichenysin biosynthesis pathway and its associated regulatory network by qRT-PCR.The results showed that the transcription of branched chain amino acids biosynthesis genes:ilvA,ilvB,leuA,bcd and alsS were up-regulated in the codY knockout;the expression of Gln and Glu biosynthesis genes glnA and rocG was obviously enhanced;the transcription of lichenysin biosynthesis gene IchAA,as well as its closely related positive regulation factors comA and degU was also increased;the transcription of Asp biosynthesis gene aspB remained unchanged;the transcription of byproducts biosynthesis gene ackA and alsD was down-regulated.In conclusion,a knockout of codY gene in the starting strain B.licheniformis WX02-Psrflch weakened overflow metabolites pathway during lichenysin fermentation,enhanced the biosynthesis of glutamine and branched chain amino acid,up-regulated the key genes involved in their biosynthesis,and improved the expression of regulators ComQXPA and DegS-DegU,thus further increased lichenysin production.This study firstly investigated the effect of codY knockout on lichenysin biosynthesis,and provides scientific insights and technological applications for lichenysin production.