Function Of The Regulatory Genes In Xantholipin Biosynthesis

Xantholipin was isolated from the fermentation of Streptomyces flavogriesus during the screening process for inhibitors of heat shock proteins HSP47.Structurally,xantholipin possesses the characteristic xanthone ring nucleus and the hexacyclic molecular skeleton,belonging to the polycyclic xanthone antibiotics.Xantholipin possesses strong antibacterial activity,and shows effective inhibitory effect on human fibroblasts collagen biosynthesis.Meanwhile,xantholipin exhibits cytotoxicity against carcinoma cell line KB and leukemia cell line HL60.In our previous work,the biosynthetic gene cluster of xantholipin(xan)has been cloned,the enzymatic construction of xanthone nucleus has been characterized and the biosynthetic pathway of xantholipin has been schemed.However,the regulatory mechanism of the biosynthesis remains unclear.Bioinformatics analysis of the xan cluster suggested three possible regulatory genes,named xanR1,xanR2 and xanR3.In this work,we determine to specify the function of these three genes,hoping to uncover the regulatory mechanism of xantholipin biosynthesis,setting stage for engineering construction of high yield strains.Protein sequence analysis showed that all the three genes encoded transcriptional regulatory proteins while each belonged to different family.XanR1 belonged to TenA family,XanR2 belonged to MarR family and XanR3 belonged to ArsR family.In order to specify the functions of these genes,each of them was disrupted individually in vivo,through genetic conjugation between S.flavogriseus SIIA-A02191 and Escherichia coli ET12567/pUZ8002.The yield of xantholipin in the resultant mutants was compared with that of the wild type strain.It turned out that the yield of xantholipin in all the three mutants declined.Moreover,the complementary of xanR3 in ?xanR3 mutant contributed to the productivity increasment.All these results showed that XanR1,XanR2 and XanR3 may act as positive regulators.Subsequently,the regulatory function of xanR2 and xanR3 were studied on transcriptional level.Firstly,RT-PCR was carried out to verify the number of operons in the xan cluster,and the analysis unveiled 18 cotranscription units.To define the regulated genes,all of the eighteen operons were then analyzed quantitively in ?xanR2 and ?xanR3 mutants.In ?xanR2 mutant,the transcription of xanW,xanO6-xanO8 and xanR3 significantly declined.Similarly,in ?xanR3 mutant the transcriptional levels of the four transcripts xanO1-xanS1,xanB1-xanB3,xanT-xanO9 and xanO10-xanC3 significantly decreased.The down-regulated genes abovementioned play important role in biosynthesis of xantholipin,either supporting blocking units or tailoring molecular skeleton.These findings were consistent with the decreased xantholipin yield in the two mutants,further confirming the positive regulatory functions of xanR2 and xanR3.In conclusion,following the bioinformatics hints,the three possible regulatory genes were firstly inactivated genetically,and the negative effect on xantholipin biosynthesis in the resultant mutants was analyzed.The transcriptional level of regulated genes of xanR2 and xanR3 were then analyzed,providing further support for the positive regulatory functions.This research will promote further detailed elucidation of xantholipin biosynthesis and contribute to the metabolic engineering of high yield strain.