Characterization Of Glycosyltransferase In Biosynthesis Of Candicidin

Candicidin is a heptaene macrolide produced by Streptomyces sp.FR-008. This kind of compounds mainly comes from the secondarymetabolites of bacterials and exhibits antifungal activity by formingtransmembrane pores in the fungus membrane. Polyenes have been usedin human therapy for treatment of severe fungal infections for over50years due to their broad spectrum of activity and the low frequency ofappearance of resistrant fungal pathogens. Mycosamine exists in mostpolyenes, it contributes a lot to the antifungal activity and solubility ofpolyenes. Condersiding of this, scientists exhibit great research interestsin glycosyltransferases (GTs), however, the substrate specificity andactivity affecting amino acid residues have not been elucidated due to thedifficulty of expression polyene GTs. In this study, we made use of therapid growth, simple genetic manipulation and high production of S. spFR-008and generated an in vivo biochemical detection method tocharacterize polyene GTs, illustrated that polyene GTs have loosesubstrate specificity toward aglycones and found the activity affectingamino acid residues of polyene GTs, thus providing an opportunity to generate libraries of polyene derivatives with improved pharmaceuticalactivity.Disruption of GT gene fscMI in the biosynthesis of candicidin led tothe accumulation of deglycosylated candicidin aglycone andcomplementation experiment confirmed FscMI is a polyene GT. Besides,expression of homologous polyene GTs (NysDI、AmphDI、PimK) infscMI mutant could all restore the production of candicidin. These resultsillustrated that polyene GTs have some tolerance toward their substrates.In order to identify which moiety of polyene structures is essentialfor GTs recognition, we constructed a DH11point mutation mutant LX8.DH11is located in FscD and is accredited for the formation of the doublebond between C22and C23, which lies in the conserved region ofpolyenes and adjacent to mycosamine. The fermentation culture extractsof LX8were analyzed by LC-MS、Q-TOF and characteritic UV detection,two new hexaene derivatives with the same mass of1,127.3([M+H]+)and formular appeared. The [M+H]+of1,127.3suggests that the sugarmoiety is still present in the compounds and the double bond betweenC22and C23has changed to a hydroxyl group at C23, thus the hydroxylgroups at C21and C23may provide two possible attachment site formycosamine, leading to the two new peaks in the fermentation cultureextracts of LX8. These results further confirm that polyene GTs havesome tolerance toward their substrates. To gain insight into the amino acid residues that contribute to theglycosylation process of FscMI, we used homology modeling and finallygot a set of site-directed mutated FscMI. Quantitative HPLC analysis ofthe site-directed mutants illustrated that Ser346, Ser361, His362andCys387contribute a lot to the catalytic activity of FscMI. This is the firsttime to report the critical amino acid residues in polyene GTs. In thefuture, we could expect to change the critical amino acid residues toappropriate ones and make use of the loose substrate specificity featuresof polyene GTs, generating novel polyene derivatives with improvedbioactivities by manipulating with an ample pool of sugar donors andacceptors with different structural originality.Also, we analyzed the ABC transporter genes fscTI and fscTII thatexist in the biosynthetic gene cluster of candicidin. pJTU4137wasconstructed for disruption of fscTI and fscTII and it was transferred intoStreptomyces sp. FR-008derived strain ZYJ-6by conjugation. Theresulting mutant LX10was unable to produce polyenes. In addition, thetransporter genes were overexpressed in strain ZYJ-6and the productionof candicidin in the resultant engineered strain LX11increased to1.5-foldwith that of the control. We confirmed that FscTI and FscTII are putativeABC(ATP-binding cassette)transporters and overexpression of transportergenes increased candicidin production provides a positive example forimproving other polyene production.