Combinatorial Synthesis Of Plipastatin Non-ribosomal Peptide Synthetases And Study Of Novel Lipopeptides Synthesis

The lipopeptides,produced by Bacillus have a variety of biologic activity,such as anti-bacterial,anti-fungal,anti-virus and anti-tumor,even have a good inhibition on some muti-resistant pathogens.Lipopeptide Plipastatin,named Fengycin has strong antifungal activity,especially against on filamentous fungi,which was applied into the bio-control of plantal pathogens.Plipastatin also could inhibit Rhizopus stolonifer and Botrytis cinerea,as well as reduce the risk caused by accumulation of my cotoxins,which have a great potential for application on fruits preservation and feed storage.Thus it is of great significance to exploit the lipopeptide synththesis metabolism and to screen novel lipopeptides for inhibiting foodborne pathogens.Compared with the traditional methods by microbial fermentation,purification,physical or chemical mutagenesis,modificating the lipopeptide synthetase complexes through strategies of combinatorial biosynthesis could induce to produce novel lipopeptides,and expand the types of lipopeptides for screening natural lipopptides with impoved biological activity,which broaden the source of natural green food preservatives.In this work,genetic engineering was used to convert a non-production model strain B.subtilis pB2 into an engineered strain coproduced the lipopeptides Surfactin and Plipastatin.Effects of Surfactin and Plipastatin on microorganisms were described by microscopic,cytoskeleton analysis and flow-cytometry,respectively.Three strategies of biocombinatorial synthesis,composed of translocation of thioesterase TE and subunit PPSE,as well as deletions of module or individual domain were utilized to the modified the Plipastatin synthetases,thus generating and identified a series of novel lipopetide derivatives.Additionally,the catalytic activity and selectivity of the TE domain in vivo,and effects of selective interaction of COM domain and deletions of module or single domain on Plipastatin assembly line all were investigated and discussed.The main results are as follows:1.Plipastatin and Surfactin coproduction by Bacillus subtilis pB2-L and their effects on microorganisms.A gene expression cassette composed of a constitutive promoter(P43),functional gene sfp,and pleiotropic regulatory gene degQ was integrated into the chromosomal amyE locus of strain B.subtilis pB2 by homologous recombination,which generated a Plipastatin and Surfactin co-producer.Plipastatin homologues with C14-21 saturated and C14-18 unsaturated β-OH fatty acid chain,as well as C9-18 Surfactin homologues and linear C12-16 Surfactin homologues were identified in lipopeptide extracts using analytical techniques.The effects of lipopeptide Plipastatin and Surfatin on microorganisms were study using SEM,TEM,cytoskeleton analysis and flow-cytometry,respectively.Plipastatins isolated from the engineered strain pB2-L exhibited strong antifungal activity(MIC 16 μg·mL-1)by cell abnormality,vacuolization and disrupting the cytoskeleton of Fusarium oxysporum f.sp.cucumerinum hyphae.Surfactins affected the cell membrane of Staphylococcus aureus(MIC 20 μg·mL-1),resulting in nucleic acid leakage and ultimately,cell death.Based on the convenience of genetic manipulation in the engineering strain,this work lay the foundation for further study of the combinatorial biosynthesis of Plipastatin synthetases using molecular engineering approaches and generating novel lipopeptide derivatives.2.Translocation of the thioesterase domain in NRPS to synthesize novel lipopetides.The intrinsic thioesterase TE region was respectively moved forward to the end of internal Module 7,8 and 9 of the thiolation(T)domains in the Plipastatin synthetase.7ProT-C linker,8GlnT-C linker and 9TyrT-TE linker were respectively used to connect the TE domains in the different constructs.The three recombinants(LP1,LP2,LP3)were genereated and were unable to produce any lipopeptide related to Plipastatin.Additionally,another three mutants(LP4,LP5 and LP6)were generated by using 10IleT-TE linker linked the T domains from Module 7,8,9 with TE domain.The results of high resolution LC-ESI-MS/MS showed that mutant strain LP4 could produce novel linear and cyclic heptapeptides:(C16-19)β-OHFA-E-O-Y-T-E-V-P and(C16-19)β-OHFA-E-O-cyclo(Y-T-E-V-P).The mutant strain LP5 could produce novel linear and cyclic octapeptides:(C16-17)β-OHFA-E-O-Y-T-E-V-P-Q and(C17-19)β-OHFA-E-O-cyclo(Y-T-E-V-P-Q).The mutant strain LP6 could produce novel linear nonapeptides(C15-17)P-OHFA-E-O-Y-T-E-V-P-Q-Y,and linear and cyclic octapeptides.This study revealed that thioesterase TE domain has relaxed substrate selectivity,and the native 10IleT-TE linker is necessary and sufficient for hybrid enzymes to synthesize the lipopeptides.3.Translocation of subunit PPSE and Swapping COM in NRPS to synthesize novel lipopetides.A mutant strain LP7,generated by moving the subuntit PPSE forward to the end of subunit PPSB in the Plipastatin synthetase,could produce a novel pentapeptides followed the designed assembly line(PPSA-B-E).This finding demonstrated that acceptor COMdppsB could communicate and interact with non-cognate donor COMappsE.Mutant strain LP8,generated by moving the PPSE forward to the end of subunit PPSC in the Plipastatin synthetase,has poor production on Plipastatin derivative.This result indicated that acceptor COMdppsc and non-cognate donor COMappsE were incompatible,and unable to communicate.The incompatible COMdppsC/COMappsE was replaced with native matched COMdppsC/COMappsD,generating a mutant strain LP9,which could produce truncated hexapeptide(C16-17)β-OHFA-Glu-Orn-Tyr-Thr-Glu-Ala/Val.While,substituted with native matched COMdppsD/COMappsE,the mutant strain LP10 could produce novel linear heptapeptide(C17-18)β-OHFA-Glu-Orn-Tyr-Thr-Glu-Ala-Ile.4.Module and individual domain deletions in NRPS to synthesize novel lipopetides.Module 6 and 7 were deleted in Plipastatin synthetases respectively,resulting in that the corresponding mutant strain BM6 and BM7 both were unable to producing any Plipastatin derivative.Whereas,individual A-domain or T-domain deletion within Module 7 generated mutant BA7 and BT7,resulting in truncated linear hexapeptides(C16-17β-OHFA-Glu-Orn-Tyr-Thr-Glu-Ala/Val).On the contrary,individual T-domain deletion with Module 6 resulted in a nonproductive strain BT6.Interestingly,deletion of the A-domain within Module 6 resulted in multiple assembly lines via module-skipping mechanism,generating three novel types of Plipastatin derivatives,pentapeptides(C16-17β-OHFA-Glu-Orn-Tyr-Thr-Glu),hexapeptides(C16-17β-OHFA-Glu-Orn-Tyr-Thr-Glu-Ile),and octapeptides(C16-17β-OHFA-Glu-Orn-Tyr-Thr-Glu-Gln-Tyr-Ile).This study revealed a unique module-skipping process in engineering NRPS biosynthetic pathways,which provides a new insight into the lipopeptides engineering for generating novel lipopeptides.