Combinatorial Biosynthesis Of Complex Lipopeptides And Polyketides Natural Products Of Burkholderia

Polyketides and nonribosomal peptides are critical library of substitute drugs.Nowadays,manipulating the biosynthetic pathways of natural products through combinatorial biosynthetic methods,such as insertion,deletion,and substitution of genes or domains,to rationally modify natural products,has become an important means of drug development.Combinatorial biosynthesis can not only greatly expand the diversity of related natural products(NPs),but also give a rational way to directed modify its active functional groups,and thus obtaining high-quality alternative drugs.It breaks through the limitation of functional groups in chemical synthesis or modification,and is a reasonable means for comprehensive reconstruction of NPs.At present,people have made good progress in the combinatorial biosynthesis of nonribosomal peptides,but there are few studies on combinatorial biosynthesis of nonribosomal lipopeptides.The reason is that control mechanism and initiation process have not yet been elucidated.Therefore,it is a reasonable strategy to combinatorial biosynthesis of lipopeptide on the basis of initiation process elucidation.For polyketides,current research of combinatorial biosynthesis mainly focuses on the cis-AT PKS,and there are few studies on the trans-AT PKS.Therefore,The systematic modification of trans-AT PKS may promote the development of combinatorial biosynthesis in this catergory PKS.Provide new ideas for the study of combinatorial biosynthesis rules,and improve the efficiency of drug dicovery.1.Modification the fatty acyl groups of nonribosomal lipopeptides in BurkholderiaRhizomides and holrhizins are lipopeptides in NRPS categories.This kind of products contain a fatty acyl group(also named as fatty acid chain),in which a condensation starter domain(Cs domain)is responsible for integrating fatty acyl groups into the downstream amino acid backbones.Studies have shown that the length or structure of the lipo chain of lipopeptides is important for their functions,so it has great significance for combinatorial biosynthesis of the Cs domain which is responsible for the specificity of their lipo chains.So far,people have limited knowledge of the Cs domain which involving initial condensation of fatty acyl and amino acid substrates,and thus it is difficult to effectively perform combinatorial biosynthesis research on related products.Therefore,if the Cs domain’mechanism of substrate specificity can be elucidated,it will provide the effective guidance for the modification of lipopeptide products,and based on this,rational combinatorial biosynthesis of lipopeptides can be conducetd to obtain high-efficiency and active derivative products,and improve the properties or yield of drugs.In this study,the Red/ET homologous recombination technology was used to directly clone the biosynthetic gene clusters of rhizomide(rzmA)and holrhizin(holA)by one step.After inserting transposable elements and replacing them with constitutive promoters,they were transferred into Burkholderia strain DSM7029 and heterologous expressed successfully.Combinatorial biosynthetic methods were used to modify the rzmA and holA gene clusters,including point mutation,module exchange,promoter replacement,heterologous expression and homologous modification etc.The modified product with improved activity was successfully obtained.i.e.C8-RzmA,C10-RzmA,C12-RzmA,C14-RzmA and C16-RzmA etc.We have used the reported crystallization to perform homology modeling analysis on the Cs domain of rhizomides and holrhizins,and preliminary screened the key amino acid in Cs that determine the specificity of their lipo chains(for the RzmA-Cs domain is:R148),which is speculated to be located at the entrance of the fatty acyl substrate binding pocket,and the key role is further confirmed by point mutations in the full-length gene cluster in vivo and in vitro enzymatic experiments.Furthermore,we conducted crystallographic analysis on the Cs domain(RzmA-Cs)of rhizomide,including the unbound form of RzmA-Cs,the bound form of fatty acyl substrate,and the ternary complex of fatty acyl and aminoacyl substrates.By analyzing the co-crystal structure of RzmA-Cs with its fatty acyl substrate,the specificity of its fatty acyl substrate was clarified.The first key amino acid R148 is located at the entrance of the binding pocket,Q36,Q136,Y138 and M143 are located inside the pocket,of which Q36 and Y138 play a decisive role,and Q136 and M143 play an auxiliary role.Through point mutations of full-length gene cluster in vivo and in vitro enzymatic studies,the acyl of rhizomide A has finally been modified to C16 from C2.Combined with the analysis of multiple crystal structures of RzmA-Cs,we further explained the initiation process of lipo chain,and constructed reaction cycle i.e.the "Unbound state" to "Bound state of with aminoacyl and fatty acyl substrates",and finally to "Release state".At the same time,we have also explored the use of Cs domain exchange methods to transform fatty acyl chains.For example,swapping the full length of Cs domains of rhizomide and holrhizin,and the exchanged products were successfully obtained.Combined with enzymatic experiments in vitro and the crystal structure,the versatility potential of this method were confirmed,and it has also been applied in other works successfully.In summary,we have established two methods for directed modification of the lipopeptides’lipo chain.1.Combined with Cs domains’ sequence data of known lipopeptides to point mutation its key amino acids which determine the specificity of fatty acyl substrates.2.Perform the full-length exchange of Cs domain to modify the length of lipo chain.In the above exploration,we obtained the co-crystal structure of the Cs domain with its fatty acyl substrate for the first time,and further identified the critical sites in the Cs domain that could determine the specificity of acyl substrates.Using these key amino acid data,we have established a strategy for targeted modification of the lipo chain length by the way of point mutation.At the same time,we have also established a full-length exchange method of the Cs domain,which complements the point mutation method.These two new methods described above will promote development of targeted modification of lipopeptides and exploration of drugs.Compared with mehod of full-length domain exchange,the point mutation method had less structural disturbance of Cs domain,and thus did not restricted by species.The full-length Cs domain exchange was dominant in yield,which is more suitable for close species.The elucidation of the above crystal structure and the establishment of corresponding methods may provide great reference for the modification of potential lipopeptides or drugs.2.Heterologous expression and combinatorial biosynthesis of rhizoxinPolyketides are one of the important sources of drugs.They are catalyzed by polyketide synthase(PKS).And can be mainly divided into three types.Among them,type I PKS is composed of non-reusable modules,and each module can be divided into three basic domains.The ketone synthase domain(KS),acyltransferase domain(AT),and acyl carrier protein domain(ACP).It can also contains one or more modification domains,such as the ketoreductase domain(KR),dehydratase domain(DH),and carbon methyl transferase domain(cMT).The linearized domain catalyzes the gradual extension of carbon units,and the final product is released by the thioesterase domain(TE).trans-AT PKS is an important class of type I modular polyketide synthase.The acyl groups of the entire product are transferred by an independent acyltransferase domain.Compared with cis-AT PKS,the evolutionary relationship of trans-AT PKS between modules is relatively independent.Unlike cis-AT PKS,there are currently few reported in the area of combinatorial biosynthesis.Rhizoxin is synthesized by hybrid NRPS/trans-AT PKS in Burkholderia DSM19002(Paraburkholderia rhizoxinica HKI 454),which can inhibit the polymerization of microtubules and therefore has antitumor potential.Because of instability of its structure and large cytotoxicity to patients,it was suspended in the second-stage clinical practice.Combinatorial biosynthetic practice may effectively reduce its biological toxicity and improve its stability.On the other hand,it can also provide reference for the subsequent modification of trans-AT PKS.In this study,the Red/ET homologous recombination technology was used to directly clone the biosynthetic gene cluster of rhizoxin(rhi).After inserting the transposable element,it was transferred into Burkholderia DSM 7029 and heterologous expressed successfully,and then method of point mutation and module replacement,are used to combinatorial biosynthesis of rhi gene cluster.1.Point mutation of active sites to inactivation of modification domains,including 11 KR domains,8 DH domains and 5 MT domains.Inactivation of key amino acids of the third and fifth MT domains successfully obtained corresponding derivates,that is,product without methyl group at the corresponding position on the backbone.MT is a suitable object for modification in trans-AT PKS.The remaining MT,all DH and KR mutations did not obtain derivative products.On the one hand,the specificity of upstream and downstream domains in the stage of intermediate processing,such as keto synthase domains(KS domain),KR or DH domains,might be also a critical factor to success.From results,the trans-AT PKS may have strong specificity of KS,it may not be recognized by downstream’s domains once modified DH and KR domain from upstream to changed structure of intermediates On the other hand,it also shows that there are differences between domains with the same function,and the importance of its active center may need to be further refined.In method 2,the full-length NRPS module from rhizoxin(trans-AT PKS)was swapped by the NRPS module of epothilone which was belong to cis-AT PKS category,and derivative with thiazoline ring was successfully obtained.In the process of exploring the fusion site,no exchange products were obtained without retaining the original acyl carrier protein(ACP).The swapping yield that retains most of the adjacent KS upstream connection area(linker)was about six times higher than the exchange that retains only a small part,and about ten times higher than the exchange that retained both ACP/PCPs from donor and acceptor.This is the first time as we konw that NRPS module in a cis-AT PKS has been used in a trans-AT PKS.We have proved through practice that the NRPS of trans-AT PKS can be functionally replaced with the NRPS of cis-AT PKS,and the appropriate replacement position has been determined as,retaining its original ACP/PCP domain as an integrated unit(functional unit)and most of the connection area adjacent to the KS domain in acceptor PKS should be retained.The above point mutation and module exchange exploration may provide reference for the modification of similar clusters.In summary,we explored the combinatorial biosynthesis strategy of rhizoxin,a natural product produced by trans-AT PKS,mainly including 1.Systematic point mutations of their modification domains,including KR,DH and MT,and finally confirmed that MT is suitable objects to modification which has relative higher success rate in practice.2.NRPS module exchange,using NRPS module of epothilone gene clusters which was belonging to cis-AT PKS to replace the NRPS module of rhizoxin,and successfully transforming the oxazole ring of rhizoxin into a thiazole ring.This is the first time as we know that the NRPS module of trans-AT PKS was successfully swapped by NRPS module of cis-AT PKS.