Heterologous Expression Of Biosynthetic Gene Cluster Of Hybrid Antibiotic Polynik A

Nikkomycins and polyoxins are peptidyl nucleoside antibiotics. They have similar chemical structures consisting of peptidyl and nucleoside moieties which are synthesized separately and then linked together via peptide bonds. Both nikkomycins and polyoxins act as competitive inhibitors of fungal chitin synthasesand show high fungicidal activity, since their chemical structures are similar to UDP-N-acetylglucosamine, which is the natural substrate of chitin synthases.In our previous works, a hybrid antibiotic (polynik A) with novel chemical structure was generated via combinatorial biosynthetic approach by introducing 5 genes required for the biosynthesis of the dipeptidyl moiety of polyoxin from Streptomyces cacaoi into a Streptomyces ansochromogenes mutant that has ability to produce nucleoside moiety, while lacks the biosynthesis of dipeptidyl moiety of nikkomycin. This hybrid antibiotic exhibited merits from both parents with better bioactivity and stable structure. However, the low yield of the hybrid antibiotics is limited for application. Taking the advantages of the development of technology in combinatorial biosynthesis and synthetic biology, we aimed to improve the production of polynik A by heterologous expression and synthetic biology approach on the one hand, and on the other hand, we aimed to explore and solve the adaptation problem during the heterologous expression of biological parts or functional mudules in different chassis (Escherichia coli or Streptomyces) in synthetic biology study, by using polynik A as the research object.First of all, we optimized the codon usage of the biosysthetic genes based on the bias difference between Streptomyces and E. coli, and rearranged the relative genes involved in the gene cluster to form transcriptional units in silico; The ribosome binding site (RBS) of each gene was also optimized for the efficient coupling of transcription and translation. Thus, polynik A gene clusters for heterologous expression in Streptomyces and E. coli host strains were redesigned, respectively.To test the adaptation of whole polynik A biosynthetic gene cluster as the functional mudule in E. coli system is likely to be highly challenging. Here we selected a relatively simple strategy to assemble the gene cluster for the biosynthesis of nucleoside moiety of polynik A (nikkomycin Cx) for preliminary test. The work in this thesis was carried out that followed up our previously constructed resulting strain, E. coli BW25113 containing sanO/P on its chromosome and under the control of T7 promoter. Nine subsequent genes (sanQ/R/F/A/B1/B2/C/D/X), which involved in the gene cluster with optimized codons for the expression in E. coli, were completely chemical synthesized. The DNA fragment of these genes was electrotransferred and intergrated into the chromosome of BW25113 to be adjacent to sanO/P sequentially by "knock-in" procedure, which is set up based on our previous work to achieve scarless assembly. The resulting strain contains sanO/P/Q/R/F/A/Bl/B2/C/D/X transcriptional unit, which is under the control of T7 promoter. Furthermore, the T7 RNA polymerase gene (polTT) under the control of arabinose promoter was constructed on BW25113 chromosome that located adjacent to sanO-X cluster by "knock-in" procedure as well. The resulting strain, BW25113/polT7/sanO-X, was used to test the expression profile of the gene cluster. During fermentation, the culture was induced by arabinose to express T7 RNA polymerase, by which the transcription of sanO-X gene cluster could be triggered. RNA isolation was performed, and reverse transcription PCR (RT-PCR) were taken place to detect the signal of the transcripts of the cluster. The results indicated that all of genes in the cluster were co-transcribed. The whole proteome of the culture was analyzed by obitrap mass analyzer, and the mass spectrum indicated that 9 out of 11 proteins encoded within this cluster were translated, and 4 of them were as soluble protein. Whereas, the product related to the biosynthesis of this gene cluster was not detected by HPLC, The results revealed that some of the genes in this cluster were not expressed properly, and the genes need to be further optimized in the next stage.To optimize the expression of the functional module in Streptomyces host, we redesigned and constructed a gene cluster for biosynthesis of nikkomycin Cz. The plasmid pIJ8660 was used as the vecter, and 8 genes (sanF/A/Bl/B2/C/D/X/Y) from a nikkomycin high producer TH322 were amplified by PCR, rearranged and assembled to form two transcriptional units under the control of hrdB promoters by Gibson assembly procedure. This construct (pIJ8660::PhrdB::sanF-Y) was introduced into Streptomyces coelicolor M145 and M1146 heterologous hosts, respectively. The preliminary comparative tests for gene expression and metabolites of the recombinant strains against host strains were performed. HPLC analysis results didn’t show the difference between the tested strain and the control. Further transcriptional analysis was carried out by RT-PCR. The results showed that 4 out of 5 tested genes can be detected except the last gene, sanY, in the cluster, which indicated that the expression of this functional module need to be further optimized.In conclusion, our work in this thesis constructed two gene clusters for the biosynthesis of the intermediate metabolites of polynik A by synthetic biology approach, which include rational design, optimization of biological parts, scarless assembly of gene cluster and characterization of the behavior of the functional module. The information for expression profile of individual gene cluster in different chassis was obtained. These results laid the foundation for the further optimization study.