Establishing One Method Regarding Preparation Of The Standard And Quantification Of The Antibiotic, Zwittermicin A, And Function Study On Some Key Genes In The Zwittermicin A Biosynthesis Gene Cluster

The antibiotic, zwittermicin A, was one kind of linear aminopolyol compound which had been firstly found in the nature. It was mainly produced by Bacillus cereus group, including Bacillus thuringiensis. The bioactivity of this antibiotic was very interesting, not only because its suppression of disease in plant caused by protists and inhibition of fungal and bacteria growth, but also its broad-spectrum amplification of the insecticidal activities of the Bt toxic protein.Since its chemical structure was elucidated in 1994, with the joint efforts of laboratories home and abroad, the complete gene cluster about 70kb was founded to locate between two transposase encoding genes, including biosynthesis, immunity, transportation, regulation genes in 2011. Kevany group put foward a model of zwittermicin A biosynthesis in 2009, while some key genes, the zmaS and zmaM for instance, within the gene cluster were only predicted by bioinformatics, so the infact function of them needed to be convinced in the genetic and biochemical level. In this study, my interest focused on this thing. The wide use of Bt pesticides and Bt crops enhances the application potential of this antibiotic, but it is very hard to detect the contents of it when Bt pesticides are produced. The main reason is that there is no zwittermicin A standard for sale and the quantification method has not been established. What’s more, the research about metabolite regulation of this antibiotic also needs zwittermicin A standard and quantification method. So I try to solve this problem in this study.Since 2000, the purification method of zwittermicin A reported is firstly two-times cation exchange chromatography and then one-times HPLC to obtain the pure zwittermicin A. However, it can’t be used in quantification of the zwittermicin A content in Bt products because its purity is very low. On the other hand, those purification methods are so complex that they can not be used in accurate quantification. With my own exploration and based on the reported methods, silica gel column chromatography and thin-layer chromatography for instance, this study obtains 125mg zwittermicin A standard (Peak areas ratio in HPLC is 75.25%),3.2mg pure zwittermicin A (Peak areas ratio in HPLC is 97.15%). Moreover, this study used the cation exchange chromatography as pretreatment procedure and then quantified the zwittermicin A contents in the fermentation samples of Bacillus cereus UW85 and its mutants with HPLC and MS respectively.When the bottleneck of quantification of zwittermicin A was solved, we could do something about zwittermicin A biosynthesis. This research mainly focused on the function study of zmaL, zmaM and the releasing mechanism of zwittermicin.The zmaS gene within zwittermicin A gene cluster of UW85 genome close to kanosamine biosynthesis gene cluster and encoding one kind of Sfp-like PPTase was predicted to catalyze the apo-ACP and apo-PCP to form holo-ACP and holo-PCP respectively. We apartment knocked out this gene before, but the inhibition zone of the mutant was becoming larger than the wild type and it still produced zwittermicin A. With coexpression of this gene and its adjacent Kanosamine gene cluster in heterologous host-BMB171, we found that zmaS could negatively regulate the kanosamine production. In this study, we quantify the yields of zwittermicin A and kanosamine of UW86. Compared to UW85, the kanosamine production of UW86 was fifteen-times more than UW85 but the zwittermicin A production of UW86 was only 2.5% of UW85. So we are sure that zmaS can negatively regulate the kanosamine production but we don’t know how it happened. With the analyzing the sequence characteristic around zmaS, we find that this gene is adjacent to the positive regulation gene of kanosamin, kanR, and there is one apparent hairpin loop between these two genes. We given that zmaS gene blocked the nomal transcription of kanR to negatively regulate the production of kanosamine. In this study we created the zmaS and zmaS# (mutation of zmaS) overexpression mutants to confirm this kind of negative regulation but the result didn’t go along with the hypothesis. So it still needs deeper analysis. Because zmaS gene encodes one Sfp-like PPTase and the yields of zwittermicin A of zmaS mutants is only 2.5% of UW85, so we think this gene is sure related with zwittermicin A biosynthesis. Therefore we purified the ZmaS, ACP and Sfp protein separately to do the catalytic experiments in vitro. We successfully confirmed that zmaS gene was one functional Sfp-like PPTase encoding gene in the end.The zmaM gene also within the zwittermicin A biosynthesis gene cluster encodes one two-function protein with a peptides domain in its N-terminal and a transporter domain in its C-terminal. It is predicted to process the zwittermicin A precursor and then transport the mature zwittermicin A molecular out of the cell membrane. We apartment knocked out this complete gene and its transporter domain with in-frame deletion method respectively, but both the mutants still produce zwittermicin A and the bacterostatic abilities of them are no more less than the wild type at 72h. This result indicated zmaM gene may be not related with zwittermicin A biosynthesis and the biosynthesis mechanism of it was sure to be explained in one new way. However, this study chooses 36h as the sampling point when the endospore has formed but the mother cell didn’t bust up and we detected the obvious inhibition activitiy differences between UW85 and the mutants. With quantification, we found that the yields of zwittermicin A of the two mutants were only 60% of UW85 and there was no obvious difference between the first two. So we confirm that zmaM gene is directly related to zwittermicin A biosynthesis. What’s more, the alignment result of zmaM with YBT-1520 genome suggests that there may be similar genes in the UW85 genome. We also confirmed our speculation by PCR amplication of the most likely gene in UW85 genome. The right result may indicate that we have found the zmaM complementary mechanism in UW85.The classic antibiotic releasing mechanism is catalyzed by type I Te domain. There is such a domain in zwittermicin A gene cluster, too. However, the precursor of zwittermicin A isn’t released with it. It is predicted to be released by a monooxygenase like protein named ZmaL. By knockout test, this gene was confirmed to be related with zwittermicin A production before but there was no biochemical evidence to support it. Moreover, the chemical synthesis of this antibiotic has obtained many stereocenters and truncated analogs, but none of them keep the activity of zwittermicin A. So there may need more analogs, especially the natural compounds produced by the cell itself, to confirm the active site of zwittermicin A. In this study, we want to create one mutant in which zwittermicin A is released by the Te domain. If this mutant could produce the bioactive zwittermicin A analogs, we could use it as the substrate to react with ZmaL and compare it with zwittermicin A to find the structural differences. Finally, we successfully screened three kinds of mutant which all contented Te fusion gene. Through the test of inhibition activity to LS005, we found that both BMB1964 and BMB1965 could produce active substance but BMB1963 had no obvious inhibitory phenotype. This study mainly focuses on the BMB1965 and finds this strain can’t produce the predicted zwittermicin A analog but its zwittermicin A yield is 7.25 times more than the wild type UW56. We presume that the second Te domain may accelerate the generation of metabolite B to rise up the production of zwittermicin A because these two compounds are in the same assembly line. There may be new clues when we have time to study the other two mutants.In summary, this research mainly focus on two parts:establishing one method regarding preparation of standard and quantification of the antibiotic, zwittermicin A, in the purification level, and studying the real function of some key genes within zwittermicin A biosynthesis gene cluster in the quantification, genetic and biochemical level.