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Studies On Xiamenmycin Biosynthesis In Streptomyces Xiamenensis 318

Posted on:2015-07-27Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y YangFull Text:PDF
GTID:1220330476953932Subject:Microbiology
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Streptomyces xiamenensis 318 was originated from mangrove sediments in Fujian, China. It was used to produce xiamenmycin which was reported to be an inhibitor of ICAM-1/LFA-1 interaction with a possible anti-inflammatory function. The compounds of amino acid substituted benzopyran derivatives were rarely found in nature. However, the biosynthetic gene cluster responsible for producing prenylated benzopyran derivatives remains unknown.The chemical structure of xiamenmycin can be divided into three parts: L-threonine, 4-hydroxybenzoic acid(4HB) and a geranyl group. Based on the structural features of xiamenmycin, a prenyltransferase was thought to play a key role in the prenylation of 4HB and could thus be used as a target for screening the xiamenmycin biosynthetic gene cluster. Moreover, some enzymes, which could form pyran ring and amide linkage, could exist in xiamenmycin gene cluster.The isotope feeding experiment showed 4-hydroxybenzoic acid was one of the building blocks of xiamenmycin. The 5.9 M bp draft genome sequence of S. xiamenensis 318, was annotated using the RAST server(http://rast.nmpdr.org/). From this analysis, we identified six homologues of 4-hydroxybenzoate polyprenyltransferase(Ubi A). Transcription of three ubi A genes(ORF4925, ORF5065, ORF5313) was confirmed using real-time reverse-transcription-PCR. The DNA fragment containing both the ubi A gene and a putative chorismate lyase gene that is responsible for generating 4-hydroxybenzoic acid was chosen for further characterization.We constructed a genomic library of S. xiamenensis 318 in E. coli using the fosmid vector p CC2 FOS. One fosmid(p9A11), which has been shown to cover the complete biosynthetic gene cluster, was obtained by PCR screening. Subcloning of a 7.5 kb DNA fragment from p9A11 generated the plasmid p LMO09403, which contained five open reading frames(ORF5311, ORF5313, ORF5314, ORF5315, ORF5316) used for further genetic analysis. Heterologous expression of the five genes(ORF5311~ORF5316) in S. lividans 1326 was then attempted.To verify the involvement of this DNA fragment in the biosynthesis of xiamenmycin, five gene replacement plasmids were constructed and introduced to S. xiamenensis 318. We individually replaced xim A(ORF5313), xim B(ORF5311), xim C(ORF5314), xim D(ORF5315), and xim E(ORF5316) with an apramycin resistance cassette. This analysis of UPLC revealed that xim A inactivation mutants produced an intermediate(xiamenmycin B) instead of xiamenmycin, while xiamenmycin production was abolished in the other four gene disruption mutants without accumulation of detectable intermediate. NMR data comfirmed the structure of xiamenmycin B. We proposed a biosynthetic pathway for xiamenmycin.On the basis of the structure of the accumulated compound, feeding studies, biochemical characterizations, and bioinformatics analysis of each gene, we describe a gene cluster consisting of five genes that is responsible for the biosynthesis of xiamenmycin and propose a biosynthetic pathway for xiamenmycin. We show that 4-hydroxybenzoic acid is the first intermediate for xiamenmycin biosynthesis. Through biochemical characterization, we also demonstrate that Xim C is responsible for the generation of 4HB. Xim B catalyzes 4HB and geranyl diphosphate(GPP) to produce 3-geranyl-4-hydroxybenzoic acid. The prenylated 4HB is then processed by Xim D to generate an epoxide intermediate, followed by catalysis of pyran ring formation by Xim E, a Snoa L-like polyketide cyclase, to generate xiamenmycin B. Finally, Xim A was biochemically characterized to be responsible for catalyzing the amide formation of xiamenmycin B and L-threonine to produce xiamenmycin. The Km value of Xim A was determined to be 474.38 m M for the substrate xiamenmycin B.These studies were firstly reported to reveal the mechanism of benzopyran derivatives biosynthesis, and confirmed that xiamenmycin gene cluster could be introduced in other Streptomyces for heterologous expression. These studies provide opportunities to use genetic and chemo-enzymatic methods to create new benzopyran derivatives as potential therapeutic agents.
Keywords/Search Tags:Streptomyces, xiamenensis, benzopyran, derivative xiamenmycin, biosynthesis
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