The tallysomycin biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 unveiling new insights into the biosynthesis of the bleomycin family of antitumor antibiotics

The tallysomycins (TLMs) belong to the bleomycin (BLM) family of antitumor antibiotics. The BLM biosynthetic gene cluster has been cloned and characterized previously from Streptomyces verticillus ATCC 15003, but engineering BLM biosynthesis for novel analogs has been hampered by the lack of a genetic system for S. verticillus. We now report the cloning and sequencing of the TLM biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 and the development of a genetic system for S. hindustanus, demonstrating the feasibility to manipulate TLM biosynthesis in S. hindustanus by gene inactivation and mutant complementation. Sequence analysis of the cloned 80.2 kb region revealed 40 open reading frames (ORFs), 30 of which were assigned to the TLM biosynthetic gene cluster. The TLM gene cluster consists of nonribosomal peptide synthetase (NRPS) genes encoding nine NRPS modules, a polyketide synthase (PKS) gene encoding one PKS module, genes encoding seven enzymes for deoxysugar biosynthesis and attachment, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The involvement of the cloned gene cluster in TLM biosynthesis was confirmed by inactivating the tlmE glycosyltransferase gene to generate a TLM non-producing mutant and by restoring TLM production to the DeltatlmE::ermE mutant strain upon expressing a functional copy of tlmE. The TLM gene cluster is highly homologous to the BLM cluster, with 25 of the 30 ORFs identified within the two clusters exhibiting striking similarities. The structural similarities and differences between TLM and BLM were reflected remarkably well by the genes and their organization in their respective biosynthetic gene clusters.     

黄脂菌素生物合成基因簇中调控基因xanR3的功能

【目的】研究黄脂菌素产生菌灰黄链霉菌中编码ArsR家族转录调控蛋白(Arsenical resistance regulator)的xanR3基因的功能。【方法】利用大肠杆菌和链霉菌双亲本接合转移的方法,构建xanR3基因缺失突变株及回补突变株。利用cDNA在相邻同方向的基因间隔区进行PCR确定黄脂菌素生物合成基因簇中的转录单元。利用荧光定量RT-PCR方法进行突变株中黄脂菌素生物合成基因簇转录水平的检测。【结果】对得到的xanR3基因缺失突变株及回补突变株进行发酵,发现xanR3基因缺失突变株产黄脂菌素能力下降,回补菌株中黄脂菌素产量相比缺失突变株有一定程度的恢复,但仍未达到野生型水平。经鉴定,黄脂菌素生物合成基因簇中共有18个共转录单元,其中4个共转录单元在?xanR3突变株中转录水平明显下降。【结论】ArsR家族转录调控基因xanR3是黄脂菌素生物合成的正调控基因。     

金霉素生物合成基因簇中调控基因ctcB的功能

【目的】研究金霉素产生菌中SARP家族转录调控基因ctc B的作用。【方法】利用大肠杆菌、链霉菌的属间接合转移和同源重组双交换的方法,构建ctc B基因缺失突变株。通过c DNA在相邻同转录方向的基因间隔进行PCR验证,确定金霉素生物合成基因簇中的转录单元。利用荧光定量RT-PCR方法进行突变株金霉素生物合成基因簇的转录水平检测。随后,生物信息学预测分析了金霉素生物合成基因簇内Ctc B与DNA的结合位点。【结果】获得了ctc B基因缺失的双交换突变株。发酵结果显示,该突变株失去产生金霉素与四环素的能力。金霉素生物合成基因簇内有6个共转录单元,其中4个共转录单元在ctc B基因缺失突变株中转录水平明显下降。软件分析预测到一致性较高的Ctc B结合重复序列。【结论】ctc B正调控金霉素生物合成结构基因ctc G-D、ctc H-K、ctc N-P、ctc W-T 4个转录单元和ctc Q,为进一步研究ctc B调控机制奠定了基础。     

Dothistroma pini,a forest pathogen,contains homologs of aflatoxin biosynthetic pathway genes

Homologs of aflatoxin biosynthetic genes have been identified in the pine needle pathogen Dothistroma pini. D. pini produces dothistromin, a difuranoanthraquinone toxin with structural similarity to the aflatoxin precursor versicolorin B. Previous studies with purified dothistromin suggest a possible role for this toxin in pathogenicity. By using an aflatoxin gene as a hybridization probe, a genomic D. pini clone was identified that contained four dot genes with similarity to genes in aflatoxin and sterigmatocystin gene clusters with predicted activities of a ketoreductase (dotA), oxidase (dotB), major facilitator superfamily transporter (dotC), and thioesterase (dotD). A D. pini dotA mutant was made by targeted gene replacement and shown to be severely impaired in dothistromin production, confirming that dotA is involved in dothistromin biosynthesis. Accumulation of versicolorin A (a precursor of aflatoxin) by the dotA mutant confirms that the dotA gene product is involved in an aflatoxin-like biosynthetic pathway. Since toxin genes have been found to be clustered in fungi in every case analyzed so far, it is speculated that the four dot genes may comprise part of a dothistromin biosynthetic gene cluster. A fifth gene, ddhA, is not a homolog of aflatoxin genes and could be at one end of the dothistromin cluster. These genes will allow comparative biochemical and genetic studies of the aflatoxin and dothistromin biosynthetic pathways and may also lead to new ways to control Dothistroma needle blight.     

Mutational analysis of nocK and nocL in the nocardicin a producer Nocardia uniformis

The nocardicins are a family of monocyclic beta-lactam antibiotics produced by the actinomycete Nocardia uniformis subsp. tsuyamanensis ATCC 21806. The most potent of this series is nocardicin A, containing a syn-configured oxime moiety, an uncommon feature in natural products. The nocardicin A biosynthetic gene cluster was recently identified and found to encode proteins in keeping with nocardicin A production, including the nocardicin N-oxygenase, NocL, in addition to genes of undetermined function, such as nocK, which bears similarities to a broad family of esterases. The latter was hypothesized to be involved in the formation of the critical beta-lactam ring. While previously shown to effect oxidation of the 2'-amine of nocardicin C to provide nocardicin A, it was uncertain whether NocL was the only N-oxidizing enzyme required for nocardicin A biosynthesis. To further detail the role of NocL in nocardicin production in N. uniformis, and to examine the function of nocK, a method for the transformation of N. uniformis protoplasts to inactivate both nocK and nocL was developed and applied. A reliable protocol is reported to achieve both insertional disruption and in trans complementation in this strain. While the nocK mutant still produced nocardicin A at levels near that seen for wild-type N. uniformis, and therefore has no obvious role in nocardicin biosynthesis, the nocL disruptant failed to generate the oxime-containing metabolite. Nocardicin A production was restored in the nocL mutant upon in trans expression of the gene. Furthermore, the nocL mutant accumulated the biosynthetic intermediate nocardicin C, confirming its role as the sole oxime-forming enzyme required for production of nocardicin A.     

Three unlinked gene clusters are involved in clavam metabolite biosynthesis in Streptomyces clavuligerus

In Streptomyces clavuligerus, three groups of genes are known to be involved in the biosynthesis of the clavam metabolites. Since antibiotic biosynthetic genes are invariably clustered on the chromosome in prokaryotes, chromosome walking was undertaken in an attempt to show that the three groups of clavam genes would resolve into a single super-cluster when analyzed at larger scale. However, no evidence of linkage between the three groups was obtained. Furthermore, Southern analysis of macro-restriction fragments of genomic DNA separated by pulsed-field gel electrophoresis also indicated that the three groups of genes are not linked. Despite the structural and biosynthetic relatedness of the clavam metabolites, our results suggest that the genes involved in their production lie in three unlinked gene clusters. We believe that this represents the first instance in bacteria of genes involved in the biosynthesis of a single family of antibiotics sharing a common biosynthetic pathway and yet residing in three separate locations on the chromosome.     

Cloning and analysis of the simocyclinone biosynthetic gene cluster of <Emphasis Type="Italic">Streptomyces antibioticus</Emphasis> Tü 6040

The biosynthetic gene cluster of the aminocoumarin antibiotic simocyclinone D8 was cloned by screening a cosmid library of Streptomyces antibioticusTü 6040 with a heterologous probe from a gene encoding a cytochrome P450 enzyme involved in the biosynthesis of the aminocoumarin antibiotic novobiocin. Sequence analysis of a 39.4-kb region revealed the presence of 38 ORFs. Six of the identified ORFs showed striking similarity to genes from the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin and coumermycin A(1). Simocyclinone also contains an angucyclinone moiety, and 12 of the ORFs showed high sequence similarity to biosynthetic genes of other angucyclinone antibiotics. Possible functions within the biosynthesis of simocyclinone D8 could be assigned to 23 ORFs by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by a gene inactivation experiment, which resulted in the abolishment of the formation of the aminocoumarin moiety of simocyclinone. Feeding of the mutant with the aminocoumarin moiety of novobiocin led to a new, artificial simocyclinone derivative.     

Molecular cloning and identification of the laspartomycin biosynthetic gene cluster from Streptomyces viridochromogenes

The biosynthetic gene cluster for laspartomycins, a family of 11 amino acid peptide antibiotics, has been cloned and sequenced from Streptomyces viridochromogenes ATCC 29814. Annotation of a segment of 88912bp of S. viridochromogenes genomic sequence revealed the putative lpm cluster and its flanking regions which harbor 43 open reading frames. The lpm cluster, which spans approximately 60 kb, consists of 21 open reading frames. Those include four NRPS genes (lpmA/orf18, lpmB/orf25, lpmC/orf26 and lpmD/orf27), four genes (orfs 21, 22, 24 and 29) involved in the lipid tail biosynthesis and attachment, four regulatory genes (orfs 13, 19, 32 and 33) and three putative exporters or self-resistance genes (orfs 14, 20 and 30). In addition, the gene involved in the biosynthesis of the nonproteinogenic amino acid Pip was also identified in the lpm cluster while the genes necessary for the biosynthesis of the rare residue diaminopropionic acid (Dap) were found to reside elsewhere on the chromosome. Interestingly, the dabA, dabB and dabC genes predicted to code for the biosynthesis of the unusual amino acid diaminobutyric acid (Dab) are organized into the lpm cluster even though the Dab residue was not found in the laspartomycins. Disruption of the NRPS lpmC gene completely abolished laspartomycin production in the corresponding mutant strain. These findings will allow molecular engineering and combinatorial biosynthesis approaches to expand the structural diversity of the amphomycin-group peptide antibiotics including the laspartomycins and friulimicins.     

肽类抗生素生物合成基因簇在Escherichia coli中的异源表达

微生物能够产生众多结构和生物活性多样的次生代谢产物,而其生物合成基因簇的挖掘和异源表达是药物创新和产量提高的必要前提. 在过去20年里,大量重要天然产物的生物合成基因簇在微生物中被不断的发现. 在这些被挖掘的基因簇中,肽类抗生素的生物合成基因簇占了很大比重.肽类抗生素因具有抗菌、抗肿瘤、抗病毒等多种生物学活性而备受化学家和药物学家的重视. 如能了解它们的生物合成机制,实现其基因簇的异源表达,将使合理化遗传修饰生物合成通路获取结构类似物（药物开发）和提高产量成为可能. 大肠杆菌作为最广泛、最成功的表达体系,常用来表达外源基因,但一般只能表达一个或几个基因,却很少有用它来表达整个生物合成基因簇. 2001年,Khosla和Cane在E.coli中成功异源表达了一个复杂聚酮天然产物（红霉素苷原6dEB）基因簇. 这是首个有关在E.coli中异源表达天然产物生物合成基因簇的研究. 至此之后,大肠杆菌开始作为生物合成基因簇的异源表达宿主,越来越受到相关领域的重视. 紧接着核糖体肽和非核糖体肽生物合成基因簇也相继在大肠杆菌中成功异源表达. 本文对肽类抗生素生物合成基因簇在E.coli中的异源表达进行了综述.     

Characterization of the biosynthetic gene cluster for the oligosaccharide antibiotic, Evernimicin, in Micromonospora carbonacea var. africana ATCC39149

Evernimicin (EV) belongs to the orthosomycin class of antibiotics and consists of several modified L- and D-deoxysugars containing unusual orthoester and glycosyl linkages and two orsellinic acid groups, one that is halogenated. The EV biosynthetic gene cluster from Micromonospora carbonacea var. africana ATCC39149 was localized by hybridization to a dTDP-D-glucose 4,6-dehydratase probe and a 120-kb region containing the EV biosynthetic cluster and surrounding regions has been sequenced. BLAST analysis has identified a type I polyketide synthase for orsellinic acid biosynthesis as well as enzymes required for L- and D-deoxyglucose and D-deoxymannose synthesis. In addition, genes involved in glycosyltransfer and resistance were identified. Insertional mutations in several biosynthetic genes blocked EV production, indicating a role for these genes in EV biosynthesis.     

Production of mycobacterial cell wall glycopeptidolipids requires a member of the MbtH-like protein family

ABSTRACT: BACKGROUND: Glycopeptidolipids (GPLs) are among the major free glycolipid components of the outer membrane of several saprophytic and clinically-relevant Mycobacterium species. The architecture of GPLs is based on a constant tripeptide-amino alcohol core of nonribosomal peptide synthetase origin that is N-acylated with a 3-hydroxy/methoxy acyl chain synthesized by a polyketide synthase and further decorated with variable glycosylation patterns built from methylated and acetylated sugars. GPLs have been implicated in many aspects of mycobacterial biology, thus highlighting the significance of gaining an understanding of their biosynthesis. Our bioinformatics analysis revealed that every GPL biosynthetic gene cluster known to date contains a gene (referred herein to as gplH) encoding a member of the MbtH-like protein family. Herein, we sought to conclusively establish whether gplH was required for GPL production. RESULTS: Deletion of gplH, a gene clustered with nonribosomal peptide synthetase-encoding genes in the GPL biosynthetic gene cluster of Mycobacterium smegmatis, produced a GPL deficient mutant. Transformation of this mutant with a plasmid expressing gplH restored GPL production. Complementation was also achieved by plasmid-based constitutive expression of mbtH, a paralog of gplH found in the biosynthetic gene cluster for production of the siderophore mycobactin of M. smegmatis. Further characterization of the gplH mutant indicated that it also displayed atypical colony morphology, lack of sliding motility, altered capacity for biofilm formation, and increased drug susceptibility. CONCLUSIONS: Herein, we provide evidence formally establishing that gplH is essential for GPL production in M. smegmatis. Inactivation of gplH also leads to a pleiotropic phenotype likely to arise from alterations in the cell envelope due to the lack of GPLs. While genes encoding MbtH-like proteins have been shown to be needed for production of siderophores and antibiotics, our study presents the first case of one such a gene proven to be required for production of a cell wall component. Furthermore, our results provide the first example of a mbtH-like gene with confirmed functional role in a member of the Mycobacterium genus. Altogether, our findings demonstrate a critical role of gplH in mycobacterial biology and advance our understanding of the genetic requirements for the biosynthesis of an important group of constituents of the mycobacterial outer membrane.