Engineered Thiomarinol Antibiotics Active against MRSA Are Generated by Mutagenesis and Mutasynthesis of Pseudoalteromonas SANK73390
Engineered Thiomarinol Antibiotics Active against MRSA Are Generated by Mutagenesis and Mutasynthesis of Pseudoalteromonas SANK73390
复制标题
DOI：
10.1002/anie.201007029
复制
发表时间：
2011-01-01
影响因子：
16.6
通讯作者：
Simpson, Thomas J.
中科院分区：
文献类型：
--
作者：
Murphy, Annabel C.;Fukuda, Daisuke;Simpson, Thomas J.
专性海洋细菌假交替单胞菌属（Pseudoalteromonas spp.）SANK 73390产生一系列杂合抗生素，硫代马林醇A-G（方案1），[1-3]其中吡咯硫部分通过酰胺连接到由荧光假单胞菌产生的具有临床意义的抗生素[4]莫匹罗星（假单胞菌酸，例如，7-9）的紧密类似物。[5-8]含吡咯烷酮的全霉素（10）、[9] N-丙酰全息霉素（11）、[10]硫羟芸香苷（12）和金丝菌素（13）[11]也是抗生素，但硫代马林醇和莫匹罗星对金黄色葡萄球菌（包括耐甲氧西林金黄色葡萄球菌）表现出特别有效的活性。金黄色葡萄球菌（MRSA）（MIC< 0.01 μg/mL）。假单胞菌酸A（7）是由“trans-AT”类模块化聚酮酶（PKS）产生的广泛抗生素家族中的第一个。[12]通过对SANK 73390全基因组测序，鉴定了硫代玛丽醇（tml）生物合成基因簇，结果表明，它包含在一个97 kb的质粒上，几乎完全由硫代玛丽醇生物合成基因组成。[13]这些由trans-AT PKS和与莫匹罗星（mup）簇具有高度同源性的相关剪裁基因组成，沿着有与一组剪裁酶连接的非核糖体肽合成酶（NRPS），所述剪裁酶类似于最近显示的在棒状链霉菌中控制全霉素生物合成的酶。[14]与thiomarinol A（1）相反，莫匹罗星的主要成分假单胞菌酸A（7）具有环氧化的9，10-烯烃，这使得它在狭窄的pH范围外容易发生分子内重排，并限制了其临床应用。莫匹罗星抑制异亮氨酸转移RNA合成酶。[4]在硫代马林醇A中附加的吡咯部分改善了对该靶点的抑制，[13]但尚未确定它是否也赋予了额外的抗菌作用模式。为了证实tml簇参与硫代马林醇A的产生，产生了两种突变株ΔPKS和ΔNRPS。[13]对于ΔPKS突变体，PKS基因的酮合酶区段（tmpD对应于莫匹罗星PKS mmpD）用于使用载体PAKE 604的自杀诱变。[15]NRPS基因的内部片段（holA cf. orf 3488）[14]类似地用于产生ΔNRPS菌株。在每种情况下，硫代马林醇生产被废除，但当两种菌株共发酵时，硫代马林醇生产恢复。[13]我们现在描述的确定的全部代谢概况和一些以前未检测到的代谢产物在野生型（WT）和突变株的假交替单胞菌SANK 73390的表征。剪裁酶TmlU的框内缺失突变体（ΔtmlU），
The obligate marine bacterium Pseudoalteromonas spp. SANK73390 produces a series of hybrid antibiotics, thiomarinols A–G (Scheme 1),[1–3] in which a pyrrothine moiety is linked through an amide to close analogues of the clinically significant antibiotic [4] mupirocin (pseudomonic acids, for example, 7–9) produced by Pseudomonas fluorescens.[5–8] The pyrrothine-containing holomycin (10),[9] N-propionylholothin (11),[10] thiolutin (12), and aureothricin (13)[11] are also antibiotics but the thiomarinols and mupirocin display particularly potent activity against Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA)(MIC< 0.01 μg mLÀ1). Pseudomonic acid A (7) was one of the first of an extensive family of antibiotics produced by the “trans-AT” class of modular polyketide synthases (PKSs).[12] Identification of the thiomarinol (tml) biosythetic gene cluster by full genome sequencing of SANK73390 showed that it is contained on a 97kb plasmid consisting almost entirely of the thiomarinol biosynthetic genes.[13] These consist of trans-AT PKSs and associated tailoring genes with high homology to the mupirocin (mup) cluster, along with a nonribosomal peptide synthetase (NRPS) linked to a set of tailoring enzymes similar to that recently shown to control holomycin biosynthesis in Streptomyces clavuligerus.[14] In contrast to thiomarinol A (1), the major mupirocin component, pseudomonic acidA (7) has the 9, 10-alkene epoxidized which makes it susceptible to intramolecular rearrangements outside a narrow pH range and limits its clinical utility. Mupirocin inhibits isoleucyl-transfer RNA synthetase.[4] The appended pyrrothine moiety in thiomarinol A improves inhibition of this target,[13] but it is yet to be established whether it also imparts an additional mode of antibacterial action.To confirm involvement of the tml cluster in thiomarinol A production, two mutant strains ΔPKS and ΔNRPS were generated.[13] For the ΔPKS mutant a ketosynthase segment of the PKS gene (tmpD corresponding to mupirocin PKS mmpD) was used for suicide mutagenesis using vector pAKE604.[15] An internal segment of the NRPS gene (holA cf. orf3488)[14] was used similarly to generate the ΔNRPS strain. In each case, thiomarinol production was abolished, but when the two strains were co-fermented thiomarinol production was restored.[13] We now describe determination of the full metabolic profiles and characterization of a number of previously undetected metabolites in wild-type (WT) and mutant strains of Pseudoalteromonas SANK73390. An inframe deletion mutant (ΔtmlU) of tailoring enzyme TmlU,