达托霉素生物合成研究进展

达托霉素是由玫瑰孢链霉菌(Streptomyces roseosporus)生产的一种环脂肽类抗生素, 具有强大的抗革兰氏阳性致病细菌的作用, 是继“抗生素最后一道防线”万古霉素后的新型抗生素。本文主要对达托霉素的结构、作用机制、合成基因簇及合成机制等当前的研究成果进行综述, 并且总结了利用组合生物学对达托霉素进行结构改造的策略, 以此来研究结构与活性之间的关系, 并寻找更广谱高效的抗生素。最后, 本文总结了提高达托霉素产量的策略, 为工业上降低达托霉素生产成本提供理论参考。     

达托霉素耐药分子机制研究进展

环脂肽抗生素达托霉素抗菌活性强,致病菌不容易产生耐药性,已成为治疗革兰氏阳性菌特别是耐药菌感染的一线药物。但由于广泛使用,仍然出现了达托霉素耐药菌。细胞膜磷脂代谢和细胞壁结构动态与致病菌达托霉素耐药密切相关。文中综述了达托霉素作用机制和耐药机制,以期对药物研发和临床用药有所裨益。     

达托霉素作为新的胰蛋白酶激活剂的初探

首次发现达托霉素是一种新的胰蛋白酶激活剂,当胰蛋白酶与达托霉素的物质的量的比在反应时达到34.05时,达托霉素对胰蛋白酶比活力的激活率平均达到32.92%。通过计算机模拟技术对达托霉素与胰蛋白酶以及达托霉素对胰蛋白酶底物复合物分别进行分子对接,并利用等温滴定量热法(Isothermal titration calorimetry,ITC)验证模拟结果。研究发现达托霉素与胰蛋白酶的活性中心位置很靠近,达托霉素链状中的天冬氨酸的R基与酶活性中心的组氨酸-57发生了氢键相互作用,达托霉素使酶和底物复合物结构更加稳定,从而有利于催化作用。ITC结果表明胰蛋白酶上具有1个达托霉素结合位点,解离常数Kd为17.83μM,摩尔结合焓△H为237.9±28.17 cal/mol,摩尔结合熵△S为22.5 cal/mol·deg,这些结果从热力学角度支持了分子对接结果。研究发现达托霉素除具有抗革兰氏阳性耐药性细菌的功能外,还能促进胰蛋白酶比活这一新的功能,是一种新的胰蛋白酶激活剂。     

环境放线菌中的达托霉素抗性机制

[目的]研究环境放线菌中的达托霉素抗性机制,为临床耐药机制的出现提供预警.[方法]通过测定土壤放线菌(49株)和药用植物内生放线菌(10株)的达托霉素耐受谱,筛选达托霉素抗性菌株;通过达托霉素灭活实验,确定抗性菌株的灭活能力;通过形态观察和16S rRNA序列分析分类鉴定达托霉素降解菌.通过PCR扩增检测达托霉素去酰基化酶基因在降解菌株中的分布情况.[结果]本研究中所有的环境放线菌均耐受达托霉素.在土壤放线菌中和药物植物内生放线菌中,分别有24株(49.0％)和4株(40％)能够灭活达托霉素,25 (51.0％)株和6株(60％)通过其他机制耐受达托霉素.序列测定表明,链霉菌属(Streptomyces)、小单孢菌属(Mcromonospora)和诺卡氏菌属(Nocardia)的部分菌株有灭活达托霉素的能力.PCR扩增表明,5株(17.9％)放线菌含有编码达托霉素去酰基酶的基因.[结论]环境放线菌具有超高的达托霉素抗性频率,灭活达托霉素是主要抗性机制之一.     

应用响应面法优化发酵培养基提高达托霉素产量

[背景]达托霉素来自玫瑰孢链霉菌NRRL 11379的发酵产物,是重要的临床用抗生素.其原始产生菌发酵周期长,影响达托霉素的生产效率.本实验室前期在天蓝色链霉菌中重构了达托霉素的生物合成途径,有效地缩短了发酵周期,但重组菌株K10中达托霉素发酵产量很低,制约了后续的研究和开发.[目的]利用响应面法优化产达托霉素的重组菌...     

芳香聚酮后修饰氧化酶

氧化酶在芳香聚酮生物合成后修饰中普遍存在并对终产物的结构产生关键影响。本文简要总结了芳香聚酮后修饰氧化酶中几类最常见的氧化酶的结构和功能,并以杰多霉素生物合成途径中的后修饰氧化酶为例,阐明这些氧化酶在后修饰反应中发生作用的方式。并对后修饰氧化酶在组合生物学中的应用做了展望。     

纳他霉素的生物合成基因研究

纳他霉素是一种多烯大环内酯类抗真菌抗生素,能专一地抑制酵母和霉菌,作为天然的防腐剂用于食品和饲料行业。概述了纳他霉素的化学结构,作用机理以及基因调控方面的研究,包括合成基因,修饰基因和调节基因。并展望了在纳他霉素基因工程研究方面的前景。     

高GC含量DNA模板的PCR扩增

目的：探索高GC含量DNA的PCR扩增条件,为扩增达托霉素生物合成基因簇及拼接奠定基础。方法：在PCR扩增体系中,使用高保真的聚合酶及添加不同浓度的DMSO、7-deaza-dGTP等增强剂,并选择合适的PCR循环程序,优化富含GC的DNA的PCR扩增条件。结果：向反应体系中额外添加1%~4%的DMSO可以显著提高富含GC的DNA的PCR扩增产物量,但会降低其特异性;7-deaza-dGTP可以提高扩增产物的特异性及保真度,但产量会有所下降。应用touch down PCR并在体系中添加7-deaza-dGTP能够提高扩增产物的特异性和产率,增加扩增的保真度。结论：应用优化的PCR扩增条件将所有达托霉素生物合成基因簇分段扩增出来,并可扩增出长达6 kb的片段,且序列完全正确,可以进行后续拼接。     

链霉菌来源的苯并异色烷醌家族抗生素的生物合成

苯并异色烷醌(benzoisochromanequinones,BIQs)家族抗生素是由链霉菌产生的聚酮类抗生素,其芳香聚酮母核结构中含有并联的两个芳香环和一个吡喃环,具有抗菌、抗肿瘤等多种生物学活性。BIQ抗生素聚酮链的早期生物合成过程代表了芳香聚酮抗生素母核的典型合成机制,而不同的后期修饰则决定了它们结构和生物学活性的多样性。在过去的二十几年中,以放线紫红素和美达霉素为研究重点,BIQ家族抗生素的生物合成机制逐渐得到揭示,但在后期结构修饰方面仍有许多问题有待解决。本文对BIQ家族抗生素的生物合成机制研究进行了综述,比较了不同BIQ家族抗生素结构特点、生物学活性,并重点阐述了它们生物合成中的后期结构修饰和调控过程的研究进展,并对BIQ抗生素在代谢工程方面的研究进行了展望。     

多重耐药性金黄色葡萄球菌(MRSA)的临床药物治疗及耐药机制研究

近年来,由多重耐药性金黄色葡萄球菌引起的院内感染不仅是临床医师必须经常面临的棘手问题,而且已对全人类的健康构成极大威胁。一直以来,万古霉素都是临床治疗革兰阳性菌感染的首选药物,曾被称为临床抗感染的"最后底线"。但目前,万古霉素耐药性金黄色葡萄球菌已经在许多国家分离出来,这就对开发新型抗感染治疗药物提出了迫切要求。就万古霉素及新型替代药物利奈唑胺和达托霉素的作用机制、临床应用情况及耐药状况做一综述。     

Lipid-specific binding of the calcium-dependent antibiotic daptomycin leads to changes in lipid polymorphism of model membranes

Daptomycin is a cyclic anionic lipopeptide with an antibiotic activity that is completely dependent on the presence of calcium (as Ca2+). In a previous study [Jung et al., 2004. Chem. Biol. 11, 949-957], it was concluded that daptomycin underwent two Ca2+-dependent structural transitions, whereby the first transition was solely dependent on Ca2+, while the second transition was dependent on both Ca2+ and the presence of negatively charged lipids that allowed daptomycin to insert into and perturb bilayer membranes with acidic character. Differences in the interaction of daptomycin with acidic and neutral membranes were further investigated by spectroscopic means. The lack of quenching of intrinsic fluorescence by the water-soluble quencher, KI, confirmed the insertion of the daptomycin Trp residue into the membrane bilayer, while the kynurenine residue was inaccessible even in an aqueous environment. Differential scanning calorimetry (DSC) indicated that the binding of daptomycin to neutral bilayers occurred through a combination of electrostatic and hydrophobic interactions, while the binding of daptomycin to bilayers containing acidic lipids primarily involved electrostatic interactions. The binding of daptomycin to acidic membranes led to the induction of non-lamellar lipid phases and membrane fusion.     

Oligomerization of daptomycin on membranes

Daptomycin is a lipopeptide antibiotic that kills Gram-positive bacteria by membrane depolarization. While it has long been assumed that the mode of action of daptomycin involves the formation of membrane-associated oligomers, this has so far not been experimentally demonstrated. We here use FRET between native daptomycin and an NBD-labeled daptomycin derivative to show that such oligomerization indeed occurs. The oligomers are observed in the presence of calcium ions on membrane vesicles isolated from Bacillus subtilis, as well as on model membranes containing the negatively charged phospholipid phosphatidylglycerol. In contrast, oligomerization does not occur on membranes containing phosphatidylcholine only, nor in solution at micromolar daptomycin concentrations. The requirements for oligomerization of daptomycin resemble those previously reported for antibacterial activity, suggesting that oligomerization is necessary for the activity.     

Daptomycin-mediated reorganization of membrane architecture causes mislocalization of essential cell division proteins

Daptomycin is a lipopeptide antibiotic used clinically for the treatment of certain types of Gram-positive infections, including those caused by methicillin-resistant Staphylococcus aureus (MRSA). Details of the mechanism of action of daptomycin continue to be elucidated, particularly the question of whether daptomycin acts on the cell membrane, the cell wall, or both. Here, we use fluorescence microscopy to directly visualize the interaction of daptomycin with the model Gram-positive bacterium Bacillus subtilis. We show that the first observable cellular effects are the formation of membrane distortions (patches of membrane) that precede cell death by more than 30 min. Membrane patches are able to recruit the essential cell division protein DivIVA. Recruitment of DivIVA correlates with membrane defects and changes in cell morphology, suggesting a localized alteration in the activity of enzymes involved in cell wall synthesis that could account for previously described effects of daptomycin on cell wall morphology and septation. Membrane defects colocalize with fluorescently labeled daptomycin, DivIVA, and fluorescent reporters of peptidoglycan biogenesis (Bocillin FL and BODIPY FL-vancomycin), suggesting that daptomycin plays a direct role in these events. Our results support a mechanism for daptomycin with a primary effect on cell membranes that in turn redirects the localization of proteins involved in cell division and cell wall synthesis, causing dramatic cell wall and membrane defects, which may ultimately lead to a breach in the cell membrane and cell death. These results help resolve the longstanding questions regarding the mechanism of action of this important class of antibiotics.     

Manipulation of kynurenine pathway for enhanced daptomycin production in Streptomyces roseosporus

Daptomycin is a cyclic lipopeptide natural product produced by Stretptomyces roseosporus, displaying good bactericidal activity against a wide range of gram‐positive pathogens. Daptomycin contains a 13 amino acid and kynurenine (Kyn) is essential for optimal activity of daptomycin. In this study, we characterized the Kyn pathway in S. roseosporus and investigated its role in supplying precursor for daptomycin biosynthesis. Two genes (dptJ and tdo) coding for tryptophan‐2,3‐dioxgenase existed in the chromosome. dptJ is located in the daptomycin biosynthetic gene cluster, while tdo is in other locus. Disruption of dptJ or tdo resulted in reduced yield by ～50%. The introduction of an additional copy of dptJ but not tdo led to enhanced production of daptomycin by 110%. Furthermore, disruption of kyn encoding kynureninase showed improved daptomycin productivity by 30%. Our results demonstrated that the enhancement of Kyn supply through metabolic engineering approach is an efficient way to increase daptomycin production. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:847–852, 2013     

The action mechanism of daptomycin

Daptomycin is a lipopeptide antibiotic produced by the soil bacterium Streptomyces roseosporus that is clinically used to treat severe infections with Gram-positive bacteria. In this review, we discuss the mode of action of this important antibiotic. Although daptomycin is structurally related to amphomycin and similar lipopeptides that inhibit peptidoglycan biosynthesis, experimental studies have not produced clear evidence that daptomycin shares their action mechanism. Instead, the best characterized effect of daptomycin is the permeabilization and depolarization of the bacterial cell membrane. This activity, which can account for daptomycin’s bactericidal effect, correlates with the level of phosphatidylglycerol (PG) in the membrane. Accordingly, reduced synthesis of PG or its increased conversion to lysyl-PG promotes bacterial resistance to daptomycin. While other resistance mechanisms suggest that daptomycin may indeed directly interfere with cell wall synthesis or cell division, such effects still await direct experimental confirmation. Daptomycin’s complex structure and biosynthesis have hampered the analysis of its structure activity relationships. Novel methods of total synthesis, including a recent one that is carried out entirely on a solid phase, will enable a more thorough and systematic exploration of the sequence space.     

An evaluation of the P450 inhibition and induction potential of daptomycin in primary human hepatocytes

Two in vitro studies assessed the potential of daptomycin (Cubicin), a newly marketed antibiotic, to affect the cytochrome P450 (CYP450) isoforms in primary cultured human hepatocytes. Both induction and inhibition of isoforms 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 were evaluated. The highest concentrations of daptomycin used in both the induction and inhibition assays were approximately eight-fold higher than the peak total drug concentration (50-60 microg/mL), or the peak free drug concentration (estimated 5-6 microg/mL), in plasma at the clinical dose regimen of 4 mg/kg qd. Results in primary human hepatocytes indicate that daptomycin, at concentrations up to 400 microg total drug/mL, demonstrated no biologically significant induction of any of the CYP450 isoform activities in comparison with the negative control or known inducers. At daptomycin concentrations up to 40 microg free drug/mL, no biologically significant inhibition of the activities of these CYP450 isoforms was observed as compared with known inhibitors. The human hepatocyte results demonstrate that daptomycin has no effects on hepatic CYP450-mediated drug metabolism and, therefore, suggest that daptomycin is unlikely to show potential for pharmacokinetic interactions with concomitantly administered drugs that are metabolized by CYP450 isoforms.     

NMR structural studies of the antibiotic lipopeptide daptomycin in DHPC micelles

Daptomycin is a cyclic anionic lipopeptide that exerts its rapid bactericidal effect by perturbing the bacterial cell membrane, a mode of action different from most other currently commercially available antibiotics (except e.g. polymyxin and gramicidin). Recent work has shown that daptomycin requires calcium in the form of Ca2+ to form a micellar structure in solution and to bind to bacterial model membranes. This evidence sheds light on the initial steps in the mechanism of action of this novel antibiotic. To understand how daptomycin goes on to perturb bacterial membranes, its three-dimensional structure has been determined in the presence of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micelles. NMR spectra of daptomycin in DHPC were obtained under two conditions, namely in the presence of Ca2+ as used by Jung et al. [D. Jung, A. Rozek, M. Okon, R.E.W. Hancock, Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin, Chem. Biol. 11 (2004) 949-57] to solve the calcium-conjugated structure of daptomycin in solution and in a phosphate buffer as used by Rotondi and Gierasch [K.S. Rotondi, L.M. Gierasch, A well-defined amphipathic conformation for the calcium-free cyclic lipopeptide antibiotic, daptomycin, in aqueous solution, Biopolymers 80 (2005) 374-85] to solve the structure of apo-daptomycin. The structures were calculated using molecular dynamics time-averaged refinement. The different sample conditions used to obtain the NMR spectra are discussed in light of fluorescence data, lipid flip-flop and calcein release assays in PC liposomes, in the presence and absence of Ca2+ [D. Jung, A. Rozek, M. Okon, R.E.W. Hancock, Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin, Chem. Biol. 11 (2004) 949-57]. The implications of these results for the membrane perturbation mechanism of daptomycin are discussed.