海洋芽孢杆菌Bacillus methylotrophicus B-9987中difficidins生物合成基因簇的分析鉴定

目的 对海洋芽孢杆菌B-9987中difficidins生物合成基因簇及其关键基因进行分析、鉴定及功能研究。 方法 通过生物信息学手段对difficidins基因簇的基因组成和功能结构域进行了分析；结合其结构特征及聚酮化合物的生物合成原理,初步推导其生物合成途径；构建了烯酰辅酶A水合酶基因difO双交换阻断突变株,对其功能进行初步验证并对基因簇进行确认。 结果 从B-9987发酵液中分离纯化得到化合物oxydifficidin,鉴定了B-9987中该化合物的生物合成基因簇,其是由位于同一个操纵子的15个基因difA-O所编码的反式酰基转移酶聚酮合酶体系组装而成；difO基因阻断后,oxydifficidin不再产生。 结论 difficidins生物合成基因簇的鉴定为后续研究其生物合成途径及相关基因的功能奠定了基础。     

Difficidins生物合成基因簇中蛋白激酶基因difB功能研究

目的 对海洋芽孢杆菌Bacillus methylotrophicus B-9987 difficidins化合物生物合成基因簇中difB基因功能进行探究。方法 通过生物信息学手段对difB的功能进行预测;构建difB双交换阻断突变株及高表达株;通过高效液相色谱法（HPLC）分析野生株和突变株发酵产物的差异;在大肠杆菌中表达DifB蛋白,以脱磷酸difficidin（3）作为底物,探究DifB的体外功能。结果 得到了ΔdifB双交换阻断突变株及difB高表达株;HPLC分析结果表明,相比较野生株,ΔdifB突变株中difficidins化合物峰不再产生,difB高表达株中difficidins的产量与野生株无显著差别;DifB蛋白不能催化脱磷酸化difficidin（3）发生磷酸化反应。结论 difB是difficidins生物合成所必须的,可能与磷酸基团的组装相关,但磷酸化机制有待进一步研究。     

海洋芽孢杆菌B-9987中抗MDR菌活性成分研究

挖掘具有优良多重耐药菌(MDR)抗菌活性的次级代谢产物,为海洋新药研发提供分子多样性。方法 对海洋芽孢杆菌B-9987进行大发酵,将发酵产物进行提取分离,每一步分离过程进行MDR活性追踪。结果 B-9987粗提物对4株MDR菌(金黄色葡萄球菌CCARM 3090、沙门氏菌CCARM 8250、大肠杆菌CCARM 1009、粪肠球菌CCARM 5203)具有良好的抗菌活性。通过HPLC分离纯化及MS、NMR等波谱分析获得了化合物1(Macrolactin F)和化合物2(Bacillaene A)。化合物1对MDR菌没有抗菌活性。由于化合物2对光、温度、氧气十分不稳定,无法直接测试活性。对含有化合物2的组分进行活性测试,确定了B-9987的MDR菌抗菌活性来自于Bacillaene类化合物。结论 鉴定了B-9987中的抗MDR菌活性成分。     

浸麻芽孢杆菌环糊精葡糖基转移酶的克隆与表达

目的获取浸麻芽孢杆菌环糊精葡糖基转移酶基因并在大肠杆菌中表达。方法构建浸麻芽孢杆菌环糊精葡糖基转移酶表达质粒,转化大肠杆菌DH5α,筛选获得阳性重组菌株。经42℃诱导后,检测酶活性。结果浸麻芽孢杆菌环糊精葡糖基转移酶在大肠杆菌中成功表达,表达量达3 U/mL。结论浸麻芽孢杆菌环糊精葡糖基转移酶可以在大肠杆菌中高效表达。     

新型酰基转移酶AT-EF080951的重组表达及底物结合分析

目的 研究新型聚酮合酶(PKS)基因簇EF568935(Gen Bank登录号)中酰基转移酶(AT)EF080951(GenBank登录号)底物特异性,为PKS的功能研究及组合生物合成研究提供新组件.方法 从酰基转移酶AT-EF080951结构域两端的连接肽区设计引物,通过PCR克隆其结构域基因at-EF080951;连接入原核表达载体pMAL-c2X,与麦芽糖结合蛋白(MBP)融合表达;直链淀粉树脂柱亲和层析纯化MBP-AT-EF08095l;融合蛋白用Xa因子切除MBP,得到AT-EF080951单体;以MBP、小牛血清白蛋白(BSA)为对照蛋白,用紫外分光光度法测定MBP-AT-EF080951与AT-EF080951对底物的结合能力,计算结合常数(Ka,μmol/L),分析其结合底物的特异性.结果 (1)MBP-AT-EF080951与底物的结合常数为甲基丙二酰辅酶A,0.0049±0.001;丙二酰辅酶A,0.0049±0.003;乙酰辅酶A,0.107±0.002;辅酶A,0.005±0.003.(2)AT-EF080951与底物的结合常数为甲基丙二酰辅酶A,0.005±0.002;丙二酰辅酶A,0.0041±0.002;乙酰辅酶A,0.120±0.001;辅酶A,0.0042±0.003.结论 MBP-AT-EF080951和AT-EF080951都特异性结合乙酰辅酶A,AT-EF080951可能是乙酰转移酶.     

微生物次级代谢产物生物合成基因簇异源表达研究进展

微生物天然产物的异源表达在药物的发展过程中发挥着越来越大的作用。通过基因工程技术,将目的生物合成基因簇转移至不同的异源宿主中表达,不仅能够激活沉默的生物合成基因簇,而且可将异源表达体系作为一个非常有用的工具通过组合生物合成生产更多结构新颖、功能独特的化合物。本文结合当今该领域的最新研究动态,概述了基因簇异源表达宿主的选择依据及生物合成基因簇在不同宿主表达的研究进展,以期为天然产物的研究提供一定的借鉴作用。     

海洋芽孢杆菌大环内酯糖基转移酶BmmGT1底物广谱性探究

目的 对海洋芽孢杆菌B-9987中糖基转移酶BmmGT1糖基受体的广谱性进行探究。方法 以UDP-D-葡萄糖为糖基供体,与不同糖基受体进行体外酶促反应,通过高效液相色谱(HPLC)检测反应产物,并采用高分辨质谱(HRMS)手段对糖基化产物进行初步鉴定。结果 糖基转移酶BmmGT1能够以UDP-D-葡萄糖为糖基供体,识别两性霉素B以及氯霉素,分别生成两性霉素B双糖基化和单糖基化衍生物以及氯霉素单糖基化衍生物得到三个糖基化产物。结论 糖基转移酶BmmGT1具对糖基受体的选择具有灵活性,可作为潜在的工具酶用于化合物结构多样性研究。     

海洋细菌B—9987发酵条件的优化及胞外抑菌物质的理化特性

从海泥中分离获得的芽孢杆菌B-9987,其胞外代谢产物对植物病原真菌有很强的拮抗作用。以改良的2216E为基础培养基,通过正交试验,得到B-9987放大培养的最佳配方。胞外抑菌物质在碱性条件下活性大,酸性条件下具有良奶的热稳定性,且水溶性、醇溶性和脂溶性都较大。纸电泳表明此物质呈弱酸性。     

海洋放线菌Streptomyces costaricanus SCSIO ZS0073中fungichromin生物合成基因簇的分析鉴定

目的 对分离自我国广西红沙红树林湿地公园的放线菌菌株Streptomyces costaricanus SCSIO ZS0073进行基因组测序分析,并对其生产的制霉色基素(fungichromin)的生物合成基因簇进行分析鉴定。 方法 对S. costaricanus SCSIO ZS0073菌株进行基因组提取,利用Highseq4000和PacBio SMRT技术相结合的手段对该菌株进行了基因组完成图测序;利用生物信息学方法对基因组进行注释并寻找fungichromin的生物合成基因簇,对fungichromin生物合成骨架基因fgmJ1进行置换突变,确定fungichromin生物合成基因簇,结合fungichromin结构特征和其基因簇内遗传信息及聚酮化合物的生物合成原理,对其生物合成途径进行推测。 结果 全基因组测序发现S. costaricanus SCSIO ZS0073菌株基因组含有一条线性染色体和一个圆形质粒,基因组含有36个次级代谢产物生物合成基因簇。利用基因敲除手段对fungichromin的生物合成基因簇进行了确认并进行了生物合成途径推导。 结论 fungichromin生物合成基因簇的确定和生物合成途径的推导为该化合物的基因工程改造研究奠定了分子基础。     

脱氧糖组合生物合成的研究进展

天然活性物质的糖基对其发挥生物活性十分重要。目前已从许多抗生素产生菌中分离出各种糖生物合成基因簇以及糖苷转移酶基因，对其生物合成途径的认识也不断深入。近年的研究结果表明抗生素的糖合成酶和糖苷转移酶具有一定的底物宽泛性。本文在总结脱氧糖生物合成基因及糖苷转移酶基因的发展概况基础上，重点综述了运用组合生物合成技术改造脱氧糖分子结构的研究进展。     

Butirosin-biosynthetic gene cluster from Bacillus circulans

Butirosin is an interesting 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotic produced by non-actinomycete Bacilli. Recently we were successful in purification of 2-deoxy-scyllo-inosose synthase from butirosin-producer Bacillus circulans as the key enzyme for the biosynthesis of DOS, in cloning of the responsible gene (btrC), and in its overexpression in Escherichia coli. The present study involved gene-walking approach, which allowed us to find a gene cluster around btrC. The function of each gene was further investigated by gene disruption, and the disruptants of btrB, btrC, btrD and btrM showed no antibiotic producing activity. Therefore, the gene cluster found so far was determined to be a part of the butirosin biosynthetic gene cluster. Functions of some ORFs are also discussed in terms of butirosin biosynthesis on the basis of database search.     

Antibacterial activities of macrolactin A and 7-O-succinyl macrolactin A from Bacillus polyfermenticus KJS-2 against vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus

The principal objective of this study was to evaluate the antibacterial activities of macrolactin A (MA) and 7-O-succinyl macrolactin A (SMA) generated from Bacillus polyfermenticus KJS-2 against vancomycin-resistant enterococci (VREs) and methicillin-resistant Staphylococcus aureus. The minimal inhibitory concentrations (MICs) of MA and SMA against VREs were 16 and 2∼1 μg/mL, respectively, and the MICs of MA and SMA against methicillin-resistant Staphylococcus aureus were 2 and < 0.25 μg/mL, respectively. Their MIC values were comparable or superior to those of teicoplanin. In evaluating the inhibitory effects of intestinal VRE colonization in mice, the oral MA and SMA effected a rapid inhibition of intestinal VRE colonization in mice, and the intraperitoneal SMA also inhibited VRE colonization, whereas intraperitoneal MA did not.     

Prediction of the binding modes between macrolactin N and peptide deformylase from Staphylococcus aureus by molecular docking and molecular dynamics simulations

Macrolactin N is a novel lactone compound against Staphylococcus aureus peptide deformylase (PDF) with an IC₅₀ value of 7.5 μM, while its binding mode with PDF still remains largely unknown. In this study, the binding mechanism of macrolactin N to PDF was investigated using molecular docking, molecular dynamics (MD) simulations, and free energy calculations. Four typical binding modes were obtained by FlexX docking and cluster analysis, which are named as Model A, Model B, Model C, and Model D. The predicted binding free energy of Model A is more stable than those of the other three models. Besides, the free energy decomposition and structure analysis, as well as the hydrogen bond occupancy analysis further demonstrate that Model A is the most appropriate conformation for ligand binding. We found that the Zn²⁺ ion in Model A has positive contribution with the ligand, which implies the introduction of a metal chelating functional group on this model could further improve the binding affinity to PDF. The feasibility of the results of our molecular docking and MD simulation work was examined by the result about PDF-actinonin in terms of the theoretical simulation and the experimental results (e.g., crystal structure). Meanwhile, four predicted binding modes are validated by means of comparing their binding modes with actinonin, and the comparison result shows that the macrolactin N in Model A may have the highest similarity to the binding mode of actinonin. This work might be useful in designing more promising PDF inhibitors.     

Overexpression of the Trichoderma brevicompactum tri5 Gene: Effect on the Expression of the Trichodermin Biosynthetic Genes and on Tomato Seedlings

Trichoderma brevicompactum IBT 40841 produces trichodermin, a trichothecene-type toxin that shares most of the steps of its biosynthesis with harzianum A, another trichothecene produced by several Trichoderma species. The first specific step in the trichothecene biosynthesis is carried out by a terpene cylcase, trichodiene synthase, that catalyzes the conversion of farnesyl pyrophosphate to trichodiene and that is encoded by the tri5 gene. Overexpression of tri5 resulted in increased levels of trichodermin production, but also in an increase in tyrosol and hydroxytyrosol production, two antioxidant compounds that may play a regulatory role in trichothecene biosynthesis, and also in a higher expression of three trichothecene genes, tri4, tri6 and tri10, and of the erg1 gene, which participates in the synthesis of triterpenes. The effect of tri5 overexpression on tomato seedling disease response was also studied.     

A plasmid which does not encode the aminoglycoside phosphotransferase in the butirosin-producing strain of Bacillus circulans

Bacillus circulans NRRL B-3312 produces the aminoglycoside antibiotic butirosin and encodes an aminoglycoside 3'-phosphotransferase. We detected a 48 kilobase plasmid, pIP850, in this strain; this was analyzed by agarose gel electrophoresis following digestion with EcoRI restriction endonuclease and by nucleic acid hybridization. The results obtained indicate that plasmid pIP850 does not carry the structural gene for the aminoglycoside modifying enzyme.     

Rational design of macrolides by virtual screening of combinatorial libraries generated through in silico manipulation of polyketide synthases

Bacterial secondary metabolites display diverse biological activities, thus having potential as pharmacological agents. Although most of these compounds are discovered by random screening, it is possible to predict and re-design their structures based on the information on their biosynthetic pathways. Biosynthesis of macrolides, governed by modular polyketide synthases (PKS), obeys certain rules, which can be simulated in silico. PKS mode of action theoretically allows for a huge number of macrolides to be produced upon combinatorial manipulation. Since engineering of all possible PKS variants is practically unfeasible, we created Biogenerator software, which simulates manipulation of PKS and generates virtual libraries of macrolides. These libraries can be screened by computer-aided prediction of biological activities, as exemplified by analysis of erythromycin and macrolactin libraries. This approach allows rational selection of macrolides with desired biological activities and provides instructions regarding the composition of the PKS gene clusters necessary for microbial production of such molecules.