PS-5, a new beta-lactam antibiotic. I. Taxonomy of the producing organism, isolation and physico-chemical properties.

Antibiotic PS-5 is a new beta-lactam antibiotic isolated from fermentation broths of Streptomyces sp. strain A271. The strain was considered to be a new subspecies of Streptomyces cremeus and the name, Streptomyces cremeus subsp. auratilis, was proposed. Fermentative production, isolation and physico-chemical properties of PS-5 are described.     

Cloning and expression of the FR901379 acylase gene from Streptomyces sp. no. 6907

FR901379 acylase, an enzyme that catalyzes the hydrolysis of the palmitoyl moiety of the antifungal lipopeptide FR901379, was purified from the culture broth of Streptomyces sp. no. 6907 (FERM BP-5809), revealing the 80?kDa, two-subunit heterodimeric protein characteristic of the β-lactam acylase family. Using oligodeoxyribonucleotide primers constructed on the basis of the N-terminal amino acid sequence of each purified subunit, the gene was identified from a cosmid library of Streptomyces sp. no. 6907 DNA. The deduced 775 amino acid sequence corresponded to a single polypeptide chain containing two subunits, and it shared 41.7% identity with aculeacin A acylase from Actinoplanes utahensis NRRL12052. FR901379 acylase activity was found to be 250-fold higher in the recombinant Streptomyces lividans 1326 carrying the cloned gene than in the original Streptomyces sp. no. 6907 strain.     

PPtase高表达菌株Streptomyces ghanaensis的发酵及其产物cytoxazone的发现

天然产物是新药发现的重要源泉。然而，由于大多数天然产物的生物合成途径在实验室培养的情况下处于沉默状态，因此需要对其生物合成调控环节进行干预，从而使微生物激活其自身的生物合成。在前期工作中，本研究发展了一种高效激活沉默天然产物的方法。通过在微生物体内过表达磷酸泛酰巯基乙胺基转移酶(phosphopantetheinyl transferase, PPtase)，可以高效激活一系列沉默天然产物的生物合成。基于该工作基础，本研究从一株PPTase过表达的嘎纳链霉菌Streptomyces ghanaensis CGMCC 4.1967中，发现了一个新的激活天然产物。经分离后通过结构鉴定该产物为Th2细胞Ⅱ型细胞因子细胞特异性抑制剂cytoxazone。Cytoxazone此前是在一株分离自广岛的链霉菌(Streptomyces sp.)的发酵液中发现，其在嘎纳链霉菌中为首次发现。本研究的结果证实了S. ghanaensis CGMCC 4.1967可以产生cytoxazone。这部分工作将对阐明此类重要天然产物的生物合成机制，并对其进行合成生物学改造以获得更好活性的细胞因子抑制剂建立基础。     

委内瑞拉链霉菌中氯霉素生物合成基因的克隆

目的克隆委内瑞拉链霉菌(Streptomyces venezuelae)ISP5230中的氯霉素生物合成基因。方法以pabAB基因片段为探针,通过菌落杂交技术从该菌的基因文库中钓取相关基因。结果得到一7.5kbBamHIBamHI DNA片段,突变株的互补实验表明,该7.5kb DNA片段使氯霉素生物合成阻断变株CML-4恢复了氯霉素的生物合成。从野生菌株的染色体上删除该7.5 kb DNA片段中的一段3.5 kbNcoINcoI DNA片段后,野生菌株丧失了氯霉素的生物合成能力。结论该7.5 kb DNA片段含有氯霉素生物合成所需的基因。     

Genetic and biochemical studies on the regulatory mechanism of the self-resistance and biosynthesis of antibiotics

The following topics are described: 1. chemistry of beta-lactamases; 2. beta-lactamases from Streptomyces including distribution of beta-lactamases in actinobacteria, properties of beta-lactamases from Streptomyces, cloning and regulatory mechanism of expression of beta-lactamase genes from Streptomyces and evolution and classification of beta-lactamases in general; 3. penicillin-binding proteins from Streptomyces including beta-lactam-producing- and non-producing strains and 4. eukaryotic-type protein kinases from Streptomyces including cloning of the genes and evolution and classification of eukaryotic-type protein kinases in general.     

新型强效免疫抑制剂西罗莫司F904的研究

从福建省福州市的土壤中分离到的链霉菌 FC90 4 ,经分类鉴定、菌株培养确定为吸水链霉菌。其发酵代谢产物 F90 4具有抗真菌、抗增殖和比环孢素强的免疫抑制活性。分离、纯化、结晶 ,得 F90 4 ,经理化性质、光谱分析和 X-射线单晶衍射研究证明 F90 4为三烯大环内酯新型强效免疫抑制剂西罗莫司。     

头状链霉菌中丝裂霉素C抗性基因mcrAB的克隆及其作用研究

丝裂霉素 C属于苯醌类抗肿瘤抗生素 ,在生物体内作为前药 ,经酶催化还原后可阻碍 DNA的复制。我们从头状链霉菌中克隆出丝裂霉素 C抗性基因 mcr AB,测序结果与另一丝裂霉素产生菌—淡紫灰链霉菌的 mcr AB基因序列进行了比较 ,结果表明此序列是高度保守的。同时还构建了带有 mcr AB基因的质粒载体 p YG2 0 6 ,将 p YG2 0 6用原生质体转化法导入变青链霉菌 TK6 4 ,并在此菌中表达 ,大大提高了该菌对丝裂霉素 C的抗性。进一步研究表明 :在有氧条件下 ,mcr AB基因表达的蛋白可抑制菌体对丝裂霉素 C的转化作用。     

他克莫司产生菌的选育和生产工艺研究

产他克莫司的原始菌株Streptomyces hzsw-1-25经紫外、60Co、HNO2等方法诱变,筛选得高产突变菌株hzsw-8-1123,优化发酵培养基后,进行摇瓶和2m3罐的发酵试验,并研究了产物的分离提取工艺.     

Exploiting Streptomyces coelicolor A3(2) P450s as a model for application in drug discovery

One of the surprising discoveries about the genomics of the cytochrome P450 (CYP) superfamily is the large number of CYPs in the bacterial class of actinomycetes. It had previously been imagined that bacteria have small numbers of CYPs or none at all. Particularly intriguing is that the bacterial genus Streptomyces, which produce a large number of secondary metabolites with important medical application, has a large CYP complement reflecting the ecological niche that the organism finds itself in. In 2001 the first complete Streptomyces species genome (Streptomyces coelicolor A3[2]) was published, revealing the presence of 18 CYP genes. Subsequently, genomes for Streptomyces avermitilis, with 33 CYPs, and Streptomyces peucetius, with 15 CYPs, have been reported. Although a certain number of these CYPs have known functions in secondary metabolism, as identified biochemically or through gene locus organisation, in the vast majority of Streptomyces species, CYP functions are unknown. The first detailed analysis of the CYP complement from a Streptomyces species genome has begun in the laboratories of Waterman et al. The long-term goal of this effort is to identify orphan CYP function, to establish their high resolution structure and to establish a strategy for producing novel secondary metabolites that have new biomedical function. This chapter provides an overview of CYP systems in Streptomyces species and provides a plan of how new drugs might be generated from streptomycetes by modifying the structure of specific CYPs.     

Cloning of midecamycin(MLS)-resistance genes from Streptomyces mycarofaciens, Streptomyces lividans and Streptomyces coelicolor A3(2)

DNA containing genes for midecamycin(Mdm)-resistance was cloned from Streptomyces mycarofaciens ATCC 21454 (mdmA gene), Streptomyces lividans 66 (lrm gene) and Streptomyces coelicolor A3(2). The phenotype imparted to S. lividans and Streptomyces griseofuscus transformants by the cloned DNA segments indicates that they encode an MLS-type of resistance activity. The mdmA and lrm genes could be distinguished by the phenotype they conferred in S. lividans and S. griseofuscus, whereas the S. lividans lrm and S. coelicolor MLS genes appears to be identical on the basis of their restriction maps and behavior in S. lividans and S. griseofuscus. The DNA sequence of a 1.4-kb BamH I DNA fragment containing the mdmA gene indicates the presence of one complete orf whose deduced product exhibits a high similarity to the deduced product of the Streptomyces thermotolerans carB gene and several other bacterial MLS-resistance genes.     

紫红路南菌素A和B产生菌的分类、发酵和生物学特性研究

从我国云南省路南县采集的土壤样品中分离到一株链霉菌,编号为SIIA76-1289。它的孢子丝卷曲或螺旋,孢子球形或椭园形,表面带刺。经形态、培养特征和生理生化特性的研究和比较,认为SIIA76-1289菌株与紫色链霉菌(Streptomyces violaceus)基本相似。命名为紫色链霉菌路南变种(Streptomyces violaceus var.lunanensis var.nov.)。所产生两个蒽环类抗生素A和B,能够抗革蓝氏阳性细菌。在体外对白血病P388细胞DNA的合成有很强抑制作用。     

几种调控基因对除虫链霉菌工业生产株的抗生素效价的影响

在模式链霉菌（如天蓝色链霉菌和变铅青链霉菌）中导入许多抗生索生物合成的调控基因可以大幅度提高抗生索的含量。本文报道利用链霉菌的整合质粒克隆几种已知的调控基因。并通过接合转移从大肠埃希菌中导入产生avermectin和多拉菌素的除虫链霉菌工业生产菌株中。发现3种调控基因afiR、aveR和orfX对菌株MMR630中avermectin的含量均可以提高约1倍。但是，以上的3种，加上另外3种调控基因分别导入菌株G11后，发现除aft8提高约13％外，其余调控基因使菌株产生多拉菌素的含量反而有不同程度的降低。将调控基因币B置于链霉菌强启动子PerrnE^＊下表达降低了菌株G11中多拉菌素的含量。上述结果表明，调控基因对不同链霉菌的抗生素生物合成具有不同的影响，反映了抗生素生物合成确实受到了复杂网络的调控。     

受SnpR激活的snpA启动子调控的柔红霉素C-14羟化酶基因的克隆、表达和应用研究

尝试将柔红霉素经微生物转化法合成多柔比星。从Streptomyces sp．C5中通过PCR的方法扩增了含核糖体结合位点、大小为1.3kb的编码C-14柔红霉素羟化酶的doxA基因及受SnpR调控的snpA启动子。最终构建的重组质粒pYG908，能表达一大小为46.6ku的蛋白条带，CO结合差光谱分析表明所表达的酶在450nm有吸收峰。重组质粒转化Streptomyces lividans TK24后，工程菌能转化柔红霉素生成和多柔比星保留时间一样的产物。     

海洋链霉菌211726代谢产物的研究

目的对来自海洋环境的链霉菌211726(Streptomyces sp.211726)的代谢产物进行分离鉴定。方法采用大孔吸附树脂和硅胶柱色谱对链霉菌211726(Streptomyces sp.211726)的代谢产物进行分离,并通过理化性质及波谱学手段对所得化合物进行结构鉴定。结果与结论分离并鉴定了2个化合物:Nocardamine(1)及其铁络合物Ferrioxamine E(2),化合物2为首次从链霉菌中分得,化合物1、2为首次同时从同一微生物中分离获得。     

Studies on the biosynthesis of carbapenem antibiotics. II. Isolation and functions of a specific acylase involved in the depantothenylation of the OA-6129 compounds

A specific acylase designated A933 acylase was isolated and purified to 90% protein homogeneity from Streptomyces fulvoviridis A933 17M9 which produces PS-5, epithienamycins A and C and MM 17880 together with minor carbapenem analogs, penicillin N and cephamycin C. This enzyme was found to catalyze the depantothenylation of OA-6129 carbapenems; the acyl exchange of OA-6129 carbapenems with acyl CoA's; the deacetylation of N-acetyl-L-amino acids; and the acylation of NS-5 and 6-aminopenicillanate with acyl CoA's, whereas the deacetylation of PS-5 and N-acetyl-D-amino acids; and the deacylation of benzylpenicillin and cephalosporin C were not observed. Similar enzyme activities were also detected in Streptomyces cattleya, Streptomyces cremeus subsp. auratilis and Streptomyces argenteolus which are all carbapenem producers.     

Molecular cloning of a xylanase gene from Streptomyces sp. No. 36a and its expression in streptomycetes

The gene for an extracellular xylanase from Streptomyces sp. No. 36a was cloned into Streptomyces lividans TK21 using pIJ702 as a vector plasmid. The smallest DNA fragment encoding the xylanase gene and its possible promotor was determined to be a 1.04 kb Sph I-Sac I fragment by sub-cloning studies. This xylanase gene fragment was transferred into the pSK2 series of plasmids and introduced into Streptomyces kasugaensis G3 protoplasts. The cloned xylanase gene was expressed in both S. lividans TK21 and S. kasugaensis G3, and these clones produced and secreted high yields of xylanase into the culture medium. The xylanase production was not detected when a foreign DNA fragment was inserted into the Bcl I site locating in the xylanase gene fragment.     

一株抗耐甲氧西林金黄色葡萄球菌(MRSA)的海洋放线菌的鉴定及系统发育分析

从海南三亚海域的海绵中分离到一株抗耐甲氧西林金黄色芎芍merhieillin-resistant Staphylococcus aureus(MRSA)的海洋放线茵A115。该菌株在燕麦片琼脂上插片培养孢子丝螺旋形，孢子长圆形，表面有稀疏的短刺；菌株细胞壁含LL—DAP，糖型C；16S rDNA序列分析及系统发育分析表明，分离菌株A115与Streptomyces albogriseolus属同一分支．同源性为99．0％．但形态学、生理生化鉴定及活性情况存在一些差异。由此推断菌株A115为S．albogriseolus的海洋变种．命名为Streptomyces albogriseolus var．marine。     

Phenacein--an angiotensin-converting enzyme inhibitor produced by a streptomycete. II. Isolation, structure determination and synthesis

Phenacein, an inhibitor of angiotensin-converting enzyme, has been isolated from the fermentation broth of a Streptomyces species belonging to the Streptomyces tanashiensis-zaomyceticus group. The inhibitor was shown to be 3,6-dihydroxy-1-phenazinecarboxylic acid by spectroscopic, degradative and synthetic methods.     

Cloning from Streptomyces cellulosae of the gene encoding beta-lactamase, a blue-dextran binding protein.

A beta-lactamase gene was cloned from Streptomyces cellulosae as a 2.3-kb DNA fragment using Streptomyces lividans 1326 and PIJ385 as a host-vector system. During the course of cloning, a part of the chromosomal DNA fragment cloned together with a part of the vector plasmid were deleted, indicating instability of this contiguous DNA region. The enzyme from the clone showed similar properties with respect to binding of blue dextran and isoelectric point to the enzyme from S. cellulosae. The cloned gene hybridized not only to DNA of S. cellulosae, the source of DNA, but also to DNAs of several Streptomyces species, irrespective of their formation of beta-lactamase. These results suggest that this gene may have homology to genes other than the one for beta-lactamase.