黑暗链霉菌安普霉素生物合成基因aprG的克隆和功能研究

从安普霉素产生菌S．tenebrarius H6总DNA中已克隆得到8．2kb的安普霉素抗性基因连锁片段。对距离抗性基因下游4．5kb的SacI-Sau3AI片段进行测序分析，发现了一个新的891bp开放阅读框架。推测其编码为296个氨基酸的蛋白质。经BLASTX程序分析，没有发现与之同源的基因，是一个新基因，该基因命名为aprG。构建基因阻断穿梭载体pSPU58，经接合转移导入S．tenebrarius H6，筛选单交换阻断变株，并用Southern blot验证阻断变株的aprG已经被破坏。经发酵分析，阻断变株不再合成安普霉素，表明aprG与安普霉素生物合成直接相关。     

利用基因工程技术选育氨甲酰妥布霉素高含量菌种

目的以产生安普霉素、氨甲酰妥布霉素和氨甲酰卡那霉素3个组分的黑暗链霉菌为出发菌株,利用基因工程技术选育氨甲酰妥布霉素含量高的菌种。方法克隆安普霉素生物合成基因,通过基因阻断技术破坏安普霉素生物合成基因,阻断安普霉素生物合成。结果获得了氨甲酰妥布霉素含量高的菌种。结论利用基因工程技术可以有效地改良抗生素组分。     

反相高效液相谱型分析检测吸水链霉菌17997变株发酵新产物

以吸水链霉菌17997为出发菌株。分别阻断格尔德霉索（geldanamycin，GA）生物合成酶基因簇中的Ⅰ型聚酮合酶（type Ⅰ polyketide synthase，pks）基因的第6模块，单加氧酶（monooxygenase，gdmM）基因和氨甲酰基转移酶（carbamoyltransferase，et）基因得到3种变株（pks^-）、（gdmM^-）和（ct^-）。比较变株与原株发酵产物的HPLC谱型，结合紫外吸收图谱分析。检测结果表明各变株的GA生物合成均被阻断。并产生3个不同于原始菌株的新物质。这证明基因操作所涉及的pks，gdmM和ct基因是GA生物合成的必需基因；这些基因的阻断可产生不同于原株的新物质。HPLC谱型比较可以在多样化代谢产物的产生菌中快速、准确地发现基因工程变株发酵产物的变化。指导新化合物的分离鉴定，样品用量甚微。是化学早期鉴别的有力工具。     

4,5-双氢-7-去氨甲酰基-7-羟基-19-S-甲基格尔德霉素及其与格尔德霉素生物合成PKS后修饰的关系

目的在吸水链霉菌17997格尔德霉素生物合成PKS后修饰gdmN-变株发酵产物中寻找新格尔德霉素衍生物。方法 gdmN-变株发酵上清液乙酸乙酯提取后,进行硅胶板TLC分析;对一个红色目标化合物进行了HPLC和高分辨质谱分析,并对其进行了1H-和13C-核磁共振(NMR)分析;对红色目标化合物在吸水链霉菌17997格尔德霉素PKS基因阻断变株中进行了生物转化实验。结果在gdmN-变株发酵产物中发现了1个预期的红色目标化合物(4,5-双氢-7-去氨甲酰基-7-羟基-19-S-甲基格尔德霉素);该目标化合物可被格尔德霉素生物合成PKS后修饰系统7-O-氨甲酰化,生成4,5-双氢-19-S-甲基格尔德霉素。结论在gdmN-变株中发现了4,5-双氢-7-去氨甲酰基-7-羟基-19-S-甲基格尔德霉素;该化合物可被格尔德霉素PKS后修饰系统继续7-O-氨甲酰化,但不能C-4,5氧化。     

井冈霉素及其分解产物的开发利用

井冈霉素可酶解为井冈霉亚基胺A、井冈胺和井冈霉胺。井冈霉亚基胺A是昆虫的海藻糖酶抑制剂,可开发为生物杀虫剂;井冈胺和井冈霉胺是糖苷酶抑制剂,是合成其他新酶抑制剂类降糖药的重要医药中间体。本文就井冈霉素的高附加值产品井冈霉亚基胺A、井冈胺和井冈霉胺的结构、特性以及制备方法作一综述。     

2-脱氧链霉胺类抗生素生物合成基因的研究进展

含2-脱氧链霉胺类（2-DOS）氨基糖苷抗生素是临床常用抗生素。近几年对2-脱氧链霉胺类抗生素的分子水平研究取得重大进展，得到了2-DOS合成中的关键酶基因，以及丁酰苷菌素、妥布霉素、卡那霉素、庆大霉素等部分生物合成基因簇克隆。本文对2-脱氧链霉胺类抗生素生物合成基因研究进展进行了综述。     

庆大霉素C_(1a)单组份产生菌─—棘孢小单孢菌JIM-202的筛选和研究

利用紫外光和氯化锂复合因子处理小诺霉素产生菌——棘孢小单孢菌JIM－401获得了JIM－202突变株，其发酵产物经阳离子交换树脂提取、洗脱、脱色、浓缩和经冷冻干燥可得白色粉末。用薄层层析检验其发酵产物为1个单组份；通过红外光谱、紫外光谱、核磁共振谱及质谱测定表明该抗生素与庆大霉素C1a同质，因而可用其进行新衍生物合成（如新抗生素89－07）。根据小诺霉素和庆大霉素生物合成途径推测，该突变株在最后一步的6’－N甲基化部位发生了阻断所致。     

2，4－二硝基氟苯柱前衍生化反相快速HPLC测定核糖霉素

本文选用２，４－二硝基氟苯（ＤＮＦＢ）柱前衍生化，二极管阵列紫外检测器检测，建立了核糖霉素及杂质新霉胺的反相快速ＨＰＬＣ测定方法。按ＵＳＰ（ＸＸⅡ）版附录分析方法评价的要求，对本方法的重现性、灵敏度、准确度、线性范围等指标作考察，结果满意。因采用高效细颗粒短柱进行分离，整个分析时间于５ｍｉｎ内，实现了高效快速测定。运用所建立的分离测定方法，对硫酸核糖霉素原料药及注射液中核糖霉素及新霉胺的含量分别进行了测定。     

两种小单孢菌的障碍突变株混合发酵时的抗生素生物合成

小单孢菌 M.purpurea SC1121和 M.inyoensis SC 1550F 均系2-脱氧链霉胺(2-DOS)阴性突变株,且在生物合成氨基环醇的不同阶段发生障碍。它们在单独培养时都不能产生抗生素。在有外源的2-DOS 存在时,突变株1121的主要产物为艮他霉素 C_(1a),并产生少量的艮他霉素 A、X_2与 C_(2b)。在同样条件下,突变株1550F 产生紫苏霉素(Sisomicin)和少量极性较强     

新型强效免疫抑制剂雷帕霉素的生物合成

雷帕霉素是吸水链霉菌产生的新型强效免疫抑制剂,本文综述了雷帕霉素的生物合成,探讨了雷帕霉素构成单元、相关酶及雷帕霉素效价三者之间的关系,指出以雷帕霉素生物合成相关酶或前体含量为指标可能是筛选雷帕霉素高产突变株的又一途径。     

Biosynthesis of astromicin and related antibiotics. I. Biosynthetic studies by bioconversion experiments

Biosynthesis of astromicin, a unique pseudodisaccharide aminoglycoside antibiotic containing 1,4-diaminocyclitol component, was investigated by isolating a variety of possible precursor compounds from mutants of Micromonospora olivasterospora in which biosynthetic pathways for astromicin were blocked. Washed mycelia of M. olivasterospora mutants converted these compounds to astromicin, which was detected by thin-layer chromatography. Since astromicin possesses one glycyl and three methyl groups, [14C]glycine and [14C]methionine should be incorporated into precursors to form astromicin. To confirm the biosynthetic pathway, formation of labeled astromicin from the precursors was examined using [1-14C]-glycine or [methyl-14C]methionine. From above results, we propose the biosynthetic pathway for astromicin as shown in Fig. 2.     

Nitrogen incorporation in the biosynthetic pathway of the nitrogen-containing polyketide, pamamycin in Streptomyces alboniger

The biosynthetic pathway of pamamycin (1), a nitrogen-containing polyketide, was investigated using blocked mutants of Streptomyces alboniger. Hydroxy acids K (3), L (4) and S (5) were found in cultured materials of blocked mutants and the wild type strain, but no PM-ketone (2) was detected. Hydroxy acids 3, 4, 5 and de-N-methylhydroxy acid L (7) were converted into 1, but 2 nor de-N-methylpamamycin (6) were not. We also confirmed that 3 and 7 were converted into 4. These results showed that an amino group was introduced into the carbonyl group of 3 by transamination, and subsequent N-methylation led to 4 in the pamamycin biosynthetic pathway. Quantitative analyses of hydroxy acid intermediates 3, 4, and 5, and pamamycin (1) suggested that transamination was the rate-determining step in pamamycin biosynthesis.     

Biosynthesis of kalafungin in Streptomyces tanashiensis

The mutants of Streptomyces tanashiensis strain Kala, which were specifically blocked in the synthesis of the benzoisochromanequinone antibiotic kalafungin, were isolated and classified into seven phenotypic classes on the basis of the antibiotic activity and cosynthetic properties. The polarity of cosynthetic reactions and the production of kalafungin by a converter strain showed that the seven mutant classes could be arranged in the most probable linear sequence of biosynthetic blocks. Since kalafungin, which closely resembles the undimerized form of actinorhodin, was accumulated in one of the biosynthetically blocked mutants of the actinorhodin-producing Streptomyces coelicolor A3(2), the cosynthesis between kalafungin-nonproducing mutants of S. tanashiensis and actinorhodin-nonproducing mutants of S. coelicolor was performed. The results of these experiments showed that the early steps in kalafungin biosynthesis in S. tanashiensis and actinorhodin biosynthesis in S. coelicolor were similar, but the entire biosynthetic pathway of kalafungin in these two streptomycetes was not identical.     

Biosynthesis of 3'-deoxy-carbamoylkanamycin C in a Streptomyces tenebrarius mutant strain by tacB gene disruption

Streptomyces tenebrarius H6 mainly produces three kinds of antibiotics: apramycin, carbamoyltobramycin and some carbamoylkanamycin B. In our present study, a dehydrogenase gene tacB in the tobramycin biosynthetic gene cluster was disrupted by in-frame deletion. The result of TLC bio-autograph analysis demonstrated the disruption mutant strain produced apramycin and a new antibiotic. The new antibiotic was identified as 3'-deoxy-carbamoylkanamycin C by MS and NMR analysis after isolation and purification. The disruption mutant was restored to produce carbamoyltobramycin in a complementation experiment by the intact tacB gene. Our studies suggested that the tacB gene encodes a 6'-dehydrogenase, which reduces the 6'-hydroxyl group of paromamine to a keto group, thus facilitating the transfer of an aminogroup to form neamine. This study is the first report on the generation of a tobramycin derivative by gene engineering, and will contribute to clarify the complete biosynthetic pathway of tobramycin.     

Biosynthesis of astromicin and related antibiotics. II. Biosynthetic studies with blocked mutants of Micromonospora olivasterospora

An inosamine-idiotrophic mutant, KY11559, which produced no astromicin unless scyllo-inosamine was added to the fermentation medium, was isolated from Micromonospora olivasterospora. Biotransformation studies were performed with resting cells of this mutant and compounds assumed to be precursors of 1,4-diaminocyclitol (fortamine). Scyllo-inosose, scyllo-inosamine and FU-10 were converted to astromicin. A number of mutants blocked in the biosynthesis of astromicin were developed from M. olivasterospora, and the intermediates accumulated by these mutants were isolated and identified. Twenty-five blocked mutants were classified into 10 groups, based on their complementation patterns by cosynthesis experiments. Further, utilizing these blocked mutants and the isolated compounds, biotransformation analyses were performed. The results showed that the amination at position 4 in fortamine occurred after formation of the pseudodisaccharide. Subsequently, the aminosugar and aminocyclitol moieties were aminated, methylated, dehydroxylated, epimerized and acylated to produce astromicin. Thus it was demonstrated that the astromicin biosynthetic pathway has a unique feature which is not found in the biosynthesis of other aminoglycoside antibiotics.     

Studies on the biosynthesis of basic 16-membered macrolide antibiotics, platenomycins. IV. Biosynthesis of platenomycins.

To elucidate the biosynthetic pathway of platenomycin (PLM), biosynthetic relationships of platenolides I (PL-I) and II (PL-II), 3-O-propionyl-5-O-mycaminosyl platenolides I (PPL-I-MC) and II (PPL-UU-MC), 9-dehydro demycarosyl platenomycin (DDM-PLM) were examined with growing cultures or the washed mycelium of blocked mutants of Streptomyces platensis subsp. malvinus MCRL0388, a platenomycin-producing organism. As a result, it was revealed that PLM was biosynthesized from PL-I via DM-PLM and DA-PLM along the pathways shown in Chart 1. 4'-Isovaleroyl unit of PLM-A and 4'-propionyl unit of PLM-B were respectively derived from L-leucine and L-isoleucine.     

Biosynthesis of hibarimicins. II. Elucidation of biosynthetic pathway by cosynthesis using blocked mutants

The biosynthetic pathway of hibarimicin (HBM) was proposed on the basis of the experimental results obtained by using blocked mutants of Microbispora rosea subsp. hibaria TP-A0121, the HBM producer. In its biosynthesis, the oxidative coupling of the aromatic undecaketide unit generates a symmetrical aglycon HMP-Y1 (hibarimicin-mutant product Y1), which is oxidatively modified to hibarimicinone, the HBM aglycon. The following glycosylation of hibarimicinone gives rise to the HBM complex. We identified that HMP-Y1 prepared by methanolysis of HMP-Y6, a glycosylated metabolite from a blocked mutant, was the key intermediate: transformation of 13C-labeled HMP-Y1 to HBM B was confirmed by NMR measurements. Mutant strain produced another type of aglycon HMP-P1 in which the coupled polyketide units were intramolecularly bridged by the ether bond. This metabolite also arose by the spontaneous elimination of methanol molecule from hibarimicinone.     

Replacement of Streptomyces hygroscopicus genomic segments with in vitro altered DNA sequences

We have developed a method for gene replacement in Streptomyces hygroscopicus which permits introduction of an in vitro derived mutation carried on a plasmid into the chromosome. We constructed the plasmid pMSB212 which can replicate in S. hygroscopicus and contains the step5 gene of the bialaphos biosynthetic pathway which was inactivated by a frame-shift mutation caused by filling in the cohesive ends of the EcoR I site in the structural gene. pMSB212 was introduced into a bialaphos producer strain and by protoplast regeneration of the primary thiostrepton-resistant transformants, non-producing mutants, were obtained. Biochemical and genetical analyses indicated that these mutants were specifically blocked by introduction of the frame-shift mutation in the step5 gene on the chromosome. This method will enable us to obtain isogenic mutants of known genes and to identify new genes encoded on a cloned fragment.     

Studies on the biosynthesis of basic 16-membered macrolide antibiotic, platenomycins. I. Selection of and cosynthesis by non-platenomycin-producing mutants.

In a search for blocked mutants which may produce a biosynthetic intermediate, mutation by N-methyl-N'-nitro-N-nitrosoguanidine treatment and/or ultra-violet irradiation were performed on platenomycin-producing Streptomyces platensis subsp. malvinus MCRL 0388 (NRRL 3761). Twenty four non-platenomycin-producing stable mutants were thus obtained and tested for cosynthesis ability. Antibiotic cosynthesis with a pair of these mutants made it possible to detect the producer of an intermediate. Among these mutants which were classified into eight groups (A to G and doubtful groups), mutants of groups A and B appeared from their complementation pattern to be the useful producers of biosynthetic intermediates of platenomycin.