The induction of antibiotic synthesis in Saccharopolyspora erythraea and Streptomyces hygroscopicus by growth rate decrease is accompanied by a down-regulation of protein synthesis rate

Abstract The relationship between antibiotic production and culture growth rate in Saccharopolyspora erythraea and Streptomyces hygroscopicus was manipulated by changing the growth-limiting substrate. Carbon- and nitrogen-limited cultures were studied and antibiotic synthesis was obtained in both cases in Saccharopolyspora erythraea cultures and in nitrogen-limited Streptomyces hygroscopicus cultures. In all cultures where antibiotic was detected, onset of antibiotic production coincided with the minimal protein synthesis rate. Further investigation in Saccharopolyspora erythraea cultures indicated that this corresponded to minimum ratio of charged to uncharged tRNA, i.e. when uncharged tRNA accumulated. This latter phenomenon was investigated in the presence of a protein synthesis inhibitor.     

Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa

Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the spinosyn gene cluster. Duplication of the spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis — the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased spinosyn yield significantly. Journal of Industrial Microbiology & Biotechnology (2001) 27, 399–402. Received 31 May 2001/ Accepted in revised form 09 July 2001     

不同碳源对须糖多孢菌生长发育及丁烯基多杀菌素生物合成的影响

本文研究了不同碳源对须糖多孢菌生长以及丁烯基多杀菌素生物合成的影响,通过寻找优势碳源优化发酵培养基配方,促进须糖多孢菌丁烯基多杀菌素的生物合成。试验共设11个处理,1个对照,通过单因素试验比较不同处理组菌体OD600值和丁烯基多杀菌素产量,筛选获得最优碳源及其发酵培养基配方。结果表明,除可溶性淀粉和木糖外,须糖多孢菌在9种碳源中都能进行生长,对不同构型碳源显示较好的利用率。在以半乳糖、葡萄糖、果糖和甘露糖作为碳源时具有较好的生长速率,而以甘露糖为碳源时能显著促进丁烯基多杀菌素的合成。选择甘露糖最佳添加浓度为5 g/L,须糖多孢菌最高菌体浓度和丁烯基多杀菌素产量分别是初始配方条件的1. 32倍和1. 78倍,显著提高了丁烯基多杀菌素的产量。上述结果为培养基碳源对丁烯基多杀菌素生物合成影响机制的研究及丁烯基多杀菌素大规模工业化发酵生产提供了科学依据和新的技术途径。     

糖多孢红霉菌多拷贝表达载体pZM的构建

对糖多孢红霉菌染色体上红霉素生物合成基因进行改造 ,已经合成了多种红霉素类似物。在糖多孢红霉菌中对红霉素类似物进行结构修饰 ,以pWOR1 0 9质粒为基础构建糖多孢红霉菌多拷贝表达载体pZM。pZM载体带有PermE启动子、fd终止子、多克隆位点、硫链丝菌肽和氨苄青霉素抗性基因、以及在大肠杆菌和糖多孢红霉菌中复制的ColE1ori和pJV1ori复制子 ,系可在大肠杆菌和糖多孢红霉菌中扩增的穿梭质粒。在糖多孢红霉菌中 ,pZM可以表达氨普霉素抗性基因和绿色荧光蛋白基因 ,从糖多孢红霉菌中提取的表达质粒酶切图谱与转化前一致 ,表明pZM是糖多孢红霉菌中多拷贝、稳定的表达载体。     

糖多孢红霉菌同源片段长度与染色体重组率关系的研究

为了探索同源片段长度与糖多孢红霉菌染色体同源重组率的关系,化学合成或用重叠PCR合成带有突变位点、在突变位点两侧长度为（26bp+27bp）、（500bp+576bp）和（1908bp+1749bp）的同源序列,克隆于糖多孢红霉菌同源重组载体pWHM3后,分别构建了pWHM1113、 pWHM1116和 pWHM1119质粒。以PEG介导转化糖多孢红霉菌A226原生质体,3个质粒分别获得每皿30个、69个和170个转化子,但pWHM1113质粒不能与染色体有效整合,pWHM1116质粒与染色体整合率为转化子的2%,而pWHM1119质粒与染色体整合率达到转化子的19%。 pWHM1116和 pWHM1119质粒均可进行有效的染色体二次重组,将突变位位点引入染色体。因此,同源片段长度为（500bp+576bp）或更长时,可与糖多孢红霉菌染色体进行有效的单重组和双重组。     

Viability, strength, and fragmentation of Saccharopolyspora erythraea in submerged fermentation.

Two fermentations of the commercially important erythromycin-producing filamentous bacterium Saccharopolyspora erythraea were conducted in defined media. One was glucose-limited and the other nitrate-limited. The viability of the hyphae was determined using the fluorescent stain BacLight (Molecular Probes, Eugene, OR). Also, the force required to strain hyphae to breakage was determined using micromanipulation and a sensitive force transducer. In both fermentations, fragmentation coincided with the appearance of regions in the mycelia with permeabilised membranes (considered nonviable). Under glucose-limitation, hyphal breaking force rose to 1,050 +/- 130 nN at the end of the growth phase and fell to an undetectable value as a result of glucose exhaustion. Under nitrate-limitation, hyphal breaking force fell from 900 +/- 160 nN during the growth phase to 550 +/- 40 nN in the stationary phase. In both cases image analysis showed that the dimensions of mycelia were of the same order, suggesting that the major factor influencing fragmentation was the appearance of nonviable regions (assumed to be weak). The location in which nonviable regions first appear within hyphae could not be determined because of their appearance coinciding with fragmentation.     

High frequency transformation of the industrial erythromycin-producing bacterium Saccharopolyspora erythraea

The DNA transformation in the industrial erythromycin-producing Saccharopolyspora erythraea was investigated as standard protoplast transformation methods are ineffective. Intergeneric conjugal transfer of DNA from E. coli demonstrated transformation efficiencies from 0.05 × 10⁻⁸ to 7.2 × 10⁻⁸ exconjugants generated per recipient. Electroporation-mediated methodologies were also established. More than 10⁵ transformants were acquired per μg DNA. The proposed protocol provides an alternative route for the introduction of DNA into industrial strains.     

多杀菌素生产菌种复壮方法及其效应的研究

用分离纯化复壮、淘汰法复壮及虫体复壮三种复壮方法对一株多杀菌素生产能力已经衰退的刺糖多孢菌进行复壮研究。结果显示：淘汰法复壮可显著提高菌株的杀虫毒力及多杀菌素的产量。通过红霉素抗性复壮筛选得到菌株杀虫死亡率达到100％,多杀菌素相对效价达1058．83％;通过NaCl抗性复壮筛选得到一菌株杀虫死亡率达到95％,多杀菌素相对效价达825．80％,因此淘汰法可作为有效的复壮方法。     

Effects of methylmalonyl-CoA mutase gene knockouts on erythromycin production in carbohydrate-based and oil-based fermentations of Saccharopolyspora erythraea

In carbohydrate-based fermentations of Saccharopolyspora erythraea, a polar knockout of the methylmalonyl-CoA mutase (MCM) gene, mutB, improved erythromycin production an average of 126% (within the range of 102–153% for a 0.95 confidence interval). In oil-based fermentations, where erythromycin production by the wild-type strain averages 184% higher (141–236%, 0.95 CI) than in carbohydrate-based fermentations, the same polar knockout in mutB surprisingly reduced erythromycin production by 66% (53–76%, 0.95 CI). A metabolic model is proposed where in carbohydrate-based fermentations MCM acts as a drain on the methylmalonyl-CoA metabolite pool, and in oil-based fermentations, MCM acts in the reverse direction to fill the methylmalonyl-CoA pool. Therefore, the model explains, in part, how the well-known oil-based process improvement for erythromycin production operates at the biochemical level; furthermore, it illustrates how the mutB erythromycin strain improvement mutation operates at the genetic level in carbohydrate-based fermentations.