A-factor and streptomycin biosynthesis inStreptomyces griseus

Accumulating data have shown that the metabolites with a -butyrolactone ring functions as an autoregulatory factor or a microbial hormone for the expression of various phenotypes not only in a variety ofStreptomyces spp. but also in the distantly related bacteria. A-factor, as a representative of this type of autoregulators, triggers streptomycin biosynthesis and cellular differentiation inStreptomyces griseus. A model for the A-factor regulatory cascade on the basis of recent work is as follows. At an early step in the A-factor regulatory relay, the positive A-factor signal is first received by an A-factor receptor protein that is comparable in every aspect to eukaryotic hormone receptors, and then, via one or more regulatory steps, transmitted to an A-factor-responsive protein that binds to the upstream activation sequence of thestrR gene, a regulatory gene in the streptomycin biosynthetic gene cluster. The StrR protein thus induced appears to activate the other streptomycin biosynthetic genes. This review summarizes the characteristics of A-factor as a microbial hormone and the A-factor regulatory relay leading to streptomycin production.     

Physiological factors affecting streptomycin production by Streptomyces griseus ATCC 12475 in batch and continuous culture

Abstract Conditions of growth are described for the production of streptomycin by Streptomyces griseus ATCC 12475 using chemically defined minimal medium and complex medium. It was found using batch cultures that early synthesis of the antibiotic occurred during growth in minimal medium but was delayed until the onset of stationary phase in complex medium. This effect was independent of whether spores or vegetative cells were used as inoculum. Stability of streptomycin biosynthesis in continuous culture was dependent on dilution rate and medium employed. Cultures were highly unstable when grown on complex medium but could be maintained in steady states in continuous culture using minimal medium when the dilution rate was increased in a stepwise manner, starting at a dilution rate of 0.02 h⁻¹ (15% of μ _max). The effect of changing dilution rate on growth, streptomycin production and the level of streptomycin phosphotransferase was examined using this technique.     

灰色链霉菌胰蛋白酶在变铅青链霉菌中的异源表达及酶学性质分析

胰蛋白酶作为一种重要的丝氨酸蛋白酶被广泛应用于食品、医药和皮革等工业领域.本文成功实现了灰色链霉菌来源的胰蛋白编码基因在变铅青链霉菌中的高效活性表达,并对其酶学性质进行分析比较.以灰色链霉菌ATCC10137基因组为模板,获得胰蛋白酶编码基因sprT并克隆至表达质粒pIJ86,成功构建了重组链霉菌工程菌TK24/pIJ86-sprT.以R2YE和SELF为发酵培养基,最高酶活分别达9.21 U/mL和8.61 U/mL.酶学性质分析表明,和牛胰蛋白酶(BT)相比,重组链霉菌胰蛋白酶(rSGT)的耐酸能力强,具有较广的pH;且rSGT对酰胺键具有更高的特异性;此外,Zn2+和有机溶剂分别对rSGT的酯酶活力和酰胺酶活力具有促进作用;本研究结果为rSGT的性质改造以及工业应用提供了依据.     

Transformation using in vivo and in vitro methylation in Streptomyces griseus

Streptomyces griseus does not readily take up foreign DNA isolated from other Streptomyces species or Escherichia coli, presumably due to its unique restriction-modification systems that function as a barrier for interspecific DNA transfer. To efficiently transform S. griseus by avoiding the restriction barriers, we methylated incoming DNA in vivo and in vitro and treated protoplasts with heat prior to transformation. Whereas heat treatment of protoplasts or methylation of the E. coli-Streptomyces shuttle vectors (pXE4 and pKK1443) did not prominently improve the transformation efficiency, HpaII methylation of the vectors from any E. coli strains tested in this study highly increased the transformation efficiency. The highest transformation efficiency was observed when the shuttle vectors were isolated from the dam, hsd strain of E. coli (GM161) and methylated by AluI and HpaII methyltransferases, and the efficiency was approximately the same as that of the vectors from S. griseus. We identified several restriction-modification systems that decrease the transformation efficiency. This research also led us to understand methylation profiles and restriction-modification systems in S. griseus.     

棘孢小单孢菌和灰色链霉菌原生质体融合的研究

用庆大霉素产生菌——棘孢小单孢菌Micromonospora echinospora 814(Gm~r,Km~r)和链霉素产生菌Streptomyces griseus No. 45(Sm~r,Lm~r)进行了原生质体融合。以抗性为选择标记,选出了融合体。其融合频率在10~(-3)—10~(-4)之间。在电镜条件下,观察了原生质体融合的详细过程,测定了融合体的产抗生素能力,其中一株融合体F106的抗生素产量比亲本菌株814高58％。用羧甲基纤维素薄层对发酵液层析表明,有一个融合体的发酵液比亲本菌株814多一个组份,但没有测出其生物活性。     

In vitro protein phosphorylation associated with cellular differentiation of Streptomyces griseus

In vitro phosphorylation reactions with crude cellular extracts revealed that phosphorylation of a 17-kDa protein is associated with the onset of aerial mycelium formation in solid culture (but not submerged spore formation in liquid culture) of Streptomyces griseus. The possible importance of the 17-kDa protein phosphorylation in cellular differentiation was further indicated by inducing aerial mycelium formation in the presence of decoyinine and in studies using certain developmental mutants (relC, afsA, and M-1). It is proposed that the 17-kDa protein may play a role in cellular differentiation of S. griseus via its phosphorylation. Received: 17 July 1997 / Accepted: 20 September 1997     

Proteins that interact with GTP in Streptomyces griseus and its possible implication in morphogenesis

Abstract By cross-linking with [α-³²P]GTP or [γ-³²P]GTP with or without UV treatment, several proteins of Streptomyces griseus were shown to interact with GTP in specific ways. After gel electrophoresis, 19 bands of radioactivity were found; 12 bands were assigned as GTP-binding proteins and 6 bands as phosphorylated proteins. One band was assumed to be a guanylylated protein. The profile of radioactive bands was similar between cells prepared from liquid or solid culture, but markedly different between growth phases. A mutant (strain M-1) defective in aerial mycelium formation, which was originally found as a decoyinine-resistant isolate, was found to have a different profile of phosphorylated proteins.     

灰色链霉菌启动子活性片段在大肠杆菌中的亚克隆及其序列分析

通过对重组质粒No.8-1的亚克隆,灰色链霉菌插入到大肠杆菌载体质粒中的序列已缩小到410bp,仍具有启动子功能。序列分析表明,启动子活性片段的G C碱基组成为50.5%。内含链霉菌启动子区域常有的正向重复序列;有1个Alul位点,1个Clal位点,2个Mbol位点,3个Nla Ⅲ位点,1个Pvu Ⅱ位点;具有类似于E.coli启动子的保守序列-10区和-35区,两者间隔18bp;在相应位置上分别有一段序列与E.coli的SD序列和在苄铅青链霉菌的SEP(Streptomyces-E.coil-type promoter)序列中存在的保守序列具有一定的相似性。     

Streptomycin biosynthesis in Streptomyces griseus I. Characterization of streptomycin-idiotrophic mutants

Abstract A total of 16 idiotrophic mutants unable to produce the aminoglycoside antibiotic streptomycin ( smi ) were isolated from Streptomyces griseus N2-3-11. Cosynthesis of streptomycin, its formation from various precursors and analysis of accumulated intermediates allowed grouping of the mutants in 3 classes, blocked: (I) in the first transamination step of the streptidine pathway; (II) in later steps of the streptidine pathway; or (III) outside streptidine biosynthesis.     

灰色链霉菌RX-17溶菌酶R1的纯化及性质研究

通过硫酸铵分级沉淀,CM-Sephadex C50、CM-Sepharose Fast Flow离子交换层析及Sephadex G-75凝胶过滤层析,从灰色链霉菌（Streptomyces griseus）RX17的发酵上清液中得到了电泳纯的溶菌酶R1,回收率6.89%。测得该酶分子量和等电点分别为16.8kD和9.10,作用于变链球菌（Streptococcus mutans）Ingbritt的最适温度和pH分别为70℃和6.6。R1酶在50℃以下及pH6～9的范围内保持稳定,60℃保温1h,残存酶活20.3％。Mg2+对酶有激活作用,而Zn2+、Cu2+、Fe2+、Cd2+、Pb2+则使酶完全丧失活性,螯合剂、盐酸羟胺、碘乙酸抑制酶活,β-巯基乙醇及表面活性剂则对溶菌有部分促进作用。R1酶溶菌谱广泛,对多种卵清溶菌酶不能作用的G+、G细菌均有溶解能力,对变链球菌、金黄色葡萄球菌（Staphylococcus aureus）、乳杆菌(Lactobacillus)等则呈现高活性。     

灰色链霉菌中乙酰木聚糖酯酶基因的克隆、表达及酶学性质研究

根据Gen Bank数据库中已报道的灰色链霉菌(Streptomyces griseus)全基因组序列,分析得到假定的乙酰木聚糖酯酶基因序列并设计引物;利用分子克隆的方法得到该菌株基因组中乙酰木聚糖酯酶基因,并构建原核表达载体p ET28a-Sgraxe,经IPTG诱导表达重组Sgr Axe,Ni-NTA亲和层析法纯化该蛋白。结果显示,克隆得到乙酰木聚糖酯酶基因axe,其序列全长1 008 bp,编码336个氨基酸。SDS-PAGE检测带有p ET28a-Sgraxe转化菌株诱导表达产物相对分子量约为37 k D,与理论值相符。纯化的重组Sgr Axe酶学性质表明,该酶最适反应温度为50℃,最适p H8.0,热稳定性较强,p H作用范围广;金属离子对酶均表现为抑制作用,尤其是Zn2+严重抑制酶活力;重组酶特征的分析揭示了其在工业中潜在的应用价值。     

响应面法优化灰色链霉菌产纤维素酶的工艺研究

以灰色链霉菌为原料,在单因素试验的基础上,采用响应面法试验,优化灰色链霉菌产纤维素酶活性的发酵条件。结果表明,单因素试验灰色链霉菌产纤维素酶活性的最适发酵条件:碳源为CMC-Na,氮源为明胶,温度为28℃,pH为7.0,转速为130 r/min。响应面法试验优化灰色链霉菌产纤维素酶活性最佳发酵条件为:温度27.7℃,pH值6.9,转数130.3 r/min,在此优化条件下,灰色链霉菌产纤维素平均酶活性为6.103 U/mL(n=3),与模型的预测值(6.217 U/mL)比较接近,误差为1.83%,证明了该响应面模型具有可靠性。     

Production of streptomycin from chitin using Streptomyces griseus in bioreactors of different configuration

Streptomyces griseus was cultured in three different bioreactors in a medium containing chitin flakes. When a conventional bioreactor stirred by two sets of Rushton impellers and operated at high speed was used, the yield of streptomycin (3.1 mg/l) was the highest observed and occurred at approximately 500 h. Cultivation of S. griseus in a bioreactor stirred at low speed by a U-shaped paddle resulted in a lower yield of streptomycin (1.8 mg/l) but this was achieved in a shorter period of time (400 h). Increasing the concentration of chitin from 5% to 10% w/v had no significant effect on either of these two parameters. The use of a novel vertical basket bioreactor in which the chitin flakes were contained within a wire mesh basket and were gently fluidised by air, enabled comparatively high yields of streptomycin (2.8 mg/l) in the relatively short time of 300 h.     

Self-cloning in Streptomyces griseus of an str gene cluster for streptomycin biosynthesis and streptomycin resistance.

An str gene cluster containing at least four genes (strR, strA, strB, and strC) involved in streptomycin biosynthesis or streptomycin resistance or both was self-cloned in Streptomyces griseus by using plasmid pOA154. The strA gene was verified to encode streptomycin 6-phosphotransferase, a streptomycin resistance factor in S. griseus, by examining the gene product expressed in Escherichia coli. The other three genes were determined by complementation tests with streptomycin-nonproducing mutants whose biochemical lesions were clearly identified. strR complemented streptomycin-sensitive mutant SM196 which exhibited impaired activity of both streptomycin 6-phosphotransferase and amidinotransferase (one of the streptomycin biosynthetic enzymes) due to a regulatory mutation; strB complemented strain SD141, which was specifically deficient in amidinotransferase; and strC complemented strain SD245, which was deficient in linkage between streptidine 6-phosphate and dihydrostreptose. By deletion analysis of plasmids with appropriate restriction endonucleases, the order of the four genes was determined to be strR-strA-strB-strC. Transformation of S. griseus with plasmids carrying both strR and strB genes enhanced amidinotransferase activity in the transformed cells. Based on the gene dosage effect and the biological characteristics of the mutants complemented by strR and strB, it was concluded that strB encodes amidinotransferase and strR encodes a positive effector required for the full expression of strA and strB genes. Furthermore, it was found that amplification of a specific 0.7-kilobase region of the cloned DNA on a plasmid inhibited streptomycin biosynthesis of the transformants. This DNA region might contain a regulatory apparatus that participates in the control of streptomycin biosynthesis.     

The nusG gene of Streptomyces griseus: Cloning of the gene and analysis of the A-factor binding properties of the gene product

Abstract The nusG gene of Streptomyces griseus was cloned and the nucleotide sequence determined. It encodes a protein with an identify of 76% to the reported receptor (VbrA) for VB-C, an autoregulatory factor in Streptomyces virginae . NusG protein was expressed in Escherichia coli . However, no binding activity for A-factor, an butyrolactone autoregulator in S. griseus very similar to VB-C, could be detected. The nusG gene of S. griseus does not seem to encode the A-factor-binding protein.     

S-腺苷甲硫氨酸合成酶对埃博霉素生物合成的影响

【目的】研究S-腺苷甲硫氨酸合成酶(SAMs)对埃博霉素生物合成的影响。【方法】通过向发酵培养基中添加抑制剂和促进剂,比较分析纤维堆囊菌中SAMs的活性变化以及埃博霉素的产量变化。【结果】在埃博霉素的合成期,SAMs的活性较高。加入抑制剂吲哚乙酸(IAA)之后,SAMs的活性和埃博霉素的产量都不同程度的降低,而加入促进剂对甲苯磺酸钠(p-TSA-Na)之后,SAMs的活性和埃博霉素的产量在不同程度上都有提高。在纤维堆囊菌的次级代谢中,SAMs活性与埃博霉素的生物合成量呈正相关。【结论】S-腺苷甲硫氨酸合成酶在纤维堆囊菌的埃博霉素生物合成过程中发挥了重要的作用。