利福霉素生产菌产生钝化RifSV物质的分离及性质研究

利福霉素ＳＶ（简称ＲｉｆＳＶ）生产菌──地中海拟无枝菌酸菌在生物合成ＲｉｆＳＶ过程中,产生一种能钝化自身产物（ＲｉｆＳＶ）的物质．实验证实,该物质是由谷氨酸、天冬氨酸、赖氨酸及缬氨酸等１４种常见氨基酸组成的蛋白质．分子量（ＭＷ）约２．５×１０４Ｄ,等电点（ＰＩ）为５．７—６．１．在１００℃下加热１０ｍｉｎ,其活性丧失．作用于ＲｉｆＳＶ的最适ｐＨ值范围为７．４—８．６,最适温度为２９℃,初步证实该物质是一种酶（暂称利福霉素钝化酶）     

Use of biodiesel‐derived crude glycerol for vancomycin production by Amycolatopsis orientalis XMU‐VS01

Crude glycerol is a primary by‐product in the biodiesel industry. Microbial fermentation on crude glycerol for producing value‐added products provides opportunities to utilize a large quantity of this by‐product. This study investigates the potential of using the crude glycerol to produce vancomycin (glycopeptide antibiotics) through fermentation of Amycolatopsis orientalis XMU‐VS01. The results show that crude glycerol was the most effective carbon source for mycelium growth and vancomycin production, with 40–60 g/L glycerol concentration as optimal range. Among other culture medium components, potato protein (nitrogen source) and the phosphate concentration had significant effects (p<0.05) for vancomycin production. A Box‐Behnken design and response surface methodology were employed to formulate the optimal medium. Their optimal values were determined as 52.73 g/L of glycerol, 17.36 g/L of potato protein, and 0.1 g/L of dipotassium phosphate. A highest vancomycin yield of 7.61 g/L with biomass concentration of 15.8 g/L was obtained after 120 h flask fermentation. The yield of vancomycin was 3.5 times higher than with basic medium. The results suggest that biodiesel‐derived crude glycerol is a promising feedstock for production of vancomycin from A. orientalis culture.     

Development of cloning vectors and transformation methods for<Emphasis Type="Italic"> Amycolatopsis</Emphasis>

The genus Amycolatopsis is of industrial importance, as its species are known to produce commercial antibiotics. It belongs to the family Pseudonocardiaceae and has an eventful taxonomic history. Initially strains were identified as Streptomyces, then later as Nocardia. However, based on biochemical, morphological and molecular features, the genus Amycolatopsis, containing seventeen species, was created. The development of molecular genetic techniques for this group has been slow. The scarcity of molecular genetic tools including stable plasmids, antibiotic resistance markers, transposons, reporter genes, cloning vectors, and high efficiency transformation protocols has made progress slow, but efforts in the past decade have led to the development of cloning vectors and transformation methods for these organisms. Some of the cloning vectors have broad host range (pRL series) whereas others have limited host range (pMEA300 and pMEA100). The cloning vector pMEA300 has been completely sequenced, while only the minimal replicon (pA-rep) has been sequenced from pRL plasmids. Direct transformation of mycelia and electroporation are the most widely applicable methods for transforming species of Amycolatopsis. Conjugational transfer from Escherichia coli has been reported only in the species A. japonicum, and gene disruption and replacements using homologous recombination are now possible in some strains. Electronic Publication     

Stability of plasmid pA387 derivatives in Amylcolatopsis mediterranei producing rifamycin

Genetic studies on the biosynthesis of rifamycins in producer strains such as Amylcolaptopsis mediterranei U-32 are severely hampered by the availability of efficient transformation procedures and stable plasmid vectors. Using an efficient electroporation procedure we have studied the replication and stability of a pA387 derivative, pDXM32. This plasmid confers enhanced plasmid stability and copy number compared to pA387 derivatives commonly used as cloning vectors in A. mediterranei. Deletion derivatives in the region previously identified as being a minimal replication origin were also examined with respect to their ability to transform A. mediterranei and at least one locus was essential for replication. A 5.4 kbp DNA fragment was sequenced and annotated encoding the replication and plasmid stability functions. A parA homologue was identified which is likely to confer plasmid stability.     

Studies on fermentative production of rifamycin using Amycolatopsis mediterranei

The fermentative production of rifamycin by Amycolatopsis mediterranei (MTCC17) has been studied. Both qualitative and quantitative aspects of the fermentation were determined by optimizing cultural conditions and medium design to improve the production of rifamycin. A pH value of 7.0, a temperature of 26°C, an aeration rate of 250rev/min for a 50ml volume, a level of inoculum of 10% grown aeration for 48h and a fermentation period of 11days were found to be optimum. Among the nitrogen sources, and culture conditions, peanut meal and aeration were found to be critical for rifamycin production respectively. The above mentioned exercise increased the yields of rifamycin from 350mg/l to 2000mg/l.     

A complex role of <Emphasis Type="Italic">Amycolatopsis mediterranei</Emphasis> GlnR in nitrogen metabolism and related antibiotics production

Amycolatopsis, genus of a rare actinomycete, produces many clinically important antibiotics, such as rifamycin and vancomycin. Although GlnR of Amycolatopsis mediterranei is a direct activator of the glnA gene expression, the production of GlnR does not linearly correlate with the expression of glnA under different nitrogen conditions. Moreover, A. mediterranei GlnR apparently inhibits rifamycin biosynthesis in the absence of nitrate but is indispensable for the nitrate-stimulating effect for its production, which leads to the hyper-production of rifamycin. When glnR of A. mediterranei was introduced into its phylogenetically related organism, Streptomyces coelicolor, we found that GlnR widely participated in the host strain’s secondary metabolism, resemblance to the phenotypes of a unique S. coelicolor glnR mutant, FS2. In contrast, absence or increment in copy number of the native S. coelicolor glnR did not result in a detectable pleiotrophic effect. We thus suggest that GlnR is a global regulator with a dual functional impact upon nitrogen metabolism and related antibiotics production.     

Bacterial Type Ⅰ Glutamine Synthetase of the Rifamycin SV Producing Actinomycete,Amycolatopsis mediterranei U32,is the Only Enzyme Responsible for Glutamine Synthesis under Physiological Conditions

The structural gene for glutamine synthetase,glnA,from Amycolatopsis mediterranei U32 wascloned via screening a genomic library using the analog gene from Streptomyces coelicolor.The clone wasfunctionally verified by complementing for glutamine requirement of an Escherichia coli glnA null mutantunder the control of a lac promoter.Sequence analysis showed an open reading frame encoding a protein of466 amino acid residues.The deduced amino acid sequence bears significant homologies to other bacterialtype Ⅰ glutamine synthetases,specifically,71% and 72% identical to the enzymes of S.coelicolor and Myco-bacterium tuberculosis,respectively.Disruption of this glnA gene in A.mediterranei U32 led to glutamineauxotrophy with no detectable glutamine synthetase activity in vivo.In contrast,the cloned glnA~ gene cancomplement for both phenotypes in trans.It thus suggested that in A.mediterranei U32,the glnA geneencoding glutamine synthetase is uniquely responsible for in vivo glutamine synthesis under our laboratorydefined physiological conditions.     

黄孢原毛平革菌木质素过氧化物酶基因在甲醇毕赤酵母中的表达

将编码黄孢原毛平革菌木质素过氧化物酶(lip)的cDNA克隆到酵母整合型质粒pMETA上,电转化Ade缺陷型甲醇毕赤酵母(Pichiamethanolica)PMAD16,通过MD平板及PCR方法筛选和鉴定重组子。重组子发酵液经SDSPAGE分析和木质素过氧化物酶活力测定等方法鉴定,表明带自身信号肽的黄孢原毛平革菌木质素过氧化物酶基因(lip)在甲醇毕赤酵母中得到表达。优化其发酵培养条件,以藜芦醇为底物进行酶活测定,其酶活可达932U/L。相应发酵指数为12.94U/h·L。比出发菌株提高了24.18%。