提高产抗生素链霉菌紫外诱变正变率的研究

将UV诱变了的产抗生素链霉菌（Streptomyces sp.）AP 19-1菌株之孢子,置于适宜的生长温度27℃与接近抑制生长的胁迫温度33℃下培养,结果表明：在33℃下生长获得的子代菌株中,产抗生素水平超过其出发菌株的正向突变体所占的比例,明显比在27℃下培养的高。27℃下培养,正向突变体占总子代菌株数的25.8%,而在33℃下培养则为58.1%。用17种随机引物对出发菌株与UV诱变子代菌株进行总DNA的RAPD测验证明,在接近抑制生长的温度33℃下培养获得的子代,发生在其DNA水平上的变异程度比在27℃的要高得多。这一方法能较大幅度提高链霉菌紫外诱变育种的工作效率,同时也为链霉菌经紫外诱变后突变形成机制的进一步研究提供了新途径。Abstract: UV irradiated spores of Streptomyces sp. AP 19 -1 strain that can produce antibiotics were incubated at 27 ℃, and 33 ℃ which is close to inhibiting growth temperature, respectively. The results showed that there were much more forward mutants, whose level of producing antibiotics is higher than that of original strain, among the offspring of UV irradiated spores grown at 33 ℃, compared to that grown at 27 ℃. The percentage of the forward mutants was 25.8 % at 27 ℃ and 58.1% at 33 ℃. The progeny strains and the original strain were tested by RAPD using total DNA with 17 primers. It was demonstrated that more variations occurred in the chromosomal DNA of the progeny strains grown at 33 ℃ than in that at 27 ℃. This method facilitates increasing the efficiency of induced mutagenesis in breeding and provides a new way to study the mechanisms of mutation formation in UV irradiated Streptomyces sp. cells.     

Isolation and identification of lipopeptide antibiotics from <Emphasis Type="Italic">Paenibacillus elgii</Emphasis> B69 with inhibitory activity against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Two lipopeptide antibiotics, pelgipeptins C and D, were isolated from Paenibacillus elgii B69 strain. The molecular masses of the two compounds were both determined to be 1,086 Da. Mass-spectrometry, amino acid analysis and NMR spectroscopy indicated that pelgipeptin C was the same compound as BMY-28160, while pelgipeptin D was identified as a new antibiotic of the polypeptin family. These two peptides were active against all the tested microorganisms, including antibiotic-resistant pathogenic bacterial strains such as methicillin-resistant Staphylococcus aureus (MRSA). Time-kill assays demonstrated that pelgipeptin D exhibited rapid and effective bactericidal action against MRSA at 4×MIC. Based on acute toxicity test, the intraperitoneal LD50 value of pelgipeptin D was slightly higher than that of the structurally related antimicrobial agent polymyxin B. Pelgipeptins are highly potent antibacterial and antifungal agents, particularly against MRSA, and warrant further investigation as possible therapeutic agents for bacteria infections resistant to currently available antibiotics.     

Identification and functional analysis of gene cluster involvement in biosynthesis of the cyclic lipopeptide antibiotic pelgipeptin produced by Paenibacillus elgii

ABSTRACT: BACKGROUND: Pelgipeptin, a potent antibacterial and antifungal agent, is a non-ribosomally synthesised lipopeptide antibiotic. This compound consists of a beta-hydroxy fatty acid and nine amino acids. To date, there is no information about its biosynthetic pathway. RESULTS: A potential pelgipeptin synthetase gene cluster (plp) was identified from Paenibacillus elgii B69 through genome analysis. The gene cluster spans 40.8 kb with eight open reading frames. Among the genes in this cluster, three large genes, plpD, plpE, and plpF, were shown to encode non-ribosomal peptide synthetases (NRPS), with one, seven, and one module(s), respectively. Bioinformatic analysis of the substrate specificity of all nine adenylation domains indicated that the sequence of the NRPS modules is well collinear with the order of amino acids in pelgipeptin. Additional biochemical analysis of four recombinant adenylation domains (PlpD A1, PlpE A1, PlpE A3, and PlpF A1) provided further evidence that the plp gene cluster involved in pelgipeptin biosynthesis. CONCLUSIONS: In this study, a gene cluster (plp) responsible for the biosynthesis of pelgipeptin was identified from the genome sequence of Paenibacillus elgii B69. The identification of the plp gene cluster provides an opportunity to develop novel lipopeptide antibiotics by genetic engineering.     

微生物发酵稻草生产饲料蛋白培养条件的研究

研究了糙皮侧耳 (Pleurotusostreatus)和康氏木霉 (TrichodermaKoningii)发酵稻草生产饲料蛋白的培养条件。在实验室条件下 ,培养基的基本组成为稻草 80g ,麸皮 2 0g ,(NH₄)₂SO₄2g ,葡萄糖 2g,KH₂ PO₄1g。原料 :水为 1∶3 ,pH5.5～6.0 ,接种量 10%,培养温度28℃～30℃ ,培养周期10d。产物分析结     

Screening and construction of Saccharomyces cerevisiae strains with improved multi-tolerance and bioethanol fermentation performance

In this study, a systemic analysis was initially performed to investigate the relationship between fermentation-related stress tolerances and ethanol yield. Based on the results obtained, two elite Saccharomyces cerevisiae strains, Z8 and Z15, with variant phenotypes were chosen to construct strains with improved multi-stress tolerance by genome shuffling in combination with optimized initial selection. After three rounds of genome shuffling, a shuffled strain, YZ1, which surpasses its parent strains in osmotic, heat, and acid tolerances, was obtained. Ethanol yields of YZ1 were 3.11%, 10.31%, and 10.55% higher than those of its parent strains under regular, increased heat, and high gravity fermentation conditions, respectively. YZ1 was applied to bioethanol production at an industrial scale. Results demonstrated that the variant phenotypes from available yeast strains could be used as parent stock for yeast breeding and that the genome shuffling approach is sufficiently powerful in combining suitable phenotypes in a single strain.     

Drug resistance marker-aided genome shuffling to improve acetic acid tolerance in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Acetic acid existing in a culture medium is one of the most limiting constraints in yeast growth and viability during ethanol fermentation. To improve acetic acid tolerance in Saccharomyces cerevisiae strains, a drug resistance marker-aided genome shuffling approach with higher screen efficiency of shuffled mutants was developed in this work. Through two rounds of genome shuffling of ultraviolet mutants derived from the original strain 308, we obtained a shuffled strain YZ2, which shows significantly faster growth and higher cell viability under acetic acid stress. Ethanol production of YZ2 (within 60 h) was 21.6% higher than that of 308 when 0.5% (v/v) acetic acid was added to fermentation medium. Membrane integrity, higher in vivo activity of the H⁺-ATPase, and lower oxidative damage after acetic acid treatment are the possible reasons for the acetic acid-tolerance phenotype of YZ2. These results indicated that this novel genome shuffling approach is powerful to rapidly improve the complex traits of industrial yeast strains.     

肿瘤细胞抗TRAIL凋亡诱导的分子机制

肿瘤坏死因子相关的凋亡诱导配体(tumornecrosisfactor-relatedapoptosis-inducingligand,TRAIL)是肿瘤坏死因子(tumornecrosisfactor,TNF)超家族的成员之一,它能选择性诱导肿瘤细胞凋亡,对大多数正常细胞无杀伤作用。研究表明,某些恶性肿瘤抵抗TRAIL诱导的凋亡,且TRAIL重复作用使一些TRAIL敏感的细胞产生获得性抗性,这是TRAIL应用于肿瘤治疗的重大障碍。现对与TRAIL凋亡诱导通路直接相关的抗TRAIL机制及由Akt等途径介导的抗性分子机制进行综述。     

Comparative functional genomics to reveal the molecular basis of phenotypic diversities and guide the genetic breeding of industrial yeast strains

An understanding of the genetic basis underlying the phenotypic variations of yeast strains would guide the breeding of this useful microorganism. Here, comparative functional genomics (CFG) of two bioethanol Saccharomyces cerevisiae strains (YJS329 and ZK2) with different stress tolerances and ethanol fermentation performances were performed. Our analysis indicated that different patterns of gene expression in the central carbon metabolism, antioxidative factors, and membrane compositions of these two strains are the main contributors to their various traits. Some of the differently expressed genes were directly caused by the genomic structural variations between YJS329 and ZK2. Moreover, CFG of these two strains also led to novel insights into the mechanism of stress tolerance in yeast. For example, it was found that more oleic acid in the plasma membrane contributes to the acetic acid tolerance of yeast. Based on the genetic information particular to each strain, strategies to improve their adaptability and ethanol fermentation performances were designed and confirmed. Thus, CFG could not only help reveal basis of phenotypic diversities but also guide the genetic breeding of industrial microorganisms.