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Enhancement and selective production of avermectin B by recombinants of Streptomyces avermitilis via intraspecific protoplast fusion
Zhi Chen,Jia Wen,Yuan Song,Ying Wen,JiLun Li
Chinese Science Bulletin , 2007, DOI: 10.1007/s11434-007-0119-y
Abstract: Among eight components of avermectin, B1 fractions have the most effective antiparasitic activities and the lowest level of toxic side-effects and are used widely in veterinary and agricultural fields. Intraspecific protoplast fusion between two strains of Streptomyces avermitilis, one an avermectin high producer (strain 76-05) and the other a genetically engineered strain containing the mutations aveD and olmA (strain 73-12) was performed for enhancement and selective production of avermectin B in the absence of oligomycin. Two recombinant strains (F23 and F29) were isolated and characterized with regards to the parental merits. F23 and F29 produced only the four avermectin B components with high yield and produced no oligomycin. The avermectin production of F23 and F29 was about 84.20% and 103.45% of the parental strain 76-05, respectively, and increased about 2.66-fold and 3.50-fold, respectively, compared to that of parental strain 73-12. F23 and F29 were genetically stable prototrophic recombinants and F29 was quite tolerant of fermentation conditions compared to avermectin high producer parental strain 76-05. The ability to produce avermectin B with high yield without the production of other avermectin components and oligomycin will make F23 and F29 useful strains for avermectin production. Strain F29’s tolerance of fermentation conditions will also make it suitable for industrial applications.
Enhancement and selective production of avermectin B by recombinants of Streptomyces avermitilis via intraspecific protoplast fusion
CHEN Zhi,WEN Jia,SONG Yuan,WEN Ying,LI Jilun,
CHEN
,Zhi,WEN,Jia,SONG,Yuant,WEN,Ying,LI,JiLun

科学通报(英文版) , 2007,
Abstract: Among eight components of avermectin, B1 fractions have the most effective antiparasitic activities and the lowest level of toxic side-effects and are used widely in veterinary and agricultural fields. Intraspecific protoplast fusion between two strains of Streptomyces avermitilis, one an avermectin high producer (strain 76-05) and the other a genetically engineered strain containing the mutations aveD and olmA (strain 73-12) was performed for enhancement and selective production of avermectin B in the absence of oligomycin. Two recombinant strains (F23 and F29) were isolated and characterized with regards to the parental merits. F23 and F29 produced only the four avermectin B components with high yield and produced no oligomycin. The avermectin production of F23 and F29 was about 84.20% and 103.45% of the parental strain 76-05, respectively, and increased about 2.66-fold and 3.50-fold, respectively, compared to that of parental strain 73-12. F23 and F29 were genetically stable prototrophic recombinants and F29 was quite tolerant of fermentation conditions compared to avermectin high producer parental strain 76-05. The ability to produce avermectin B with high yield without the production of other avermectin components and oligomycin will make F23 and F29 useful strains for avermectin production. Strain F29’s tolerance of fermentation conditions will also make it suitable for industrial applications. Supported by National Basic Research Project (Grant No. 2003CB114205) and Key Technologies R&D Programme (Grant No. 2004BA713B02-03)
A Novel TetR Family Transcriptional Regulator, SAV576, Negatively Controls Avermectin Biosynthesis in Streptomyces avermitilis  [PDF]
Jia Guo, Xuan Zhang, Shuai Luo, Fei He, Zhi Chen, Ying Wen, Jilun Li
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0071330
Abstract: Avermectins produced by Streptomyces avermitilis are potent anti-parasitic agents that are useful in animal health care, agriculture, and the treatment of human infections. In a search for novel regulators that affect avermectin biosynthesis, comparative transcriptome analysis was performed between wild-type strain ATCC31267 and avermectin overproducing strain 76-02-e, revealing some differentially expressed genes. SAV576, which is downregulated in 76-02-e and encodes a TetR family transcriptional regulator (TFR), was shown to inhibit avermectin production by indirectly affecting the expression of ave genes. SAV576 directly repressed the transcription of its gene SAV576 and of adjacent genes SAV575 (encodes cytochrome P450/NADPH-ferrihemoprotein reductase) and SAV574. The SAV576-binding sites within the bidirectional SAV575-SAV576 promoter region were determined by DNase I footprinting assays. A consensus 15-bp palindromic sequence CCRTACRVYGTATGS was found in these binding sites and shown to be important for SAV576-binding activity. SAV575, an important target gene of SAV576, was shown to exert a positive effect on avermectin production. The study findings extend our limited knowledge of the complex regulation of avermectin biosynthesis and provide a basis for rational genetic manipulation of S. avermitilis to improve avermectin production through control of SAV576 and its target gene.
Two Adjacent and Similar TetR Family Transcriptional Regulator Genes, SAV577 and SAV576, Co-Regulate Avermectin Production in Streptomyces avermitilis  [PDF]
Jia Guo, Xuan Zhang, Zhi Chen, Ying Wen, Jilun Li
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0099224
Abstract: Streptomyces avermitilis is an important bacterial species used for industrial production of avermectins, a family of broad-spectrum anthelmintic agents. We previously identified the protein SAV576, a TetR family transcriptional regulator (TFR), as a downregulator of avermectin biosynthesis that acts by controlling transcription of its major target gene SAV575 (which encodes cytochrome P450/NADPH-ferrihemoprotein reductase) and ave genes. SAV577, another TFR gene, encodes a SAV577 protein that displays high amino acid homology with SAV576. In this study, we examined the effect of SAV577 on avermectin production and the relationships between SAV576 and SAV577. SAV577 downregulated avermectin biosynthesis indirectly, similarly to SAV576. SAV576 and SAV577 both directly repressed SAV575 transcription, and reciprocally repressed each other's expression. SAV575 transcription levels in various S. avermitilis strains were correlated with avermectin production levels. DNase I footprinting and electrophoretic mobility shift assays indicated that SAV576 and SAV577 compete for the same binding regions, and that DNA-binding affinity of SAV576 is much stronger than that of SAV577. GST pull-down assays revealed no direct interaction between the two proteins. Taken together, these findings suggest that SAV577 regulates avermectin production in S. avermitilis by a mechanism similar to that of SAV576, and that the role of SAV576 is dominant over that of SAV577. This is the first report of two adjacent and similar TFR genes that co-regulate antibiotic production in Streptomyces.
Inactivation of the positive LuxR-type oligomycin biosynthesis regulators OlmRI and OlmRII increases avermectin production in Streptomyces avermitilis
Qing Yu,LinQuan Bai,XiuFen Zhou,ZiXin Deng
Chinese Science Bulletin , 2012, DOI: 10.1007/s11434-011-4865-5
Abstract: Oligomycins are a group of 26 macrocyclic lactones that exhibit broad biological activities, including antifungal, anticancer and nematocidal activities. Analysis of the oligomycin biosynthetic gene cluster (olm) in S. avermitilis revealed 2 tandem LuxR-type regulators, OlmRI (931 aa) and OlmRII (941 aa), with shared identity of 38%. Gene replacement of olmRI or olmRII abolished oligomycin production, and this production could be partially restored in the disruptants by introducing cloned olmRI and olmRII with their native promoters, demonstrating the essential role of OlmRI and OlmRII for oligomycin biosynthesis. Quantitative real-time RT-PCR analysis revealed that transcription of 14 olm genes was differentially affected by the deletion of olmRI and olmRII. Unexpectedly, avermectin production in both mutants was enhanced at least 4-fold. The identification of the positive cluster-situated regulators, OlmRI and OlmRII, paves the way for the transcriptional analysis of oligomycin biosynthesis and for the enhancement of oligomycin and avermectin production through regulator engineering.
An adpA homologue in Streptomyces avermitilis is involved in regulation of morphogenesis and melanogenesis
JinLei Zhao,Ying Wen,Zhi Chen,Yuan Song,JiLun Li
Chinese Science Bulletin , 2007, DOI: 10.1007/s11434-007-0105-4
Abstract: In Streptomyces griseus, AdpA, the key transcriptional activator in the A-factor regulatory cascade, switches on the transcription of multiple genes required for secondary metabolism and morphological differentiation. Streptomyces avermitilis also contains an ortholog of adpA, which is named adpA-a. To clarify the in vivo function of adpA-a, an adpA-a-disrupted strain was constructed by double crossover recombination. No difference in avermectin production was found between the adpA-a-disruptant and the wild-type strain. However, this disruptant neither formed spores nor produced melanin and its phenotype was restored to the original wild-type by a single copy of the adpA-a gene integrated into the chromosome. This report shows that adpA-a is involved in regulation of morphological differentiation and melanin production in S. avermitilis.
Efficacy Evaluation of Fungus Syncephalastrum racemosum and Nematicide Avermectin against the Root-Knot Nematode Meloidogyne incognita on Cucumber  [PDF]
Wen-Kun Huang, Jian-Hua Sun, Jiang-Kuan Cui, Gang-Feng Wang, Ling-An Kong, Huan Peng, Shu-Long Chen, De-Liang Peng
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0089717
Abstract: The root-knot nematode (RKN) is one of the most damaging agricultural pests.Effective biological control is need for controlling this destructive pathogen in organic farming system. During October 2010 to 2011, the nematicidal effects of the Syncephalastrum racemosum fungus and the nematicide, avermectin, alone or combined were tested against the RKN (Meloidogyne incognita) on cucumber under pot and field condition in China. Under pot conditions, the application of S. racemosum alone or combined with avermectin significantly increased the plant vigor index by 31.4% and 10.9%, respectively compared to the M. incognita-inoculated control. However, treatment with avermectin alone did not significantly affect the plant vigor index. All treatments reduced the number of root galls and juvenile nematodes compared to the untreated control. Under greenhouse conditions, all treatments reduced the disease severity and enhanced fruit yield compared to the untreated control. Fewer nematodes infecting plant roots were observed after treatment with avermectin alone, S. racemosum alone or their combination compared to the M. incognita-inoculated control. Among all the treatments, application of avermectin or S. racemosum combined with avermectin was more effective than the S. racemosum treatment. Our results showed that application of S. racemosum combined with avermectin not only reduced the nematode number and plant disease severity but also enhanced plant vigor and yield. The results indicated that the combination of S. racemosum with avermectin could be an effective biological component in integrated management of RKN on cucumber.
Effect of Gene Deletion of aveD on Avermectins Production in Streptomyces avermitilis
阿维链霉菌中aveD基因缺失对阿维菌素合成的影响

Chen Zhi Wen Ying Song Yuan Li Jilun,
陈芝
,文莹,宋渊,李季伦

微生物学报 , 2002,
Abstract: 利用aveD基因的缺失载体pCZ8(pKC1139∷△aveD)对阿维菌素(Avermectin)产生菌阿维链霉菌(Streptomyces avermitilis)76\|9的aveD基因进行缺失获得aveD缺失突变株。经摇瓶发酵和HPLC检测,发现该突变株只产生阿维菌素B组分。说明将阿维链霉菌的aveD基因缺失,并不影响下游aveF的表达。缺失突变株的阿维菌素的总产量与出发菌株的总产量基本相同,突变株中B1的产量略有提高,阿维菌素B2的含量显著提高。
Diversity of Streptomyces in mangrove ecosystem of Bhitarkanika
Nibha Gupta,S Mishra,UC Basak
Iranian Journal of Microbiology , 2009,
Abstract: Background and objective: Bhitarkanika is the mangrove ecosystem of Orissa, India. It was not explored before for occurrence"nand distribution of Streptomyces. With the aim of isolation and characterization of special group of bacteria from this"nmangrove ecosystem, the present study has been made."nMaterials and Methods: Isolates of Streptomyces were obtained from plant, soil and water collected from Bhitarkanika"nmangroves on specific ISP media. Different isolates of Streptomyces were characterized for their colony characteristics,"nmorphological properties, physiological and biochemical properties and were tentatively identified."nResults: 105 isolates of Streptomyces belonging to 20 different species were isolated from 19 mangrove plants in different"nlocations of Bhitarkanika mangroves. According to physiological and biochemical data, all strains were taxonomically identified"nto the genus Streptomyces. However, all the strains were morphologically varied and exhibited different extracellular"nactivity. Maximum number of Streptomyces species was observed in the Khola region. S. xanthochromogenes was found to"nbe most prevalent species followed by S. exfoliates and S. auranticus."nConclusion: We have confirmed occurrence and distribution of Streptomyces in the Bhitarkanika mangrove environment."nThis is the first report of Streptomyces biodiversity in mangrove ecosystem of Bhitarkanika.
Chromosomal instability in Streptomyces avermitilis: major deletion in the central region and stable circularized chromosome
Wei Chen, Fei He, Xiaojuan Zhang, Zhi Chen, Ying Wen, Jilun Li
BMC Microbiology , 2010, DOI: 10.1186/1471-2180-10-198
Abstract: Thirty randomly-selected "bald" mutants derived from the wild-type strain all contained gross chromosomal rearrangements of various types. One of the bald mutants, SA1-8, had the same linear chromosomal structure as the high avermectin-producing mutant 76-9. Chromosomes of both strains displayed at least three independent chromosomal rearrangements, including chromosomal arm replacement to form new 88-kb terminal inverted repeats (TIRs), and two major deletions. One of the deletions eliminated the 36-kb central region of the chromosome, but surprisingly did not affect viability of the cells. The other deletion (74-kb) was internal to the right chromosomal arm. The chromosome of another bald mutant, SA1-6, was circularized with deletions at both ends. No obvious homology was found in all fusion sequences. Generational stability analysis showed that the chromosomal structure of SA1-8 and SA1-6 was stable.Various chromosomal rearrangements, including chromosomal arm replacement, interstitial deletions and chromosomal circularization, occurred in S. avermitilis by non-homologous recombination. The finding of an inner deletion involving in the central region of S. avermitilis chromosome suggests that the entire Streptomyces chromosome may be the target for rearrangements, which are not limited, as previously reported, to the two chromosomal ends.Streptomyces are a genus of Gram-positive, filamentous soil bacteria, which display complex morphological differentiation and produce a broad range of bioactive secondary metabolites such as antibiotics, immunosuppressants and cholesterol-lowering agents. These bacteria thus provide an important natural source of commercial products for the pharmaceutical and agricultural industries [1]. The Streptomyces genome consists of an 8- to 9-Mb linear chromosome, characterized by terminal inverted repeats (TIRs) and a protein covalently attached to 5' end [2-4]. This chromosome is inherently unstable, and frequently undergoes gross chrom
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