Transcriptome analysis of an antibiotic downregulator mutant and synergistic Actinorhodin stimulation via disruption of a precursor flux regulator in Streptomyces coelicolor

Through microarray analysis of an antibiotic-downregulator-deleted Streptomyces coelicolor ΔwblA ΔSCO1712 mutant, 28 wblA- and SCO1712-dependent genes were identified and characterized. Among 14 wblA- and SCO1712-independent genes, a carbon flux regulating 6-phosphofructokinase SCO5426 was additionally disrupted in the ΔwblA ΔSCO1712 mutant and further stimulated actinorhodin production in S. coelicolor, implying that both regulatory and precursor flux pathways could be synergistically optimized for antibiotic production.     

Streptomyces ghanaensis pleiotropic regulatory gene wblA(gh) influences morphogenesis and moenomycin production

The wblA _gh gene, encoding a homologue of the WhiB-family of proteins, was identified in the sequenced genome of moenomycin producer Streptomyces ghanaensis. Deletion of the gene blocked aerial mycelium sporulation and caused a 230% increase in moenomycins production. S. ghanaensis overexpressing SSFG-01620: a homologue of extracellular protease inhibitor SCO0762, whose expression in Streptomyces coelicolor is down-regulated by wblA: showed deficiencies in sporulation similar to that of wblA _gh knockout strain. The wblA _gh gene of S. ghanaensis appears to play a negative role in the control of moenomycin biosynthesis and is essential for sporulation.     

Transfer and expression of a Streptomyces cholesterol oxidase gene in Streptococcus thermophilus

The recombinant plasmid pNCO937 (8.1 kbp) containing a Streptomyces sp. cholesterol oxidase gene was introduced into Streptococcus thermophilus by electrotransformation. Transformation frequency was 7.2 x 10(5) colony forming units/micrograms of DNA. The presence of the cholesterol oxidase gene in S. thermophilus was confirmed with Southern blot analysis using a biotinylated probe. Thin-layer chromatographic analysis showed the expression of the Streptomyces cholesterol oxidase gene resulting in the oxidation of cholesterol to 4-cholesten-3-one. S. thermophilus may be a suitable host for the expression of other genes regulating prokaryotic cholesterol metabolism.     

Synthesis of bovine growth hormone by Streptomyces lividans

Streptomyces lividans 66 was transformed with a plasmid containing the regulatory region of the Streptomyces fradiae aph gene and a structural gene that specifies bovine growth hormone (bGH). When grown in liquid culture the transformant contained a protein identical to authentic bGH, as judged by radioimmunoassay and immuno-blotting (Western analysis). The bGH was present in cells that had been in culture for up to four weeks but was not found in the medium. The strategy employed should be generally applicable to the expression of foreign genes in actinomycetes.     

A thiostrepton-inducible expression vector for use in Streptomyces spp

A shuttle expression vector containing the thiostrepton-inducible Streptomyces lividans promoter, ptipA, and the origin of transfer from plasmid RP4 was constructed. Cassettes containing a promoterless xylE gene upstream from a hyg gene were used to demonstrate thiostrepton-inducible expression from ptipA in both S. lividans and Streptomyces ambofaciens, ptipA was estimated to be induced 60-fold or more in Streptomyces ambofaciens.     

报告基因法比较两种放线菌启动子的活性

摘要：【目的】比较启动子Psf与红霉素抗性基因启动子（PermE*）在链霉菌中的表达强度差异。【方法】本文利用卡那霉素抗性梯度以及邻苯二酚2,3-双加氧酶显色系统,比较了两个启动子的表达差异。【结果】两个启动子在棒状链霉菌(Streptomyces clavuligerus) NRRL3585、天蓝色链霉菌（Streptomyces coelicolor）M145,委内瑞拉链霉菌（Streptomyces venezuelae）ISP5230及变铅青链霉菌（Streptomyces lividans TK     

在链霉菌中表达透明颤菌血红蛋白需要异源启动子

构建了质粒pIJ4083Mpro、pIJ4083\|pro\,pWLD8和pFW3。在浅青紫链霉菌TK24中,启动子探针质粒pIJ4083上的邻苯二酚双加氧酶基因(xylE)不能被透明颤菌血红蛋白基因(vgb)的启动子带动转录,表明vgb启动子在链霉菌中无作用。TK24中,pWLD8和pFW3均能表达透明颤菌血红蛋白(VHb),pWLD8上可能是由Plac带动vgb的表达;pFW3上vgb基因去掉了非必要部分,克隆在PCR扩增得到的glnA启动子下游,两者连成嵌合基因。     

A second generation snp-derived Escherichia coli-Streptomyces shuttle expression vector that is generally transferable by conjugation

An Escherichia coli-Streptomyces shuttle vector (pJN100) was constructed, by inserting an origin of transfer (oriT), derived from the E. coli broad host range plasmid RK2, into pANT1202, a high-copy-number vector for gene expression in Streptomyces. The resulting conjugably transferable vector contains the pANT1202-derived SnpR (LysR-like protein) activated snpA promoter that drives strong heterologous expression of proteins. We initially demonstrated that plasmid pJN100 was transferred with high frequency (10(-5-7) exconjugants per recipient) into several Streptomyces strains that were refractory to transformation by other means. Plasmid pJN100 was also shown to be stable in E. coli and Streptomyces. We confirmed functional protein expression by using a pJN100 derivative to complement a mutant of Streptomyces griseus with a disrupted chromosomal copy of the gene nonM, a gene encoding an essential reductase in the nonactin biosynthesis gene cluster. High levels of protein expression were confirmed using Western blotting to assess the production of the serine esterase NonR, an enzyme responsible for nonactin resistance in the nonactin producer S. griseus.     

Development of a temperature-inducible expression system for Streptomyces spp.

PCR mutagenesis of a 0.9-kbp fragment, containing a repressor gene, traR, and its target promoter, Ptra, from Streptomyces nigrifaciens plasmid pSN22, produced Streptomyces lividans clones with temperature-inducible Ptra expression. Using the promoterless gene for the thermostable Thermus flavus malate dehydrogenase as an indicator, an induction of enzyme activity of as much as was observed in a temperature shift from 28 to 37 degrees C. Temperature downshift reestablished repression of Ptra, making these promoter cassettes very attractive for the temporally regulated expression of cloned genes in Streptomyces spp.     

High-level expression in Streptomyces lividans 66 of a gene encoding Streptomyces subtilisin inhibitor from Streptomyces albogriseolus S-3253

A secretory expression system for Streptomyces subtilisin inhibitor (SSI) was established in a heterologous host, Streptomyces lividans 66, by introducing the 1.8-kbp BglII/SalI fragment containing SSI gene into the Streptomyces multicopy vector, pIJ 702. The expression of SSI did not depend on the orientation of the 1.8-kbp BglII/SalI fragment or on the promoter for tyrosinase gene (mel) in pIJ 702, which suggested that this fragment also carries the SSI promoter. The expressed SSI in S.lividans 66 was secreted into the culture medium in a large amount, as observed with the original strain, S. albogriseolus S-3253. Amino acid sequence analysis showed that the SSI secreted from S. lividans 66 contained three additional amino acid residues in the NH2-terminal region. The inhibitory activity toward subtilisin BPN' and the antigenic activity of the SSI secreted from S. lividans 66 were found to be identical with those of authentic SSI.     

Regulation and secretion of an extracellular esterase from Streptomyces scabies.

Production of a heat-stable, extracellular esterase by Streptomyces scabies is regulated by zinc ions. The esterase-encoding gene (est) from S. scabies was cloned and expressed in Streptomyces lividans. In S. lividans, expression of the est gene is also regulated by Zn2+, and the esterase is efficiently secreted in this organism. The sequence of the est gene suggests that a 39-amino acid signal peptide is removed during secretion of this protein. Deletion analysis has indicated that the hydrophobic domain of the signal peptide is required for secretion. Gel retardation assays and DNaseI footprinting using an S-30 protein extract from S. scabies have previously identified a specific 23-bp protein-binding site upstream from the est coding sequence. Deletion of this protein-binding sequence significantly decreased expression of the est gene.     

东乡野生稻内生放线菌Streptomyces sp. PRh5的全基因组测序及序列分析

【目的】Streptomyces sp. PRh5是从东乡野生稻(Oryza rufipogon Griff.)中分离获得的一株对细菌和真菌都具有较强抗菌活性的内生放线菌。为深入研究PRh5菌株抗菌机制及挖掘次级代谢产物基因资源,有必要解析PRh5菌株的基因组序列信息。【方法】采用高通量测序技术对PRh5菌株进行全基因组测序,然后使用相关软件对测序数据进行基因组组装、基因预测与功能注释、直系同源簇(COG)聚类分析、共线性分析及次级代谢产物合成基因簇预测等。【结果】基因组组装获得290 contigs,整个基因组大小约11.1 Mb,GC含量为71.1%,序列已提交至GenBank数据库,登录号为JABQ00000000。同时,预测得到50个次级代谢产物合成基因簇。【结论】将为Streptomyces sp. PRh5的功能基因组学研究及相关次级代谢产物的生物合成途径与异源表达研究提供基础。     

High-level expression of a novel amine-synthesizing enzyme, N-substituted formamide deformylase, in Streptomyces with a strong protein expression system

N-substituted formamide deformylase (NfdA) from Arthrobacter pascens F164 is a novel deformylase involved in the metabolism of isonitriles. The enzyme catalyzes the deformylation of an N-substituted formamide, which is produced from the corresponding isonitrile, to yield the corresponding amine and formate. The nfdA gene from A. pascens F164 was cloned into different types of expression vectors for Escherichia coli and Streptomyces strains. Expression in E. coli resulted in the accumulation of an insoluble protein. However, Streptomyces strains transformed with a P(nitA)-NitR system, which we very recently developed as a regulatory gene expression system for streptomycetes, allowed the heterologous overproduction of NfdA in an active form. When Streptomyces lividans TK24 transformed with pSH19-nfdA was cultured under the optimum conditions, the NfdA activity of the cell-free extract amounted to 8.5 U/mg, which was 29-fold higher than that of A. pascens F164. The enzyme also comprised approximately 20% of the total extractable cellular protein. The recombinant enzyme was purified to homogeneity and characterized. The expression system established here will allow structural analysis and mutagenesis studies of NfdA.     

Tn5099, a xylE promoter probe transposon for Streptomyces spp.

Tn5099, a promoter probe transposon for Streptomyces spp., was constructed by inserting a promoterless xylE gene and a hygromycin resistance gene into IS493. Tn5099 transposed into different sites in the Streptomyces griseofuscus genome, and the xylE reporter gene was expressed in some of the transposition mutants. Strains containing Tn5099 insertions that gave regulated expression of the xylE gene were identified.