A ketoreductase gene from Streptomyces mycarofaciens 1748 DNA involved in biosynthesis of a spore pigment

An efficient plasmid transformation system forS. mycarofaciens 1748 has been established. In order to determine the function of MKR gene in S.mycarofaciens 1748, the gene disruption experiment was carried out. For this purpose the plasmid pKC1139 was used. A recombinant strain with white spore appeared, in contrast to the grey-colour spore of S.myarofaciens 1748. This suggested that homologous recombination between plasmidborne MKR gene sequence and the chromosome of S.mycarofaciens 1748 had occurred. A Southern hybridization experiment using a-³²P-labelled MKR gene as probe indicated that the desired integration event had occurred in the recombinant. The result of gene disruption showed that the alteration of this gene in the chromosome of S.mycarofaciens 1748 made sporulating colonies remain white instead of taking on the typical grey colour of sporulating wild type colonies, suggesting that MKR gene is involved in the biosynthesis of a spore pigment. The recombinant strain was incubated with fermentation medium optimised for midecamycin production. A TLC assay showed that the recombinant strain produced midecamycin in quantities comparable to that ofS. mycarofaciens 1748. A pCN8B12 was a clone from genomic library of midecamycin producing strain which contained a 28-kb DNA insert. The 28-kb DNA fragment contained act I-homologous and act III-homologous regions. he PKS (act I-homologous) and MKR (act III-homologous) genes that define spore pigment of midecamycin producing strain were Jocalized by restriction endonuclease digestion with pCN8B12, indicating that they are separated by about 10 kb DNA. The polyketide synthase gene cluster of simila; organization has not been reported yet. Project supported by the National Natural Science Foundation of China.     

A ketoreductase gene from Streptomyces mycarofaciens 1748 DNA involved in biosynthesis of a spore pigment

An efficient plasmid transformation system for S. mycarofaciens 1748 has been established. In order to determine the function of MKR gene in S. mycarofaciens 1748, the gene disruption experiment was carried out. For this purpose the plasmid pKC1139 was used. A recombinant strain with white spore appeared, in contrast to the grey-colour spore of S. mycarofaciens 1748. This suggested that homologous recombination between plasmid-borne MKR gene sequence and the chromosome of S. mycarofaciens 1748 had occurred. A Southern hybridization experiment using α- P-labelled MKR gene as probe indicated that the desired integration event had occurred in the re-combinant. The result of gene disruption showed that the alteration of this gene in the chromosome of S. mycarofa-ciens 1748 made sporulating colonies remain white instead of taking on the typical grey colour of sporulating wild type colonies, suggesting that MKR gene is involved in the biosynthesis of a spore pigment. The recombinant strain was in-cubated wit     

MpigE, a gene involved in pigment biosynthesis in Monascus ruber M7

Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries. However, MP biosynthesis pathway is still a controversy, and only few related genes have been reported. In this study, the function of MpigE, a gene involved in MP biosynthesis in Monascus ruber M7, was analyzed. The results revealed that the disruption, complementation, and overexpression of MpigE in M. ruber M7 had very little effects on the growth and phenotypes except MPs. The MpigE deletion strain (?MpigE) just yielded four kinds of yellow MPs and very little red pigments, while the wild-type strain M. ruber M7 produced a MP complex mixture including three (orange, red, and yellow) categories of MP compounds. Two of the four yellow MPs produced by ?MpigE were the same as those yielded by M. ruber M7. The MpigE complementation strain (?MpigE::MpigE) recovered the ability to generate orange and red MPs as M. ruber M7. The MP types produced by the MpigE overexpression strain (M7::PtrpC-MpigE) were consistent with those of M. ruber M7, while the color value was about 1.3-fold as that of M. ruber M7 (3,129 U/g red kojic). For the production of citrinin, the disruption of MpigE almost had no influence on the strain, whereas the overexpression of MpigE made citrinin decrease drastically in YES fermentation. This work will make a contribution to the study on the biosynthesis pathway of MPs in M. ruber.     

美达霉素产生菌中立体专一性酮基还原酶基因med-ORF12的表达

美达霉素是链霉菌产生的具有强抗肿瘤活性的芳香聚酮类抗生素,其砒喃环并内酯结构对于其抗癌活性非常重要。位于美达霉素生物合成基因簇中的基因med-ORF12编码立体专一性酮基还原酶,可能参与美达霉素的砒喃环并内酯结构中手性中心(C3S)的形成,但在美达霉素产生菌中的功能和表达还未曾研究。【目的和方法】为了研究med-ORF12在野生菌中的表达情况以及与美达霉素生物合成的关系,本文采用了原核表达、抗体制备、免疫杂交等技术方法对这个基因展开了体内表达研究。【结果】首先利用pET载体建立了med-ORF12的原核表达系统,在优化诱导表达条件的基础上获得了可溶性目的蛋白,制备了相应的多抗血清;然后利用多抗血清对美达霉素产生菌中的基因med-ORF12的表达情况进行了检测,表明在美达霉素产生菌中参与次生代谢的med-ORF12在稳定期大量表达,同时伴随美达霉素的大量积累。【结论】这些结果表明在美达霉素产生菌中,基因med-ORF12参与次生代谢,其表达与美达霉素生物合成有一定相关性。     

A gene cluster involved in nogalamycin biosynthesis fromStreptomyces nogalater: sequence analysis and complementation of early-block mutations in the anthracycline pathway

We have analyzed an anthracycline biosynthesis gene cluster fromStreptomyces nogalater. Based on sequence analysis, a contiguous region of 11 kb is deduced to include genes for the early steps in anthracycline biosynthesis, a regulatory gene (snoA) promoting the expression of the biosynthetic genes, and at least one gene whose product might have a role in modification of the glycoside moiety. The three ORFs encoding a minimal polyketide synthase (PKS) are separated from the regulatory gene (snoA) by a comparatively AT-rich region (GC content 60%). Subfragments of the DNA region were transferred toStreptomyces galilaeus mutants blocked in aclacinomycin biosynthesis, and to a regulatory mutant ofS. nogalater. TheS. galilaeus mutants carrying theS. nogalater minimal PKS genes produced auramycinone glycosides, demonstrating replacement of the starter unit for polyketide biosynthesis. The product ofsnoA seems to be needed for expression of at least the genes for the minimal PKS.     

Molecular cloning of a Bacillus subtilis gene involved in spore outgrowth

A lambda Charon 4A derivative carrying the outB gene of Bacillus subtilis has been identified by transformation of a B. subtilis mutant temperature-sensitive in spore outgrowth. The cloned region is a single EcoRI fragment 14 kb in length. In addition to outB, the cloned DNA includes at least part of the amyE and aroI loci.     

A hydroxylase-like gene product contributes to synthesis of a polyketide spore pigment in Streptomyces halstedii.

A gene, schC, adjacent to the sch gene cluster encoding the biosynthesis of a polyketide spore pigment in Streptomyces halstedii was sequenced. Its deduced product resembled flavin adenine nucleotide-containing hydroxylases involved in the biosynthesis of polycyclic aromatic polyketide antibiotics and in catabolic pathways of aromatic compounds. When schC was disrupted, the normally green spores of S. halstedii became lilac. An schC-like gene was located in an equivalent position next to a large gene cluster (whiE) known to determine spore pigment in Streptomyces coelicolor A3(2).     

Cloning of a DNA fragment involved in pigment production in Streptomyces avermitilis

Streptomyces avermitilis has the ability to synthesize a diffusible, brown, melanin-like pigment, a common property among many Streptomyces species. A region of the S. avermitilis chromosome involved in the production of this pigment was cloned in Escherichia coli. Production of the brown pigment was attained in E. coli, and is optimal when medium is supplemented with copper ions, tyrosine and IPTG. The cloned S. avermitilis pigment-producing DNA fragment is under the control of the lac promoter carried in the E. coli vector. The gene involved in pigment production could be used as a tool to analyse gene expression in S. avermitilis, and as an alternative cloning marker in Streptomyces-Escherichia coli vectors.     

A eukaryotic alanine racemase gene involved in cyclic peptide biosynthesis

The cyclic tetrapeptide HC-toxin is an essential virulence determinant for the plant pathogenic fungus Cochliobolus carbonum and an inhibitor of histone deacetylase. The major form of HC-toxin contains the D-isomers of Ala and Pro. The non-ribosomal peptide synthetase that synthesizes HC-toxin has only one epimerizing domain for conversion of L-Pro to D-Pro; the source of D-Ala has remained unknown. Here we present the cloning and characterization of a new gene involved in HC-toxin biosynthesis, TOXG. TOXG is present only in HC-toxin-producing (Tox2(+)) isolates of C. carbonum. TOXG is able to support D-Ala-independent growth of a strain of Escherichia coli defective in D-Ala synthesis. A C. carbonum strain with both of its copies of TOXG mutated grows normally in culture, and although it no longer makes the three forms of HC-toxin that contain D-Ala, it still makes a minor form of HC-toxin that contains Gly in place of D-Ala. The addition of D-Ala to the culture medium restores production of the D-Ala-containing forms of HC-toxin by the toxG mutant. The toxG mutant has only partially reduced virulence. It is concluded that TOXG encodes an alanine racemase whose function is to synthesize D-Ala for incorporation into HC-toxin.     

Functional studies on a ketoreductase gene from Streptomyces sp. AM-7161 to control the stereochemistry in medermycin biosynthesis

Medermycin shows the same trans (3S,15R) configuration as actinorhodin in the pyran ring crucial for its bioactivity. One medermycin biosynthetic gene, med-ORF12, is assumed to be involved in the stereochemical control at C-3. Functional complementation suggested that it plays a similar role as actVI-ORF1 previously proved to determine the stereospecificity at C-3 in actinorhodin biosynthesis. Co-expression of med-ORF12 with actinorhodin early biosynthetic genes further demonstrated that med-ORF12 encodes a ketoreductase responsible for the enantioselective reduction at C-3 in the formation of the pyran ring.     

A DNA fragment fromStreptomyces fradiae increases the production of a metalloprotease inStreptomyces lividans

Streptomyces fradiae produces several extracellular proteases and many of these are inducible. An 8.8 kb DNA fragment of Streptomyces fradiae cloned on pIJ699 caused increased protease activity in Streptomyces lividans.Clones carrying this recombinant plasmid showed a significant delay in sporulation. A protein of 18 kDa was purified from the extracellular proteins secreted by the host carrying the recombinant plasmid. Further characterization showed that this protease is a metalloprotease.     

Cloning and characterization of a gene from Pasteurella haemolytica A1 involved in lipopolysaccharide biosynthesis

Abstract A Pasteurella haemolytica A1 gene involved in the biosynthesis of a moiety on the core of the lipopolysaccharide molecule has been cloned and characterized. Escherichia coli clones which carry this gene showed an alteration of its lipopolysaccharide migration profile on tricine SDS-PAGE and exhibited resistance to the core-specific phage U3. In addition, lipopolysaccharide extracted from the E. coli clones was recognized by an anti-corespecific antiserum, but not by antiserum specific for the O antigen of P. haemolytica A1 lipopolysaccharide. Nucleotide sequence analysis of the cloned DNA identified an open reading frame ( lpsA ) coding for a protein of 263 amino acids which showed significant homology with a Haemophilus influenzae type b lipooligosaccharide biosynthesis gene. PCR amplification of genomic DNA, using primers based on the P. haemolytica A1 lpsA sequence, yielded products from only the A biotypes of P. haemolytica .     

Suppression of a key gene involved in chlorophyll biosynthesis by means of virus-inducing gene silencing

The ChlH gene coding the H subunit of magnesium chelatase, an enzyme involved in chlorophyll biosynthesis, was silenced in Nicotiana benthamiana plants by infection with tobacco mosaic virus vectors (pTMV-30b) containing 67, 214 or 549 nt long ChlH inserts. Silencing of the nuclear ChlH gene induced a chimeric phenotype with green and yellow/white leaves associated with alterations of chloroplast shape and ultrastructure. The symptoms became first evident around veins of young leaves, and only later in the mesophyll tissues. The efficiency of gene silencing was not dependent on the insert orientation, but was strongly correlated with the size of the ChlH insert, providing a flexible method to modulate the level of gene suppression. Silencing efficiency seemed to be strongly dependent on endogenous ChlH mRNA level of the target tissue. Silencing of the ChlH gene with the longest fragment of 549 nt also lowered the accumulation of ChlD and chlorophyll synthetase mRNAs, i.e. other genes involved in chlorophyll biosynthesis.     

黑曲霉中生物合成赭曲霉毒素A的非核糖体肽合成酶基因的鉴定

赭曲霉毒素A(ochratoxin A,OTA)是国际癌症研究机构认定的"2B"类致癌物。黑曲霉Aspergillus niger是美国食品药品监督局认可的食品安全菌。然而近年来陆续发现某些黑曲霉菌株能够产生OTA,这会对人类健康构成潜在威胁。阐明黑曲霉生物合成OTA的关键基因有助于理解OTA生物合成机制,这对OTA污染的防控具有重要意义。本研究克隆了产OTA黑曲霉中非核糖体肽合成酶(NRPS)编码基因(An15g07910),并对其进行了生物信息学分析,在此基础上采用同源重组的方法敲除了该基因,获得了一株性能稳定的敲除突变株Δnrps。与野生株相比,Δnrps突变株的表型在CYA培养基中并无明显改变,但在7d培养期间完全失去了合成赭曲霉毒素α(ochratoxinα,OTα)和OTA的能力,而赭曲霉毒素β(ochratoxinβ,OTβ)的合成不受影响。在野生株培养过程中,该nrps基因前4d表达量逐渐增大,并在第4天达到最高,随后基因表达量逐渐下降并趋于稳定,这与OTA的含量变化基本一致。结果表明该nrps基因(An15g07910)参与OTA的生物合成,其编码的NRPS可能负责催化苯丙氨酸部分和二氢异香豆素部分的交联。     

一个甲基转移酶基因lomo3在洛蒙真菌素生物合成途径中的功能

【目的】洛蒙真菌素是在洛蒙德链霉菌(Streptomyces lomondensis)中生物合成的一种具有广谱抑菌活性的吩嗪类抗生素,但其合成机理仍不清晰。在洛蒙德链霉菌S015的洛蒙真菌素生物合成核心基因簇下游,有一甲基转移酶基因——lomo3,研究该基因对洛蒙真菌素生物合成的影响。【方法】对lomo3基因进行无痕敲除得到基因缺失突变株S015Δlomo3,再过表达重组质粒构建回补突变株S015Δlomo3::lomo3,比较两株突变株与野生型S015的发酵产物的变化。【结果】发现基因缺失菌株S015Δlomo3不能合成洛蒙真菌素,而基因回补菌株S015Δlomo3::lomo3则可恢复洛蒙真菌素的合成能力。【结论】甲基转移酶基因lomo3在洛蒙真菌素生物合成过程中起着重要的作用,但该基因的具体功能还有待深入研究。研究对于阐明洛蒙真菌素的生物合成途径具有一定的指导作用。