A ketoreductase gene fromStreptomyces 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.     

Construction of 4″-Isovalerylspiramycin-I-Producing Strain by In-Frame Partial Deletion of 3-<Emphasis Type="Italic">O</Emphasis>-Acyltransferase Gene in <Emphasis Type="Italic">Streptomyces spiramyceticus</Emphasis> WSJ-1, the Bitespiramycin Producer

Bitespiramycin (BT), a multi-component antibiotic consisted mainly of 4″-isovalerylspiramycin I, II and III, is produced by Streptomyces spiramyceticus WSJ-1, a recombinant spiramycin-production strain that harbored the 4″-O-acyltransferase gene (ist) from Streptomyces mycarofaciens 1748, which could isovalerylate the 4″-OH of spiramycin. To eliminate the production of components 4″-isovalerylspiramycin II and III, therefore reducing the component complexity of BT, inactivation of the sspA gene, which encodes the 3-O-acyltransferase responsible for the acylation of spiramycin I to spiramycin II and III, was performed in Streptomyces spiramyceticus WSJ-1, by in-frame partial deletion. The resulting strain, Streptomyces spiramyceticus WSJ-2, is a 4″-isovalerylspiramycin-I-producing strain as expected.     

Cloning and expression of a Saccharomyces diastaticus glucoamylase gene in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

A recombinant plasmid pool of the Saccharomyces diastaticus genome was constructed in plasmid YEp13 and used to transform a strain of Saccharomyces cerevisiae. Six transformants were obtained which expressed amylolytic activity. The plasmids each contained a 3.9-kilobase (kb) BamHI fragment, and all of these fragments were cloned in the same orientations and had identical restriction maps, which differed from the map of the STA1 gene (I. Yamashita and S. Fukui, Agric. Biol. Chem. 47:2689-2692, 1983). The glucoamylase activity exhibited by all S. cerevisiae transformants was approximately 100 times less than that of the donor strain. An even lower level of activity was obtained when the recombinant plasmid was introduced into Schizosaccharomyces pombe. No expression was observed in Escherichia coli. The 3.9-kb BamHI fragment hybridized to two sequences (4.4 and 3.9 kb) in BamHI-digested S. diastaticus DNA, regardless of which DEX (STA) gene S. diastaticus contained, and one sequence (3.9 kb) in BamHI-digested S. cerevisiae DNA. Tetrad analysis of crosses involving untransformed S. cerevisiae and S. diastaticus indicated that the 4.4-kb homologous sequence cosegregated with the glucoamylase activity, whereas the 3.9-kb fragment was present in each of the meiotic products. Poly(A)+ RNA fractions from vegetative and sporulating diploid cultures of S. cerevisiae and S. diastaticus were probed with the 3.9-kb BamHI fragment. Two RNA species, measuring 2.1 and 1.5 kb, were found in both the vegetative and sporulating cultures of S. diastaticus, whereas one 1.5-kb species was present only in the RNA from sporulating cultures of S. cerevisiae.     

麦迪霉素产生菌酮基还原酶基因的研究

将麦迪霉素产生菌基因文库中与放线紫红索酮基还原酶基因actⅢ有同源性的4·0kb DNA片段克隆到质粒载体pWHM3中,构成重组质粒pCB4。将质粒pCB4转入酮基还原酶基因缺陷菌株——加利利链霉菌ATCC3167l中,得到转化子。转化子发酵产物经TLC和HPLC分析证明是阿克拉菌酮,与加利利链霉菌原株ATCC31133的产物相同,说明麦迪霉素产生菌酮基还原酶基因互补了加利利链霉菌ATCC31671中缺陷的酮基还原酶基因,使其恢复了产生阿克拉菌酮的能力。4．Okb DNA片段插入方向相反的重组质粒pCBR4在加利利链霉菌ATCC31671中发酵产物经TLC分析证明也是阿克拉菌酮,这说明4．0kbDNA片段中麦迪霉素产生菌酮基还原酶基因具有自身的启动子。对4．0kb DNA片段进行了限制酶酶切分析,建立了其酶切图谱。以actⅢ基因为探针,经分子杂交以及亚克隆和DNA转化实验,将麦迪霉索产生菌酮基还原酶基因定位于BssH Ⅱ—BamH Ⅰ 1．3kb DNA片段上。对1．3kb DNA片段核苷酸序列分析结果表明：此1．3kbDNA片段中含有一个独立的ORF,起始密码ATG,终止密码TAG,含783bp;在起始密码上游有GGAGG5个核苷酸SD序列;此ORF编码260个氨基酸,与actⅢ基因编码的261个氨基酸相似性为77．4%,相同性为66．7％,对麦迪霉素产生苗酮基还原酶基因的可能作用进行了讨论。     

Leucine improves the component of isovalerylspiramycins for the production of bitespiramycin

Bitespiramycin, a group of 4″-O-acylated spiramycins with 4″-O-isovalerylspiramycins as the major components, is produced by recombinant spiramycin-producing strain Streptomyces spiramyceticus harboring a 4″-O-acyltransferase gene from a carbomycin-producing strain S. mycarofaciens 1748. The effects of leucine feeding on the bitespiramycin fermentation, especially the synthesis of isovalerylspiramycin components, were investigated. The experiment was initially performed in flask culture under the condition of feeding 15.4 mmol/l of leucine at 72 h fermentation, and the culture without leucine feeding was used as control. When 15.4 mmol/l leucine was fed at 72 h, 51.3 ± 0.33% total isovalerylspiramycins was recorded compared to 40.9 ± 0.26% under the control condition after 96 h of fermentation. The improvement of total isovalerylspiramycin content was further achieved in 15 l fermentation when 15.4 mmol/l of leucine was supplemented from 65 to 72 h. These results indicated that isovaleryl group derived from leucine catabolism could act as the precursor of the 4″ side chain of bitespiramycin, which profoundly enhanced the synthesis of isovalerylspiramycins in the bitespiramycin complex.     

麦迪霉素产生菌生米卡链霉菌1748变株68的特性研究

A stable mutant No. 68 was obtained by treatment of S. mycarofaciens 1748 spores at high temperature. The electromicroscopic examination has shown that the mutant No. 68 and parent strain 1748 both have the spore chains of the spiratype. The spores of both strain are cylindrical in shape. The only difference is that the spores of the mutant No. 68 are of smooth surface, but the 1748 are of thorny. The physiological characteristics of both strains are also very similar with slight differences in utilization of few carbon sources and in cultural characters in few medium. Feeding experiment has shown that the mutant No. 68 was blocked in the formation of the macrolide lactone in the midecamycin biosynthetic pathway. This suggested that the mutant No. 68 might be a polyketide synthase genes deficient mutant. The ability of the mutant No. 68 to convert spiramycin into 4"-propionylspiramycin indicated that the mutant No. 68 contained the midecamycin 4"-propionyltransferase and could be used for microbial bioconversion of spiramycin into 4"-propionylspiramycin.     

圈卷产色链霉菌分化相关基因——saw1的结构和功能

用双脱氧链终止法进行了分化基因——saw1的双链测序.结果表明在1500bp的DNA片段中有一个完整的开读框架(ORF),其编码区是在419bp至1252bp处.其产物与已知的天蓝色链霉菌whiG的氨基酸序列有89%的同源性.当把1500bp的saw1DNA片段插入到链霉菌表达质粒载体pIJ702后,构建的重组质粒转化天蓝色链霉菌孢子形成缺陷突变株C71,可使C71形成孢子和灰色色素.用基因破坏的策略进一步研究了该基因的生物学功能,结果表明saw1在圈卷产色链霉菌气生菌丝到孢子形成的发育转变中有重要作用,是分化中控制孢子发育起始的一个重要基因.     

Heterologous Expression of the Single-Mutation Glucose Isomerase (GIG138P) Gene in Streptomyces lividans and Its Genetic Instability

A 1.3-kb PstI-BamHI fragment containing the single-mutation glucose isomerase (GIG138P, GI1) gene and its natural promoter was inserted into PstI-BglII linearized Streptomyces vector pIJ702. The ligation mixture was then introduced into Streptomyces lividans TK54 protoplasts; transformants were identified based on their thiostrepton resistance (Th^R) and insertional inactivation of the melanin phenotype; and three white colonies, XY-2, 6, and 9, harboring recombinant expression plasmid pYH703, were obtained. Enzyme assay and SDS-PAGE analysis indicated that the GI1 gene was expressed, the intracellular GI1 specific activity was 6 U/mg, and GI1 accounted for 20% of the soluble proteins in S. lividans. Restriction analysis and Southern blot of pYH703 showed the existence of plasmid deletion, presumably owing to the interaction between the mel and GI1 sequences. Continuous liquid cultures of the recombinant strain demonstrated that the GI1 specific activity and GI1 expression in S. lividans decreased, and more obviously under non-selective conditions. Received: 10 August 2000 / Accepted: 5 September 2000     

The gene for indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans : molecular cloning, nucleotide sequence, and expression in Escherichia coli

A 5.5-kb DNA fragment containing the indole-3-acetyl-aspartic acid (IAA-asp) hydrolase gene (iaaspH) was isolated from Enterobacter agglomerans strain GK12 using a hybridization probe based on the N-terminal amino acid sequence of the protein. The DNA sequence of a 2.4-kb region of this fragment was determined and revealed a 1311-nucleotide ORF large enough to encode the 45-kDa IAA-asp hydrolase. A 1.5-kb DNA fragment containing iaaspH was subcloned into the Escherichia coli expression plasmid pTTQ8 to yield plasmid pJCC2. Extracts of IPTG-induced E. coli cultures containing the pJCC2 recombinant plasmid showed IAA-asp hydrolase levels 5 to 10-fold higher than those in E. agglomerans extracts. Homology searches revealed that the IAA-asp hydrolase was similar to a variety of amidohydrolases. In addition, IAA-asp hydrolase showed 70% sequence identity to a putative thermostable carboxypeptidase of E. coli. Received: 12 March 1998 / Accepted: 30 March 1998     

Identification of cry-Type Genes on 20-kb DNA Associated with Cry1 Crystal Proteins from Bacillus thuringiensis

Heterologous DNA fragments (20-kb) associated with Cry1 crystal proteins (protoxins) from a soil-isolated Bacillus thuringiensis strain 4.0718 were isolated and analyzed. RFLP patterns of the PCR products showed that the 20-kb DNA fragments harbored cry1Aa, cry1Ac, cry2Aa, and cry2Ab genes. Furthermore, a 4.2-kb DNA fragment, which contained the promoter, the coding region, and the terminator of cry1Ac gene, was cloned from the 20-kb DNAs by PCR, and then the cry1Ac gene was expressed in an acrystalliferous B. thuringiensis strain 4Q7 by using E. coli-B. thuringiensis shuttle vector pHT3101. SDS-PAGE and microscopy studies revealed that the recombinant could express 130-kDa Cry1Ac protoxin and produce bipyramidal crystals during sporulation. Bioassay results proved that crystal-spore mixture from the recombinant was toxic to Plutella xylostella. This was the first report of cry-type genes present on 20-kb DNA associated with Cry1 protoxins of B. thuringiensis.     

A histidine kinase sensor protein gene is necessary for induction of low pH tolerance in Sinorhizobium sp. strain BL3

The aim of this investigation was to identify and isolate genes involved in acid tolerance from Sinorhizobium sp. strain BL3. It was hypothesized that acid tolerance of strain BL3 could be enhanced by high level expression of certain genes involved in acid tolerance, following insertion of these genes in a multiple copy plasmid. A cosmid clone library of BL3 was introduced into BL3, and the transconjugant colonies were selected at low pH. A single cosmid containing genes for acid tolerance was isolated from 40 different colonies. By transposon–insertion mutagenesis, subcloning and DNA sequencing, a gene involved in acid tolerance, actX, was identified in a 4.4-kb fragment of this cosmid. The actX mutant of BL3 showed increased acid sensitivity and was complemented by the 4.4-kb subcloned fragment. Phaseolus lathyroides seedlings inoculated with recombinant strains containing multiple copies of actX showed increased symbiotic performance at low pH. By constructing an actX::gus fusion, it was shown that actX was induced at low pH. actX encodes a putative histidine kinase sensor protein of a two-component regulatory system. The method of gene identification used in this study for isolation of actX may be applied for the isolation of other genes involved in tolerance to adverse environmental factors.     

Secretory overproduction of Arthrobacter simplex 3-ketosteroid Δ1-dehydrogenase by Streptomyces lividans with a multi-copy shuttle vector

The gene for 3-ketosteroid ¹-dehydrogenase (ksdD) of Arthrobacter simplex was expressed in Streptomyces lividans and the secreted enzyme was overproduced by using a multi-copy shuttle vector composed of pIJ702 and pUC19. Deletional analysis of the recombinant plasmid showed that the entire coding sequence of the ksdD gene was located within a 7-kb segment of the chromosomal DNA obtained from the enzyme-producing strain of A. simplex. When S. lividans carrying the recombinant plasmid was grown in an appropriate medium, the cells produced about 100-fold more 3-ketosteroid ¹-dehydrogenase than the original strain. Although the percentage of enzyme secreted was changed during cultivation, a maximum 55% of the enzyme was secreted into the cultured medium of S. lividans, while A. simplex did not produce the enzyme extracellularly. Secretory overproduction of 3-ketosteroid ¹-dehydrogenase in S. lividans was also identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and on native gel, and the enzyme reaction was confirmed by reverse-phase HPLC using 4-androstene-3,17-dione as a substrate.     

酵母菌色氨酸合成酶基因的克隆与表达

用RemHI酶切酿酒酵母(Saceharomyces cercuisiae) 1412-4D染色体DNA,通过蔗糖梯度分离2-4kb DNA片段并插入穿棱质粒pCN60,构成1412-4D基因文库。从基因文库中提取重组质粒,转化受体菌C9(a,trp5,adcl,ade6),用直接功能互补法,分离到9株重组质粒,它们都含有3．2kb的TRP5 DNA片段,分别命名为pCN60(trps)1-90转化体中色氨酸合成酶的酶活水平比原始菌株1412-4D高3倍。     

Phage-mediated cloning of bldA, a region involved in Streptomyces coelicolor morphological development, and its analysis by genetic complementation.

Streptomyces coelicolor bald (bld) mutants form colonies of vegetative substrate mycelium, but do not develop aerial hyphae or spore chains. The bldA strains form none of the four antibiotics known to be produced by the parent strain. With a vector derived from the temperate bacteriophage phi C31, a 5.6-kilobase fragment of wildtype DNA was cloned which restored sporulation to five independent bldA mutants when lysogenized with the recombinant phage. The cloned gene(s) was dominant over the mutant alleles. Phage integration by recombination of the cloned bldA+ DNA with the bldA region of each mutant produced mainly sporulating colonies, presumably heterozygous bldA+/bldA partial diploids for the insert DNA. However, a minority of these primary transductants were bald and were apparently homozygous bldA/bldA mutant partial diploids, formed by some homogenetization process. The phages released from the bald lysogens carried bldA mutations and were used to show that bldA+ sequences had been cloned and that fine mapping of the region could be performed.     

Gene cloning of the maoA gene and overproduction of a soluble monoamine oxidase from Klebsiella aerogenes

Summary We cloned the structural gene for monoamine oxidase (maoA) from Klebsiella aerogenes into a pKI212 vector in an maoA mutant strain of K. aerogenes. Deletion analysis and complementation tests of the recombinant plasmid showed that the maoA gene was located entirely within a 4.1-kb segment. In an maoA mutant strain harbouring the cloned maoA gene, synthesis of monoamine oxidase was induced by addition of tyramine and related compounds. Transfer of a plasmid containing the maoA gene into a monoamine oxidase-producing strain of K. aerogenes W70 resulted in about a 30- to 40-fold increase in total production of the enzyme. When cells of K. aerogenes carrying the plasmid containing the maoA gene were grown with tyramine, more than 85% of the monoamine oxidase was produced in soluble form, whereas the parent strain W70 produced most monoamine oxidase as the membrane-bound form. Offprint requests to: Y. Murooka     

Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae

A sporulating culture ofBacillus thuringiensis subsp.kenyae strain HD549 is toxic to larvae of lepidopteran insect species such asSpodoptera litura, Helicoverpa armigera andPhthorimaea operculella, and a dipteran insect,Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed inE. coli. The recombinant protein produced 92% mortality in first-instar larvae ofSpodoptera litura and 86% inhibition of adult emergence inPhthorimaea operculella, but showed very low toxicity againstHelicoverpa armigera, and lower mortality against third-instar larvae of dipteran insectsCulex fatigans, Anopheles stephensi andAedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene fromBacillus thuringiensis var.kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.     

Cloning and analysis of a genomic fragment of Dictyostelium discoideum hybridizing to an RNA specifically accumulated in spore cells

A marker for Dictyostelium discoideum spore RNA differentiation has been isolated from a genomic DNA library by differential screening and a recombinant plasmid containing a genomic sequence complementary to spore specific RNA has been characterized. Only a small portion (~1 kb) of the 4.7-kb genomic insert is transcribed. The genomic organization of this spore specific gene shows a unique sequence flanked by reiterated sequences.     

Peroxyl Radical Scavenging Activity of Caffeic Acid and Its Related Phenolic Compounds in Solution

The esterase gene (est) of Pseudomonas putida MR-2068 was cloned into Escherichia coli JM109. An 8-kb inserted DNA directed synthesis of an esterase in E. coli. The esterase gene was in a 1.1-kb PstI-ClaI fragment within the insert DNA. The complete nucleotides of the DNA fragment containing the esterase gene were sequenced and found to include a single open reading frame of 828 bp coding for a protein of 276 amino acid residues. The open reading frame was confirmed by N-terminal amino acid sequence analysis of the purified esterase. A potential Shine-Dalgarno sequence is followed by the open reading frame. The esterase activity of the recombinant E. coli was more than 200 times higher than that of parental strain, P. putida MR-2068.     

A DNA Region That Complements on Escherichia coli cysG Mutation in Thiobacillus Ferrooxidans

Thiobacillus ferrooxidans AP19-3 has a novel NADH-dependent sulfite reductase in the periplasmic space. The gene responsible for the appearance of NADH-dependent sulfite reductase activity was cloned into a vector plasmid pBR322 to give a 5.7-kb hybrid plasmid, pTHS1, which contains a 1.3-kb DNA fragment of T. ferrooxidans AP19-3. When pTHS1 was used to transform sulfite reductase deficient E. coli mutants, strain AT2455 (cysG), JM246 (cysl), and AT2427 (cysJ), it complemented only the E. coli cysG mutation. Since cysG codes for S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase, the enzyme involved in siroheme synthesis, the results indicate that the DNA region that codes for S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase is present in a T. ferrooxidans 1.3 kb DNA fragment on pTHS1.     

Gene replacement method for determining conditions in which<Emphasis Type="Italic"> Bacillus subtilis</Emphasis> genes are essential or dispensable for cell viability

We describe a method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability. This method utilizes a chloramphenicol-resistant plasmid containing a temperature-sensitive (ts) replication origin. In this method, the target gene is first cloned into the ts vector and the recombinant plasmid is used to transform wild-type B. subtilis. The second step involves transformation of the resulting strain with a linear DNA fragment containing a second antibiotic resistance marker (tet) that disrupts the gene of interest. Selection for tetracycline resistance forces a double crossover between the chromosomal and fragment-borne copies of the gene, thereby replacing the wild-type gene in the chromosome with the disrupted allele. Cells survive even if the gene is essential by virtue of the complementing plasmid. Transformants are then grown at the non-permissive temperature for plasmid replication under various growth conditions. Isolation of chloramphenicol-sensitive colonies indicates that the gene is dispensable, whereas the inability to isolate chloramphenicol-sensitive colonies indicates that the gene is essential. The general utility of this method is demonstrated by allowing disruption of mtrA and trpE under conditions that render each gene non-essential, but not under growth conditions in which each gene is essential.