Molecular cloning of tylosin-producing Streptomyces fradiae B-45 genes determining increased inducible resistance to tylosin in Streptomyces lividans TK64

DNA of S. fradiae B-45 partially cleaved by Sau3A restrictase was cloned in S. lividans TK64 in the plasmid vector pIJ702. Three recombinant plasmids pVG251, pVG262, and pVG253 with tlr1, tlr2 and tlr3 genes were isolated from the transformed clones of S. lividans TK64 with higher inducible resistance to tylosin as compared to the plasmid-free strain. DNA-DNA blot hybridization was performed between the total DNA cleaved by several restrictases from S. fradiae B-45 and some other strains and the DNA probes containing the tlr genes. It was shown that tlr1 and tlr3 genes were unique in S. fradiae B-45. Sequences homologous to tlr2 gene were present both in DNA of S. fradiae B-45 in 7 copies and in strains of S. antibiotics and S. hygroscopicus producing respectively oleandomycin and turimycin.     

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase from Streptomyces cacaoi

A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of beta-lactamase was obtained from S. lividans strain ML1, carrying the recombinant plasmid pDML51, than from S. cacaoi grown under optimal production conditions. In all respects (molecular mass, isoelectric point, kinetics of inhibition by beta-iodopenicillanate) the overproduced S. lividans ML1 beta-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of beta-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the beta-lactamase structural gene. The beta-lactamase gene was located within a 1.8 NcoI-BclI fragment but when this fragment was cloned in S. lividans pIJ702, the resulting strain produced hardly any more beta-lactamase than the original S. cacaoi.     

Molecular cloning and expression of a Streptomyces sarcosine oxidase gene in Streptomyces lividans.

A genomic library of Streptomyces sp. KB210-8SY, prepared in the plasmid vector pACYC184, was screened to obtain the gene encoding sarcosine oxidase with probes based on the amino acid sequence of the protein. A plasmid pSOXS13, which was isolated from a clone identified by hybridization with the probes, contained a 8.4-kb insert of Streptomyces DNA. When the 2.0-kb MIuI/EcoRV DNA fragment of pSOXS13 was inserted into the Streptomyces vector pIJ680 and introduced into S. lividans, the transformants produced 100-fold more sarcosine oxidase intracellularly than KB210-8SY. The nucleotides of the 1.7-kb fragment containing the sarcosine oxidase gene were sequenced. An open reading frame encoded a mature sarcosine oxidase consisting of 388 amino acids, with a calculated molecular mass of 42,107 daltons.     

The tylosin-biosynthetic genes of Streptomyces fradiae

The tylosin-biosynthetic (tyl) gene cluster occupies about 1% of the genome of Streptomyces fradiae and includes at least 43 open reading frames. In addition to structural genes required for tylosin production, the tylcluster contains three resistance determinants and several regulatory genes. Tylosin production is evidently controlled by pathway-specific and pleiotropic regulators with the likely involvement of -butyrolactone signalling factors. Accumulation of the polyketide aglycone is controlled by glycosylated macrolides and optimal performance of the complex polyketide synthase enzyme requires the activity of an editing thioesterase.     

大鼠α-酰胺酶在变铅青链霉菌中的克隆及表达研究

α－酰胺酶（α－ａｍｉｄａｓｅ,α－ＡＥ）催化神经和内分泌系统中活性多肽的Ｃ－端酰胺化,多多肽的生物活性至关重要。以大鼠心房组织的总ＲＮＡ为模板,采用ＲＴ－ＰＣＲ技术扩增获得编码α－酰胺酶的ｃＤＮＡ,并进行了克隆和测序。为了使α－酰胺酶能在链霉菌中分泌表达,将其ｃＤＮＡ与链霉菌酷氮酶酶（ｍｅｌＣ１）信号的编码序列融合得到融合ｍｅｌ／ＡＥ,将ｍｅｌ／ＡＥ插入链霉菌质粒ｐＩＪ６８０,获得重组质粒ｐＩＪ     

Deamplification and deletion of amplified DNA in Streptomyces lividans and S. fradiae

Summary Streptomyces lividans arginine auxotrophs which show amplification of a 5.7-kb DNA sequence, arose at a very high frequency, varying from 10% to 25% of Cml^s spores. The amplifiable DNA sequence was shown to be stable over many generations. However, treatment of Cml^s arg mutants with subinhibitory concentrations of antibiotics such as spectinomycin, streptomycin, chloramphenicol, thiostrepton and kanamycin, either during sporulation or during vegetative growth of mycelia, led to the deletion of the entire amplified DNA sequence, including the left and right junction sequences. Depending upon the method of antibiotic treatment a reduction in the copy number of the amplified DNA was also observed. This reduction in copy number apparently occurred without drastically affecting the basic structure of the amplifiable unit of DNA. This phenomenon appears to be universal since deamplification and deletion were observed also in S. fradiae. Further, spontaneous arg mutants arose at much lower frequency from spectinomycin-pretreated Cml^s cells compared to untreated cells. These arg mutants isolated in the presence of spectinomycin did not show amplification of the 5.7-kb sequence. Southern blot analysis using the 5.7-kb probe showed that the entire DNA sequence homologous to the amplifiable DNA sequence had been deleted. Offprint requests to: K. Dharmalingam     

Cloning and heterologous expression in Streptomyces lividans of Streptomyces rimosus genes involved in oxytetracycline biosynthesis.

The anhydrotetracycline (ATC) oxygenase enzyme which carries out the conversion of ATC to dehydrotetracycline was purified and the N-terminal amino acid sequence was determined. The sequence displays a significant similarity to that of the p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. This is consistent with the activity of the oxygenase, i.e., addition of a hydroxyl moiety to an aromatic ring structure. Oligonucleotide probes were designed and used to clone the corresponding fragment of chromosomal DNA from Streptomyces rimosus. This DNA fragment was used to screen a cosmid library, allowing the isolation of flanking DNA sequences. Surprisingly, the gene was located within the previously cloned cluster of genes involved in the synthesis of the biosynthetic intermediate ATC and not as had been expected (P. M. Rhodes, N. Winskill, E. J. Friend, and M. Warren, J. Gen. Microbiol. 124:329-338, 1981) at a separate locus on the other side of the chromosome. Subcloning of an appropriate DNA fragment from one of the cosmid clones onto pIJ916 produced Streptomyces lividans transformants which synthesized oxytetracycline.     

The mycinose-biosynthetic genes of Streptomyces fradiae, producer of tylosin

The tylE-J region of the tylosin-biosynthetic gene cluster of Streptomyces fradiae contains six open reading frames. The products of tylJ and tylD are nucleoside diphospho (NDP)-deoxyhexose 3-epimerase and NDP-deoxyhexose 4-ketoreductase, respectively, involved in the synthesis of NDP-6-deoxyallose from NDP-4-keto, 6-deoxyglucose. After incorporation of deoxyallose at C23-OH of the polyketide lactone, tylosin biosynthesis is completed by the products of tylE and tylF, which convert the deoxyallosyl moiety to mycinose via bis-O-methylation at 2-OH and 3-OH, respectively. Hydroxylation of the polyketide lactone at C23 is catalysed by the cytochrome P450 enzyme, TylHl. The product of tylHll is a ferredoxin of unknown specificity that could conceivably act together with TylHl. Received 17 March 1999/ Accepted in revised form 20 June 1999     

Cloning of Streptomyces griseus and Streptomyces lividans genes for glycerol dissimilation

Streptomyces lividans gyl DNA (for glycerol utilisation) was cloned by complementation of a Streptomyces coelicolor gyl mutant. Restriction mapping showed that the cloned DNA was highly homologous (perhaps 99%) to S. coelicolor gyl DNA. Using phage-mediated mutational cloning, an internal fragment of the S. coelicolor gyl operon was used to generate a gyl mutant of S. lividans, which subsequently served as recipient in the cloning of gyl DNA from S. griseus. A 7.5-kb SstI-generated fragment of S. griseus DNA was obtained which, as judged by analysis of restriction sites, was only perhaps 87% homologous with the S. coelicolor gyl operon. The cloned S. griseus DNA appears to contain intact gylA and gylB genes and probably also an upstream gene related to the putative gyl regulatory '0.9-kb' gene of S. coelicolor. Cloning of the fragment on a high-copy-number vector in S. lividans did not lead to high levels of the enzymes encoded by gylA and gylB. The S. griseus gylA and gylB genes were not detectably expressed in Escherichia coli glp mutants.     

Molecular cloning and expression in Streptomyces lividans of a proteinous alpha-amylase inhibitor (HaimII) gene from Streptomyces griseosporeus

The gene encoding a proteinous alpha-amylase inhibitor (HaimII) of Streptomyces griseosporeus YM-25 has been cloned in Escherichia coli K12 using a deoxyinosine-containing synthetic oligonucleotide as the probe. A 1.6 kilobases BamHI fragment was confirmed to hybridize with the probe and subcloned in an E. coli-S. lividans shuttle vector. The plasmid clone was transferred into S. lividans by transformation. An appreciable amount of alpha-amylase inhibitor activity was found in the culture medium of S. lividans harboring the plasmid. As the specificity was indistinguishable from that of HaimII produced by the original S. griseosporeus strain, we concluded that the HaimII protein was synthesized in S. lividans and excreted into the medium.     

灰色链霉菌胰蛋白酶在变铅青链霉菌中的异源表达及酶学性质分析

胰蛋白酶作为一种重要的丝氨酸蛋白酶被广泛应用于食品、医药和皮革等工业领域.本文成功实现了灰色链霉菌来源的胰蛋白编码基因在变铅青链霉菌中的高效活性表达,并对其酶学性质进行分析比较.以灰色链霉菌ATCC10137基因组为模板,获得胰蛋白酶编码基因sprT并克隆至表达质粒pIJ86,成功构建了重组链霉菌工程菌TK24/pIJ86-sprT.以R2YE和SELF为发酵培养基,最高酶活分别达9.21 U/mL和8.61 U/mL.酶学性质分析表明,和牛胰蛋白酶(BT)相比,重组链霉菌胰蛋白酶(rSGT)的耐酸能力强,具有较广的pH;且rSGT对酰胺键具有更高的特异性;此外,Zn2+和有机溶剂分别对rSGT的酯酶活力和酰胺酶活力具有促进作用;本研究结果为rSGT的性质改造以及工业应用提供了依据.     

Gene cloning and heterologous expression of a serine protease from Streptomyces fradiae var.k11

The gene sfp1, which encodes a predicted serine proteinase designated SFP1, was isolated by the screening of a gene library of the feather-degrading strain Streptomyces fradiae var.k11. The open reading frame of sfp1 encodes a protein of 454 amino acids with a calculated molecular mass of 46.19 kDa. Sequence analysis reveals that SFP1 possesses a typical pre-pro-mature organization that consists of a signal sequence, an N-terminal propeptide region, and a mature proteinase domain. The pre-enzyme of SFP1 was expressed in Escherichia coli and consequently purified. The 25.6 kDa fraction with protease activity separated by gel filtration chromatography indicated that the mature enzyme of SFP1 was formed by autolysis of the propeptide after its expression. The purified SFP1 is active under a broad range of pH and temperature. SFP1 has pH and temperature optima of pH 8.5 and 65 degrees C for its caseinolytic activity and pH 9 and 62 degrees C for its keratinolytic activity. SFP1 was sharply inhibited by the serine proteinase inhibitor phenylmethyl sulfonyl fluoride and exhibited a good stability to solvents, detergents, and salts. Comparison of the protease activity of SFP1 with other commercial proteases indicates that SFP1 has a considerable caseinolytic and keratinolytic activity as does proteinase K.