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1.
Cloning and characterization of Streptomyces galilaeus aclacinomycins polyketide synthase (PKS) cluster 总被引:1,自引:0,他引:1
We have cloned and sequenced polyketide synthase (PKS) genes from the aclacinomycin producer Streptomyces galilaeus ATCC 31,615. The sequenced 13.5-kb region contained 13 complete genes. Their organization as well as their protein sequences showed high similarity to those of other type II PKS genes. The continuous region included the genes for the minimal PKS, consisting of ketosynthase I (aknB), ketosynthase II (aknC), and acyl carrier protein (aknD). These were followed by the daunomycin dpsC and dpsD homologues (aknE2 and F, respectively), which are rare in type II PKS clusters. They are associated with the unusual starter unit, propionate, used in the biosynthesis of aklavinone, a common precursor of aclacinomycin and daunomycin. Accordingly, when aclacinomycins minimal PKS genes were substituted for those of nogalamycin in the plasmid carrying genes for auramycinone biosynthesis, aklavinone was produced in the heterologous hosts. In addition to the minimal PKS, the cloned region included the PKS genes for polyketide ketoreductase (aknA), aromatase (aknE1) and oxygenase (aknX), as well as genes putatively encoding an aklanonic acid methyl transferase (aknG) and an aklanonic acid methyl ester cyclase (aknH) for post-polyketide steps were found. Moreover, the region carried genes for an activator (aknI), a glycosyl transferase (aknK) and an epimerase (aknL) taking part in deoxysugar biosynthesis. 相似文献
2.
Nucleotide sequence of the aknA region of the aklavinone biosynthetic gene cluster of Streptomyces galilaeus. 总被引:3,自引:2,他引:1
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A 3.4-kb BamHI fragment that is assumed to be a part of the aklavinone biosynthetic gene cluster of Streptomyces galilaeus 3AR-33 and contains the genes required for the early stage of polyketide biosynthesis was sequenced. The nucleotide sequence of the region that hybridizes to the actIII probe reveals the presence of a gene, aknA, whose deduced protein product is very similar to the ActIII protein and other known oxidoreductases. The predicted AknA protein is believed to be responsible for catalyzing the reduction of the keto group at the ninth carbon from the carboxyl terminus of the assembled polyketide to the corresponding secondary alcohol. The predicted AknA protein has a calculated molecular mass of 27,197 Da (261 amino acids) and the highly conserved sequence Gly-Xaa-Gly-Xaa-Xaa-Ala commonly seen in oxidoreductases. Cloning and sequence analysis of the aknA region of the 2-hydroxyaklavinone-producing strain S. galilaeus ANR-58 identified an alteration in the gene, confirming that the aknA gene is essential for aklavinone biosynthesis. 相似文献
3.
Cosynthesis of anthracycline compounds was followed in five phenotypic groups of mutants of Streptomyces coeruleorubidus (A--E), blocked in the biosynthesis of the daunomycine complex, and in two mutant types of Streptomyces galilaeus (F, G) blocked in the biosynthesis of glycosides of epsilon-pyrromycinone and aklavinone. Glycosides of daunomycinone and 13-dihydrodaunomycinone were produced in combinations A+B, A+C, A+D, A+E and A+F, epsilon-rhodomycinone was synthesized in combinations A+E, A+F, B+E and B+F. During the cultivation of types B--E with type G or F non-anthracycline compounds, typical of S. galilaeus, were cosynthesized. No cosynthesis could be observed in other combinations of the mutant types. Negative results were also obtained with combinations of mutants of the same group and during cultivation of all mutant types with streptomycetes not producing anthracyclines. A scheme illustrating metabolic pathways leading to the biosynthesis of daunomycinone, aklavinone, epsilon-rhodomycinone in S. coeruleorubidus and S. galilaeus was constructed. 相似文献
4.
Gullón S Olano C Abdelfattah MS Braña AF Rohr J Méndez C Salas JA 《Applied and environmental microbiology》2006,72(6):4172-4183
The biosynthetic gene cluster for the aromatic polyketide steffimycin of the anthracycline family has been cloned and characterized from "Streptomyces steffisburgensis" NRRL 3193. Sequence analysis of a 42.8-kbp DNA region revealed the presence of 36 open reading frames (ORFs) (one of them incomplete), 24 of which, spanning 26.5 kb, are probably involved in steffimycin biosynthesis. They code for all the activities required for polyketide biosynthesis, tailoring, regulation, and resistance but show no evidence of genes involved in L-rhamnose biosynthesis. The involvement of the cluster in steffimycin biosynthesis was confirmed by expression of a region of about 15 kb containing 15 ORFS, 11 of them forming part of the cluster, in the heterologous host Streptomyces albus, allowing the isolation of a biosynthetic intermediate. In addition, the expression in S. albus of the entire cluster, contained in a region of 34.8 kb, combined with the expression of plasmid pRHAM, directing the biosynthesis of L-rhamnose, led to the production of steffimycin. Inactivation of the stfX gene, coding for a putative cyclase, revealed that this enzymatic activity participates in the cyclization of the fourth ring, making the final steps in the biosynthesis of the steffimycin aglycon similar to those in the biosynthesis of jadomycin or rabelomycin. Inactivation of the stfG gene, coding for a putative glycosyltransferase involved in the attachment of L-rhamnose, allowed the production of a new compound corresponding to the steffimycin aglycon compound also observed in S. albus upon expression of the entire cluster. 相似文献
5.
Biosynthesis of anthraquinones by interspecies cloning of actinorhodin biosynthesis genes in streptomycetes: clarification of actinorhodin gene functions. 总被引:4,自引:10,他引:4
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P L Bartel C B Zhu J S Lampel D C Dosch N C Connors W R Strohl J M Beale Jr H G Floss 《Journal of bacteriology》1990,172(9):4816-4826
Streptomyces galilaeus ATCC 31133 and ATCC 31671, producers of the anthracyclines aclacinomycin A and 2-hydroxyaklavinone, respectively, formed an anthraquinone, aloesaponarin II, when they were transformed with DNA from Streptomyces coelicolor containing four genetic loci, actI, actIII, actIV, and actVII, encoding early reactions in the actinorhodin biosynthesis pathway. Subcloning experiments indicated that a 2.8-kilobase-pair XhoI fragment containing only the actI and actVII loci was necessary for aloesaponarin II biosynthesis by S. galilaeus ATCC 31133. Aloesaponarin II was synthesized via the condensation of 8 acetyl coenzyme A equivalents, followed by a decarboxylation reaction as demonstrated by [1,2-13C2]acetate feeding experiments. S. coelicolor B22 and B159, actVI blocked mutants, also formed aloesaponarin II as an apparent shunt product. Mutants of S. coelicolor blocked in several other steps in actinorhodin biosynthesis did not synthesize aloesaponarin II or other detectable anthraquinones. When S. galilaeus ATCC 31671 was transformed with the DNA carrying the actI, actIII, and actVII loci, the recombinant strain produced both aloesaponarin II and aklavinone, suggesting that the actinorhodin biosynthesis DNA encoded a function able to deoxygenate 2-hydroxyaklavinone to aklavinone. When S. galilaeus ATCC 31671 was transformed with a plasmid carrying only the intact actIII gene (pANT45), aklavinone was formed exclusively. These experiments indicate a function for the actIII gene, which is the reduction of the keto group at C-9 from the carboxy terminus of the assembled polyketide to the corresponding secondary alcohol. In the presence of the actIII gene, anthraquinones or anthracyclines formed as a result of dehydration and aromatization lack an oxygen function on the carbon on which the keto reductase operated. When S. galilaeus ATCC 31671 was transformed with the DNA carrying the actI, actVII, and actIV loci, the recombinant strain produced two novel anthraquinones, desoxyerythrolaccin, the 3-hydroxy analog of aloesaponarin II, and 1-O-methyldesoxyerythrolaccin. The results obtained in these experiments together with earlier data suggest a pathway for the biosynthesis of actinorhodin and related compounds by S. coelicolor. 相似文献
6.
A glycosyltransferase gene, rhoG, involved in the biosynthesis of the anthracycline antibiotic beta-rhodomycin was isolated as a 4.1-kb DNA fragment containing rhoG and its flanking region from Streptomyces violaceus by degenerate and inverse PCR. Sequencing analysis showed that rhoG was located in a gene cluster involved in the biosynthesis of the constitutive deoxysugar of beta-rhodomycin. The function of rhoG was verified by gene disruption, which was generated by replacing the internal 0.9-kb region of S. violaceus chromosome with a fragment including the SacI-blunted region. The rhoG disruption resulted in complete loss of beta-rhodomycin productivity, along with the accumulation of a non-glycosyl intermediate epsilon-rhodomycinone. In addition, the complementation test demonstrated that rhoG restored beta-rhodomycin production in this gene disruptant. These results indicated that rhoG is the glycosyltransferase gene responsible for the glycosylation of epsilon-rhodomycinone in beta-rhodomycin biosynthesis. 相似文献
7.
S. Torkkell T. Kunnari K. Palmu P. Mäntsälä J. Hakala K. Ylihonko 《Molecular genetics and genomics : MGG》2001,266(2):276-288
Fragments spanning 20 kb of Streptomyces nogalater genomic DNA were characterized to elucidate the molecular genetic basis of the biosynthetic pathway of the anthracycline antibiotic nogalamycin. Structural analysis of the products obtained by expression of the fragments in S. galilaeus and S. peucetius mutants producing aclacinomycin and daunomycin metabolites, respectively, revealed hybrid compounds in which either the aglycone or the sugar moiety was modified. Subsequent sequence analysis revealed twenty ORFs involved in nogalamycin biosynthesis, of which eleven could be assigned to the deoxysugar pathway, four to aglycone biosynthesis, while the remaining five express products with unknown function. On the basis of sequence similarity and experimental data, the functions of the products of the newly discovered genes were determined. The results suggest that the entire biosynthetic gene cluster for nogalamycin is now known. Furthermore, the compounds obtained by heterologous expression of the genes show that it is possible to use the genes in combinatorial biosynthesis to create novel chemical structures for drug screening purposes. 相似文献
8.
Identification and characterization of a new exopolysaccharide biosynthesis gene cluster from Streptomyces 总被引:4,自引:0,他引:4
We report the identification and characterization of the ste (Streptomyces eps) gene cluster of Streptomyces sp. 139 required for exopolysaccharide (EPS) biosynthesis. This report is the first genetic work on polysaccharide production in Streptomyces. To investigate the gene cluster involved in exopolysaccharide 139A biosynthesis, degenerate primers were designed to polymerase chain reaction amplify an internal fragment of the priming glycosyltransferase gene that catalyzes the first step in exopolysaccharide biosynthesis. Screening of a genomic library of Streptomyces sp. 139 with this polymerase chain reaction product as probe allowed the isolation of a ste gene cluster containing 22 open reading frames similar to polysaccharide biosynthesis genes of other bacterial species. Involvement of the ste gene cluster in exopolysaccharide biosynthesis was confirmed by disrupting the priming glycosyltransferase gene in Streptomyces sp. 139 to generate non-exopolysaccharide-producing mutants. 相似文献
9.
Cloning, sequencing, and analysis of aklaviketone reductase from Streptomyces sp. strain C5. 总被引:4,自引:4,他引:0
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DNA sequence analysis of a region of the Streptomyces sp. strain C5 daunomycin biosynthesis gene cluster, located just upstream of the daunomycin polyketide biosynthesis genes, revealed the presence of six complete genes. The two genes reading right to left include genes encoding the potentially translationally coupled gene products, an acyl carrier protein and a ketoreductase, and the four genes reading divergently, left to right, include two open reading frames of unknown function followed by a gene encoding an apparent glycosyltransferase and dauE, encoding aklaviketone reductase. Extracts of Streptomyces lividans TK24 containing recombinant DauE catalyzed the NADPH-specific conversion of aklaviketone, maggiemycin, and 7-oxodaunomycinone to aklavinone, epsilon-rhodomycinone, and daunomycinone, respectively. Neither the product of dauB nor that of the ketoreductase gene directly downstream of the acyl carrier protein gene demonstrated aklaviketone reductase activity. 相似文献
10.
The ability to transorm biologically exogenous daunomycinone, 13-dihydrodaunomycinone, aklavinone, 7-deoxyaklavinone, epsilon-rhodomycinone, epsilon-isorhodomycinone and epsilon-pyrromycinone was studied in submerged cultures of the following strains: wild Streptomyces coeruleorubidus JA 10092 (W1) and its improved variants 39-146 and 84-17 (type P1) producing glycosides of daunomycinone and of 13-dihydrodaunomycinone, together with epsilon-rhodomycinone, 13-dihydrodaunomycinone and 7-deoxy-13-dihydrodaunomycinone; in five mutant types of S. coeruleorubidus (A, B, C, D, E) blocked in the biosynthesis of glycosides and differing in the production of free anthracyclinones; in the wild Streptomyces galilaeus JA 3043 (W2) and its improved variant G-167 (P2) producing glycosides of epsilon-pyrromycinone and of aklavinone together with 7-deoxy and bisanhydro derivatives of both aglycones; in two mutant types S. galilaeus (F and G) blocked in biosynthesis of glycosides and differing in the occurrence of anthracyclinones. The following bioconversions were observed: daunomycinone leads to 13-dihydrodaunomycinone and 7-deoxy-13-dihydrodaunomycinone (all strains); 13-dihydrodaunomycinone leads to 7-deoxy-13-dihydrodaunomycinone (all strains); daunomycinone or 13-dihydrodaunomycinone leads to glycosides of daunomycinone and of 13-dihydrodaunomycinone, identical with metabolites W1 and P1 (type A), or only a single glycoside of daunomycinone (type E); aklavinone leads to epsilon-rhodomycinone (types A and B); aklaviinone leads to 7-deoxyaklavinone and bisanhydroaklavinone (type C); epsilon-rhodomycinone leads to zeta-rhodomycinone (types C, E); epsilon-rhodomycinone leads to glycosides of epsilon-rhodomycinone (types W2, P2); epsilon-isorhodomycinone leads to glycosides of epsilon-isorhodomycinone (types W2, P2); epsilon-pyrromycinone leads to a glycoside of epsilon-pyrromycinone (types W1, P1). 7-Deoxyaklavinone remained intact in all tests. Exogenous daunomycinone suppressed the biosynthesis of its own glycosides in W1 and P1; it simultaneously increased the production of epsilon-rhodomycinone in P1. 相似文献
11.
Tao M Wang L Wendt-Pienkowski E George NP Galm U Zhang G Coughlin JM Shen B 《Molecular bioSystems》2007,3(1):60-74
The tallysomycins (TLMs) belong to the bleomycin (BLM) family of antitumor antibiotics. The BLM biosynthetic gene cluster has been cloned and characterized previously from Streptomyces verticillus ATCC 15003, but engineering BLM biosynthesis for novel analogs has been hampered by the lack of a genetic system for S. verticillus. We now report the cloning and sequencing of the TLM biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 and the development of a genetic system for S. hindustanus, demonstrating the feasibility to manipulate TLM biosynthesis in S. hindustanus by gene inactivation and mutant complementation. Sequence analysis of the cloned 80.2 kb region revealed 40 open reading frames (ORFs), 30 of which were assigned to the TLM biosynthetic gene cluster. The TLM gene cluster consists of nonribosomal peptide synthetase (NRPS) genes encoding nine NRPS modules, a polyketide synthase (PKS) gene encoding one PKS module, genes encoding seven enzymes for deoxysugar biosynthesis and attachment, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The involvement of the cloned gene cluster in TLM biosynthesis was confirmed by inactivating the tlmE glycosyltransferase gene to generate a TLM non-producing mutant and by restoring TLM production to the DeltatlmE::ermE mutant strain upon expressing a functional copy of tlmE. The TLM gene cluster is highly homologous to the BLM cluster, with 25 of the 30 ORFs identified within the two clusters exhibiting striking similarities. The structural similarities and differences between TLM and BLM were reflected remarkably well by the genes and their organization in their respective biosynthetic gene clusters. 相似文献
12.
麦迪霉素产生菌酮基还原酶基因的研究 总被引:3,自引:0,他引:3
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。 相似文献
13.
K. Ylihonko J. Tuikkanen S. Jussila L. Cong P. Mäntsälä 《Molecular genetics and genomics : MGG》1996,251(2):113-120
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. 相似文献
14.
The rdm genes B, C and E from Streptomyces purpurascens encode enzymes that tailor aklavinone and aclacinomycins. We report that in addition to hydroxylation of aklavinone to epsilon-rhodomycinone, RdmE (aklavinone-11-hydroxylase) hydroxylated 11-deoxy-beta-rhodomycinone to beta-rhodomycinone both in vivo and in vitro. 15-Demethoxyaklavinone and decarbomethoxyaklavinone did not serve as substrates. RdmC (aclacinomycin methyl esterase) converted aclacinomycin T (AcmT) to 15-demethoxyaclacinomycin T, which was in turn converted to 10-decarbomethoxyaclacinomycin T and then to rhodomycin B by RdmB (aclacinomycin-10-hydroxylase). RdmC and RdmB were most active on AcmT, the one-sugar derivative, with their activity decreasing by 70-90% on two- and three-sugar aclacinomycins. Aclacinomycin A competitively inhibited the AcmT modifications at C-10. The results presented here suggest that in vivo the modifications at C-10 take place principally after addition of the first sugar. 相似文献
15.
K. Ylihonko J. Tuikkanen S. Jussila L. Cong P. Mäntsälä 《Molecular & general genetics : MGG》1996,251(2):113-120
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. 相似文献
16.
Isolation and sequence analysis of polyketide synthase genes from the daunomycin-producing Streptomyces sp. strain C5. 总被引:7,自引:5,他引:2
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J Ye M L Dickens R Plater Y Li J Lawrence W R Strohl 《Journal of bacteriology》1994,176(20):6270-6280
A contiguous region of about 30 kbp of DNA putatively encoding reactions in daunomycin biosynthesis was isolated from Streptomyces sp. strain C5 DNA. The DNA sequence of an 8.1-kbp EcoRI fragment, which hybridized with actI polyketide synthase (PKS) and actIII polyketide reductase (PKR) gene probes, was determined, revealing seven complete open reading frames (ORFs), two in one cluster and five in a divergently transcribed cluster. The former two genes are likely to encode PKR and a bifunctional cyclase/dehydrase. The five latter genes encode: (i) a homolog of TcmH, an oxygenase of the tetracenomycin biosynthesis pathway; (ii) a PKS Orf1 homolog; (iii) a PKS Orf2 homolog (chain length factor); (iv) a product having moderate sequence identity with Escherichia coli beta-ketoacyl acyl carrier protein synthase III but lacking the conserved active site; and (v) a protein highly similar to several acyltransferases. The DNA within the 8.1-kbp EcoRI fragment restored daunomycin production to two dauA non-daunomycin-producing mutants of Streptomyces sp. strain C5 and restored wild-type antibiotic production to Streptomyces coelicolor B40 (act VII; nonfunctional cyclase/dehydrase), and to S. coelicolor B41 (actIII) and Streptomyces galilaeus ATCC 31671, strains defective in PKR activity. 相似文献
17.
18.
19.
Expression of polyketide biosynthesis and regulatory genes in heterologous streptomycetes 总被引:3,自引:0,他引:3
William R. Strohl Paul L. Bartel Yun Li Neal C. Connors Robert H. Woodman 《Journal of industrial microbiology & biotechnology》1991,7(3):163-174
Summary There are now several examples showing that hybrid secondary metabolites can be produced as a result of interspecies cloning of antibiotic biosynthesis genes in streptomycetes. This paper reviews examples of hybrid secondary metabolite production, and examines the underlying biochemical and regulatory principles leading to the formation of hybrid anthraquinones by recombinant anthracycline-producing streptomycetes carrying actinorhodin biosynthesis genes. An anthraquinone, aloesaponarin II, was produced by cloning theactI, actIII, actIV, andactVII genes (pANT12) of actinorhodin biosynthesis pathway fromStreptomyces coelicolor in anthracycline producing streptomycetes.Streptomyces galilaeus strains 31 133 and 31 671, aclacinomycin and 2-hydroxyaklavinone producers, respectively, formed aloesaponarin II as their major polyketide product when transformed with pANT12. Subcloning experiments indicated that a 2.8-kbXhoI fragment containing only theactI andactVII loci was necessary for aloesaponarin II biosynthesis byS. galilaeus 31 133. WhenS. galilaeus 31 671 was transformed with theactI, actVII, andactIV genes, however, the recombinant strain produced two novel anthraquinones, desoxyerythrolaccin and 1-0-methyldesoxyerythrolaccin. WhenS. galilaeus 31671 was transformed with only the intactactIII gene (pANT45), aklavinone was formed exclusively. These experiments indicate a function for theactIII gene, which is the reduction of the keto group at C-9 from the carboxyl terminus of the assembled polyketide to the corresponding secondary alcohol. The effects of three regulatory loci,dauG, dnrR1, andasaA, on the production of natural and hybrid polyketides were also shown. 相似文献
20.
Richard van Kranenburg Harmjan R. Vos Iris I. van Swam Michiel Kleerebezem Willem M. de Vos 《Journal of bacteriology》1999,181(20):6347-6353
Sixteen exopolysaccharide (EPS)-producing Lactococcus lactis strains were analyzed for the chemical compositions of their EPSs and the locations, sequences, and organization of the eps genes involved in EPS biosynthesis. This allowed the grouping of these strains into three major groups, representatives of which were studied in detail. Previously, we have characterized the eps gene cluster of strain NIZO B40 (group I) and determined the function of three of its glycosyltransferase (GTF) genes. Fragments of the eps gene clusters of strains NIZO B35 (group II) and NIZO B891 (group III) were cloned, and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase involved in the second step of repeating-unit synthesis. The NIZO B40 priming glucosyltransferase gene epsD was replaced with an erythromycin resistance gene, and this resulted in loss of EPS production. This epsD deletion was complemented with priming GTF genes from gram-positive organisms with known function and substrate specificity. Although no EPS production was found with priming galactosyltransferase genes from L. lactis or Streptococcus thermophilus, complementation with priming glucosyltransferase genes involved in L. lactis EPS and Streptococcus pneumoniae capsule biosynthesis could completely restore or even increase EPS production in L. lactis. 相似文献