Two genes, rif15 and rif16, of the rifamycin biosynthetic gene cluster in Amycolatopsis mediterranei likely encode a transketolase and a P450 monooxygenase, respectively, both essential for the conversion of rifamycin SV into B

Amycolatopsis mediterranei produces an important antibiotic rifamycin, the biosynthesis of which involves many unusual modifications. Previous work suggested a putative P450 enzyme encoded by rif16 within the rifamycin biosynthetic gene cluster (rif) was required for the conversion of the intermediate rifamycin SV into the end product rifamycin B. In this study, we genetically proved that a putative transketolase encoded by rif15 is another essential enzyme for this conversion. Expression of merely rif15 and rif16 in a rif cluster null mutant of A. mediterranei U32 was able to convert rifamycin SV into B. However, this Rif15- and Rif16-mediated conversion was only detected in intact cells of A. meidterranei, but not in Streptomyce coelicolor or Mycobacterium smegmatis, suggesting that yet-characterized gene(s) in A. mediterranei other than those encoded by the rif cluster should be involved in this process.     

地中海拟无枝菌酸菌U-32对硝酸盐的同化及其硝酸还原酶特性

对地中海拟无枝菌酸菌U-32菌株的研究发现,像植物及真菌硝酸还原酶一样,地中海拟无枝菌酸菌U-32硝酸还原酶也是诱导酶,其合成受铵盐阻遏,受硝酸盐的诱导。氯霉素抑制实验的结果表明,该菌株硝酸还原酶的诱导涉及到蛋白质的新合成。钼和钨的竞争实验说明U-32菌株硝酸还原酶也为一钼酶。另外在离体实验中,发现硝酸还原酶活力受到KCN和NADH的抑制,但至今未能找到其生理电子供体。此外,U-32菌株硝酸还原酶也不表现类似于植物的黄递酶等组份酶活性。该菌株硝酸还原酶和其力复霉素产量有一定相关性,但两者确切的关系尚待研究。     

Functional analysis of the Escherichia coli molybdopterin cofactor biosynthesis protein MoeA by site-directed mutagenesis

Five moeA mutants were generated by replacing some conserved amino acids of MoeA by site-directed mutagenesis. The mutants were assayed for the ability to restore in vivo nitrate reductase activity of the moeA mutant Escherichia coli JRG97 and in vitro Neurospora crassa nit-1 nitrate reductase activity. The replacements Asp59AlaGly60Ala, Asp259Ala, Pro298AlaPro301Ala abolished the function of MoeA in Mo-molybdopterin formation and stabilization, reflected in the inability to restore nitrate reductase activity. The replacements Gly251AlaGly252Ala reduced, and that of Pro283Ala had no effect, on nitrate reductase activity. E. coli JRG97 cells transformed with mutants that failed to restore nitrate reductase activity showed by HPLC analysis a decreased level of molybdopterin-derived dephospho FormA as compared to bacteria transformed with wild-type moeA. The effects of the amino acid replacements on MoeA function may be explained in correlation with the MoeA crystal structure.     

An eukaryotic-type serine/threonine protein kinase involved in the carbon source-dependent pigment biosynthesis in Amycolatopsis mediterranei U32

The structural gene, pkmA, was cloned and sequenced from a rifamycin SV-producing Amycolatopsis mediterranei U32 strain. The N-terminal portion of the deduced amino acid sequence of pkmA showed significant similarity to the family of serine/threonine protein kinases. It contains all the structural features which are highly conserved in protein kinases, including the Gly-X-Gly-X-X-Gly motif of ATP binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein specific kinases. The protein possesses a region rich in Ala and Pro residues around the middle of pkmA open reading frame, which might be involved in the transmembrane function, as suggested by PhoA fusion protein analysis. The pkmA gene was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein, and the protein was found to have the activity of autophosphorylation. A double crossover gene replacement was achieved by inserting an aparmycin resistance gene into pkmA in A. mediterranei chromosomal DNA. The phenotypic analysis of the mutant suggested that pkmA gene is involved in carbon source-dependent pigment formation in A. mediterranei U32.     

Effect of uracil on rifamycin SV production by Amycolatopsis mediterranei MV35R

The effect of different organic nitrogen compounds on the production of rifamycin SV by Amycolatopsis mediterranei MV35R and their optimum concentrations have been described. Results obtained indicate that rifamycin SV production increased from 4020 mg l-1 to 4575 mg l-1 when organic nitrogen compound uracil was added at 0.2% (w/v) concentration to the fermentation medium by A. mediterranei MV35R. The rifamycin SV yield was enhanced by 505 mg l-1 using uracil (2 g l-1) when compared with barbital.     

Improvement of industry-applied rifamycin B-producing strain,Amycolatopsis mediterranei,by rational screening

An industrially applied rifamycin B-producing strain, Amycolatopsis mediterranei XC 1-02, was used for further screening. A special mutation and screening procedure was adopted to select a strain, which can alleviate the inhibition caused by both aromatic amino acid and p-hydroxybenzoic acid in the pathway of rifamycin B biosynthesis as well as enhance the production of propionate, one of the precursors of rifamycin B biosynthesis. By the above methods, a strain A. mediterranei XC 9-25 was obtained, and its rifamycin B productivity in shaking flask reaches 10 g/L, which is 2.38 times higher than that of the ancestral strain XC 1-02. The productivity of rifamycin B fed-batch fermentation in 60000 L fermentor with A. mediterranei XC 9-25 reached 19.11 g/L.     

地中海拟无枝菌酸菌U32中amrC／amkC基因的序列分析及在大肠杆 …

从力复霉素ＳＶ产生菌－－地中海拟无枝菌酸菌（Ａｍｙｃｏｌａｔｏｐｓｉｓ ｍｅｄｉｔｅｒｒａｎｅｉ）Ｕ３２的硝酸盐同化基因簇的上游克隆了一个２．６ｋｂ的ＥｃｏＲＩ－ＸｈｏＩ ＤＮＡ片段并测定其序列。序列分析表明,该ＤＮＡ片段编码两个完整的开放阅读框架（ＯＲＦ）,ＯＲＦ２的起始密码子ＧＴＧ与ＯＲＦ１的终止密码子ＴＧＡ在ＴＧ处重叠。ＯＲＦ１编码一个含２２４个氨基酸的多肽,它同放线菌中典型的应答调节蛋白包     

地中海拟无枝菌酸菌U32染色体特性的脉冲场电泳分析

地中海拟无枝菌酸菌(Amycolatopsis mediterranei)U32是产力复霉素SV的工业生产菌株。采用脉冲场电泳分析发现,地中海拟无枝菌酸菌U32仅有一条约10 Mb的线性染色体, 没有内源性质粒。利用Southern杂交法,对11个编码力复霉素生物合成、相关初级、次级代谢关键酶以及调控蛋白的基因,在U32染色体DNA的PshBI酶切片段上进行了定位。分析发现在一条长度约700kb的PshBI酶切片段上,分别存在着力复霉素合成基因簇(rif)、氮代谢的亚硝酸还原酶小亚基基因(nasD)、衔接初级与次级代谢的甲基丙二酰变位酶基因(mcm)、脂肪酸代谢的乙酰辅酶A羧化酶生物素载体蛋白基因(accA)以及一套核糖体RNA转录单元。同时还发现U32至少有5套核糖体RNA转录单元。其余定位的基因均只出现单一杂交信号。     

Cloning and characterization of the chromosomal replication origin region of Amycolatopsis mediterranei U32

The chromosomal replication origins (oriC) of gram positive, acid-fast actinomycetes have been investigated in streptomycetes and mycobacteria. A 1339 bp DNA fragment of the putative oriC region from the rifamycin SV producer Amycolatopsis mediterranei U32 was cloned by PCR amplification employing primers designed based on the conserved flanking genes of dnaA and dnaN. The 884 bp sequence of the intergenic region between dnaA and dnaN genes consists of 19 DnaA-boxes and two 13-mer AT-rich sequences, which is similar to the oriC structure of Streptomyces lividans. A mini-chromosome constructed by cloning the putative U32 oriC DNA fragment into an Escherichia coli plasmid was able to replicate autonomously, but was unstable, in A. mediterranei U32 with an estimated copy number of two per cell. Although efficient replication of the mini-chromosome in U32 requires the complete set of DnaA-boxes and AT-rich regions, only one of the AT-rich sequences together with part of the DnaA-boxes is sufficient, suggesting the presence of combinatorial alternatives for a functional oriC region of A. mediterranei U32. Phylogenetic analysis based on definite oriC sequences among eubacteria reflects well the relationship between these species.     

Production of rifamycin SV using mutant strains of Amycolatopsis mediterranei MTCC17

Production of rifamycin SV using mutant strains of Amycolatopsis mediterranei (MTCC17) has been studied. Attempts were made to increase the productivity of rifamycin SV by ultraviolet radiation and specific screening programmes for selection of superior producers. Among 20 morphologically variant mutants of A. mediterranei MTCC17 isolated, four mutants showed increased resistance to the rifamycin SV. These selected mutant isolates increased the yield of rifamycin SV from 2000 to 3250mg/l.     

Isolation and characterization of 27-O-demethylrifamycin SV methyltransferase provides new insights into the post-PKS modification steps during the biosynthesis of the antitubercular drug rifamycin B by Amycolatopsis mediterranei S699

The gene rif orf14 in the rifamycin biosynthetic gene cluster of Amycolatopsis mediterranei S699, producer of the antitubercular drug rifamycin B, encodes a protein of 272 amino acids identified as an AdoMet: 27-O-demethylrifamycin SV methyltransferase. Frameshift inactivation of rif orf14 generated a mutant of A. mediterranei S699 that produces no rifamycin B, but accumulates 27-O-demethylrifamycin SV (DMRSV) as the major new metabolite, together with a small quantity of 27-O-demethyl-25-O-desacetylrifamycin SV (DMDARSV). Heterologous expression of rif orf14 in Escherichia coli yielded a 33.8-kDa polyhistidine-tagged polypeptide, which efficiently catalyzes the methylation of DMRSV to rifamycin SV, but not that of DMDARSV or rifamycin W. 27-O-Demethylrifamycin S was methylated poorly, if at all, by the enzyme to produce rifamycin S. The purified enzyme does not require a divalent cation for catalytic activity. While Ca(2+) or Mg(2+) inhibits the enzyme activity slightly, Zn(2+), Ni(2+), and Co(2+) are strongly inhibitory. The K(m) values for DMRSV and S-adenosyl-L-methionine (AdoMet) are 18.0 and 19.3 microM, respectively, and the K(cat) is 87s(-1). The results indicate that DMRSV is a direct precursor of rifamycin SV and that acetylation of the C-25 hydroxyl group must precede the methylation reaction. They also suggest that rifamycin S is not the precursor of rifamycin SV in rifamycin B biosynthesis, but rather an oxidative shunt-product.     

力复霉素前体甲基丙二酰CoA合成途径的研究

力复霉素合成的碳前体之一(2R)—甲基丙二酰CoA至少可以有三条酶学合成途径。三条途径中的关键酶分别为甲基丙二酰CoA转羧基酶、丙二酰CoA羧化酶、甲基丙二酰CoA变位酶和甲基丙二酰CoA消旋酶。通过比较各个酶活性的时间进程和力复霉素合成时间的相关性,以及各个酶的底物亲合力,对它们在地中海拟无枝酸菌(Amycolatopsis mediterranei)甲基丙二酰CoA合成中的贡献作了排序,发现甲基丙二酰CoA变位酶途径是主要负责酶系。但是各个途径的贡献排序并不是固定不变的,能受到环境因素的调控,丙酸盐的加入将抑制甲基丙二酰CoA变位酶活力,而使得甲基丙二酰CoA转羧基酶成为主要酶系。甲基丙二酰CoA合成途径的多样性有助于细胞对环境变化的灵活反应。此外,对各个酶的调控特性也进行了研究。     

Cloning and partial characterization of the putative rifamycin biosynthetic gene cluster from the actinomycete Amycolatopsis mediterranei DSM 46095.

The actinomycete Amycolatopsis mediterranei produces the commercially and medically important polyketide antibiotic rifamycin, which is widely used against mycobacterial infections. The rifamycin biosynthetic (rif) gene cluster has been isolated, cloned and characterized from A. mediterranei S699 and A. mediterranei LBGA 3136. However, there are several other strains of A. mediterranei which also produce rifamycins. In order to detect the variability in the rif gene cluster among these strains, several strains were screened by PCR amplification using oligonucleotide primers based on the published DNA sequence of the rif gene cluster and by using dEBS II (second component of deoxy-erythronolide biosynthase gene) as a gene probe. Out of eight strains of A. mediterranei selected for the study, seven of them showed the expected amplification of the DNA fragments whereas the amplified DNA pattern was different in strain A. mediterranei DSM 46095. This strain also showed striking differences in the banding pattern obtained after hybridization of its genomic DNA against the dEBS II probe. Initial cloning and characterization of the 4-kb DNA fragment from the strain DSM 46095, representing a part of the putative rifamycin biosynthetic cluster, revealed nearly 10% and 8% differences in the DNA and amino acid sequence, respectively, as compared to that of A. mediterranei S699 and A. mediterranei LBGA 3136. The entire rif gene cluster was later cloned on two cosmids from A. mediterranei DSM 46095. Based on the partial sequence analysis of the cluster and sequence comparison with the published sequence, it was deduced that among eight strains of A. mediterranei, only A. mediterranei DSM 46095 carries a novel rifamycin biosynthetic gene cluster.     

A Novel Two-Component System <Emphasis Type="Italic">amrB-amkB</Emphasis> Involved in the Regulation of Central Carbohydrate Metabolism in Rifamycin SV-Producing <Emphasis Type="Italic">Amycolatopsis mediterranei</Emphasis> U32

A novel two-component signal transduction system amrB-amkB was cloned from rifamycin SV-producing Amycolatopsis mediterranei U32, and their biochemical functions as a response regulator and a histidine protein kinase, respectively, were proven. The amrB disruption mutant was generated by insertional inactivation with the aparmycin resistance gene. The metabolic response to the absence of amrB gene was determined by a biochemical profiling technique in which the concentration changes of metabolic intermediates were measured by gas chromatography with time-of-flight mass spectrometry (GC/TOF-MS). Although the phenotype analyses of the amrB gene disruption mutant showed no significant change with respect to rifamycin SV production and morphological differentiation, the global metabolomic analyses found the concentration levels of some key intermediates in the TCA cycle and glycolysis pathway were affected by an amrB gene disruption event. The primary results suggested that amrB-amkB genes might be involved in the regulation of central carbohydrate metabolism in A. mediterranei U32.     

Mutational analysis and reconstituted expression of the biosynthetic genes involved in the formation of 3-amino-5-hydroxybenzoic acid, the starter unit of rifamycin biosynthesis in amycolatopsis Mediterranei S699

To investigate a novel branch of the shikimate biosynthesis pathway operating in the formation of 3-amino-5-hydroxybenzoic acid (AHBA), the unique biosynthetic precursor of rifamycin and related ansamycins, a series of target-directed mutations and heterologous gene expressions were investigated in Amycolatopsis mediterranei and Streptomyces coelicolor. The genes involved in AHBA formation were inactivated individually, and the resulting mutants were further examined by incubating the cell-free extracts with known intermediates of the pathway and analyzing for AHBA formation. The rifL, -M, and -N genes were shown to be involved in the step(s) from either phosphoenolpyruvate/d-erythrose 4-phosphate or other precursors to 3,4-dideoxy-4-amino-d-arabino-heptulosonate 7-phosphate. The gene products of the rifH, -G, and -J genes resemble enzymes involved in the shikimate biosynthesis pathway (August, P. R., Tang, L., Yoon, Y. J., Ning, S., Müller, R., Yu, T.-W., Taylor, M., Hoffmann, D., Kim, C.-G., Zhang, X., Hutchinson, C. R., and Floss, H. G. (1998) Chem. Biol. 5, 69-79). Mutants of the rifH and -J genes produced rifamycin B at 1% and 10%, respectively, of the yields of the wild type; inactivation of the rifG gene did not affect rifamycin production significantly. Finally, coexpressing the rifG-N and -J genes in S. coelicolor YU105 under the control of the act promoter led to significant production of AHBA in the fermented cultures, confirming that seven of these genes are indeed necessary and sufficient for AHBA formation. The effects of deletion of individual genes from the heterologous expression cassette on AHBA formation duplicated the effects of the genomic rifG-N and -J mutations on rifamycin production, indicating that all these genes encode proteins with catalytic rather than regulatory functions in AHBA formation for rifamycin biosynthesis by A. mediterranei.