Metabolic network analysis of Streptomyces tenebrarius, a Streptomyces species with an active entner-doudoroff pathway

Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin. (13)C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.     

Heterologous production of paromamine in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> TK24 using kanamycin biosynthetic genes from Streptomyces kanamyceticus ATCC12853

The 2-deoxystreptamine and paromamine are two key intermediates in kanamycin biosynthesis. In the present study, pSK-2 and pSK-7 recombinant plasmids were constructed with two combinations of genes: kanABK and kanABKF and kacA respectively from kanamycin producer Streptomyces kanamyceticus ATCC12853. These plasmids were heterologously expressed into Streptomyces lividans TK24 independently and generated two recombinant strains named S. lividans Sk-2/SL and S. lividans SK-7/SL, respectively. ESI/ MS and ESI-LC/MS analysis of the metabolite from S. lividans SK-2/SL showed that the compound had a molecular mass of 163 [M + H]⁺, which corresponds to that of 2-deoxystreptamine. ESI/MS and MS/MS analysis of metabolites from S. lividans SK-7/SL demonstrated the production of paromamine with a molecular mass of 324 [M + H]⁺. In this study, we report the production of paromamine in a heterologous host for the first time. This study will evoke to explore complete biosynthetic pathways of kanamycin and related aminoglycoside antibiotics.     

Biosynthesis of the beta-lactam antibiotic, thienamycin, by Streptomyces cattleya

Radioactive- and stable isotope-containing substrates were used to identify the biosynthetic precursors of the beta-lactam antibiotic, thienamycin, in Streptomyces cattleya. Acetate is utilized by the organism to form C(6) and C(7) of the beta-lactam ring. The two carbons of the hydroxyethyl group attached to C(6) are both derived from the methyl of methionine. The cysteaminyl side chain attached to C(2) is derived from cysteine. Selective inhibition of thienamycin and cephamycin C biosynthesis has been achieved either through the addition of metabolic inhibitors or through manipulation of the growth medium. These results suggest that the two beta-lactam antibiotics, thienamycin and cephamycin C, are formed by different biosynthetic pathways.     

Metabolic Network Analysis of Streptomyces tenebrarius, a Streptomyces Species with an Active Entner-Doudoroff Pathway

Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin. ¹³C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.     

Biosynthesis of epsilon-rhodomycinone from glucose by Streptomyces C5 and comparison with intermediary metabolism of other polyketide-producing streptomycetes

The catabolism of glucose by Streptomyces C5, a producer of anthracycline antibiotics, was investigated to determine the pathways that supply precursors for anthracycline biosynthesis. Carbons for the biosynthesis of epsilon-rhodomycinone, an anthracycline aglycone, from radiolabelled glucose were derived primarily from the Embden-Meyerhof-Parnas pathway, with a minor contribution from the pentose phosphate pathway. Furthermore, the anthracycline-producing strain, Streptomyces C5, as well as Streptomyces aureofaciens and Streptomyces lividans, strains that produce nonanthracycline polyketide antibiotics, displayed enzyme activities indicative of the Embden-Meyerhof-Parnas and pentose phosphate glycolytic pathways. As determined from labelling patterns, Streptomyces C5 apparently has a complete tricarboxylic acid cycle, but does not have a glyoxylate bypass pathway.     

Occurrence of two 5-aminolevulinate biosynthetic pathways in Streptomyces nodosus subsp. asukaensis is linked with the production of asukamycin

We report the results of cloning genes for two key biosynthetic enzymes of different 5-aminolevulinic acid (ALA) biosynthetic routes from Streptomyces. The genes encode the glutamyl-tRNAGlu reductase (GluTR) of the C5 pathway and the ALA synthase (ALAS) of the Shemin pathway. While Streptomyces coelicolor A3(2) synthesizes ALA via the C5 route, both pathways are operational in Streptomyces nodosus subsp. asukaensis, a producer of asukamycin. In this strain, the C5 route produces ALA for tetrapyrrole biosynthesis; the ALA formed by the Shemin pathway serves as a precursor of the 2-amino-3-hydroxycyclopent-2-enone moiety (C5N unit), an antibiotic component. The growth of S. nodosus and S. coelicolor strains deficient in the GluTR genes (gtr) is strictly dependent on ALA or heme supplementation, whereas the defect in the ALAS-encoding gene (hemA-asuA) abolishes the asukamycin production in S. nodosus. The recombinant hemA-asuA gene was expressed in Escherichia coli and in Streptomyces, and the encoded enzyme activity was demonstrated both in vivo and in vitro. The hemA-asuA gene is situated within a putative cluster of asukamycin biosynthetic genes. This is the first report about the cloning of genes for two different ALA biosynthetic routes from a single bacterium.     

Biosynthesis of spectinomycin: heterologous production of spectinomycin and spectinamine in an aminoglycoside-deficient host, Streptomyces venezuelae YJ003

Aims:  To obtain spectinomycin and spectinamine by heterologous expression into the biosynthetic deoxysugar (desosamine) gene-deleted host Streptomyces venezuelae YJ003.
Methods and Results:  The 17-kb spectinomycin biosynthetic gene cluster from Streptomyces spectabilis ATCC 27741 was heterologously expressed into Streptomyces venezuelae YJ003. Furthermore, the speA , speB and spcS2 encoded in the spectinomycin biosynthetic gene cluster of cosmid pSPC8 were also heterologously characterized to be responsible for the production of spectinamine.
Conclusions:  The results of this study indicated that pSPC8 contains all the genes necessary for the biosynthesis of spectinomycin. We also concluded that SpeA, SpeB and SpcS2 are sufficient for the biosynthesis of spectinamine. We also verified that SpeB and SpcS2 show dual character in the biosynthetic pathway of spectinomycin in Streptomyces spectabilis .
Significance and Impact of the Study:  This is the report regarding the expression of a biosynthetic gene cluster that gives rise to the production of aminoglycoside antibiotics in Streptomyces venezuelae YJ003. Therefore, this work may serve as a foundation for further research on spectinomycin biosynthesis and other aminoglycosides.     

Molecular engineering approaches to peptide, polyketide and other antibiotics

Molecular engineering approaches to producing new antibiotics have been in development for about 25 years. Advances in cloning and analysis of antibiotic gene clusters, engineering biosynthetic pathways in Escherichia coli, transfer of engineered pathways from E. coli into Streptomyces expression hosts, and stable maintenance and expression of cloned genes have streamlined the process in recent years. Advances in understanding mechanisms and substrate specificities during assembly by polyketide synthases, nonribosomal peptide synthetases, glycosyltransferases and other enzymes have made molecular engineering design and outcomes more predictable. Complex molecular scaffolds not amenable to synthesis by medicinal chemistry (for example, vancomycin (Vancocin), daptomycin (Cubicin) and erythromycin) are now tractable by molecular engineering. Medicinal chemistry can further embellish the properties of engineered antibiotics, making the two disciplines complementary.     

Identification of the biosynthetic gene cluster and an additional gene for resistance to the antituberculosis drug capreomycin

Capreomycin (CMN) belongs to the tuberactinomycin family of nonribosomal peptide antibiotics that are essential components of the drug arsenal for the treatment of multidrug-resistant tuberculosis. Members of this antibiotic family target the ribosomes of sensitive bacteria and disrupt the function of both subunits of the ribosome. Resistance to these antibiotics in Mycobacterium species arises due to mutations in the genes coding for the 16S or 23S rRNA but can also arise due to mutations in a gene coding for an rRNA-modifying enzyme, TlyA. While Mycobacterium species develop resistance due to alterations in the drug target, it has been proposed that the CMN-producing bacterium, Saccharothrix mutabilis subsp. capreolus, uses CMN modification as a mechanism for resistance rather than ribosome modification. To better understand CMN biosynthesis and resistance in S. mutabilis subsp. capreolus, we focused on the identification of the CMN biosynthetic gene cluster in this bacterium. Here, we describe the cloning and sequence analysis of the CMN biosynthetic gene cluster from S. mutabilis subsp. capreolus ATCC 23892. We provide evidence for the heterologous production of CMN in the genetically tractable bacterium Streptomyces lividans 1326. Finally, we present data supporting the existence of an additional CMN resistance gene. Initial work suggests that this resistance gene codes for an rRNA-modifying enzyme that results in the formation of CMN-resistant ribosomes that are also resistant to the aminoglycoside antibiotic kanamycin. Thus, S. mutabilis subsp. capreolus may also use ribosome modification as a mechanism for CMN resistance.     

链霉菌Streptomyces tenebrarius H6中与抗生素有关的糖生物合成基因的克隆

链霉菌S.tenebrarius H6产生多种氨基糖甙类抗生素,主要有阿普霉素、妥普霉素及卡那霉素B,其中阿普霉素因含有8碳糖的一种特殊结构令人注目,它的抗菌谱广,特别是对革兰氏阴性菌有较强的抗菌活性,不容易产生耐药性,对已有的耐药菌产生的氨基糖苷转移酶等失活酶仍有抵抗力.主要用于牛、猪、鸡等的大肠杆菌、沙门氏菌和支原体所引起的白痢、腹泻和肺炎等疾病.迄今有关八碳糖生物合成基因簇的研究在国内外尚无报道,在该菌株开展有关糖合成代谢基因的研究有着一定的意义.     

多烯大环内酯类抗生素生物合成基因簇中调控因子的研究进展

多烯大环内酯类抗生素具有良好的抗真菌活性,广泛应用于医疗卫生、食品加工和农业生产领域。随着高通量测序技术和生物信息学技术的发展,越来越多的链霉菌抗生素生物合成基因簇被发现和鉴定,调控因子作为生物合成基因簇中的重要组成部分,在庞大复杂的调控网络中起着至关重要的作用。本文总结了链霉菌中重要的调控因子类型,综述了多烯大环内酯类抗生素生物合成基因簇中调控因子的生物学功能、结合位点、作用机制等研究进展,并展望了后续研究工作。     

Biosynthesis of Polyketide Antibiotics by Various StreptomycesSpecies that Produce Actinomycins

A collection of actinomycin-producing Streptomycesstrains, their variants with different levels of antibiotic biosynthesis, and recombinant strains were screened in order to select new strains that produce polyketide antibiotics. Screening with the use of the cloned actgene encoding a component of actinorhodin polyketide synthase (PKS) multienzyme complex from Streptomyces coelicolorrevealed that many strains tested can synthesize polyketide antibiotics along with actinomycins. A relationship between the biosynthetic pathways of actinomycins and polyketides is discussed.     

Carbon flux distribution in antibiotic-producing chemostat cultures of Streptomyces lividans

The carbon metabolism of derivatives of Streptomyces lividans growing under phosphate limitation in chemostat cultures and producing the antibiotics actinorhodin and undecylprodigiosin was investigated. By applying metabolic flux analysis to a stoichiometric model, the relationship between antibiotic production, biomass accumulation, and carbon flux through the major carbon metabolic pathways (the Embden Meyerhoff Parnas and pentose-phosphate pathways) was analyzed. Distribution of carbon flux through the catabolic pathways was shown to be dependent on growth rate, as well as on the carbon and energy source (glucose or gluconate) used. Increasing growth rates promoted an increase in the flux of carbon through glycolysis and the pentose-phosphate pathway. The synthesis of both actinorhodin and undecylprodigiosin was found to be inversely related to flux through the pentose-phosphate pathway.     

Structural and functional dissection of aminocoumarin antibiotic biosynthesis: a review

Aminocoumarin antibiotics are natural products of soil-dwelling bacteria called Streptomycetes. They are potent inhibitors of DNA gyrase, an essential bacterial enzyme and validated drug target, and thus have attracted considerable interest as potential templates for drug development. To date, aminocoumarins have not seen widespread clinical application on account of their poor pharmacological properties. Through studying the structures and mechanisms of enzymes from their biosynthetic pathways we will be better informed to redesign these compounds through rational pathway engineering. Novobiocin, the simplest compound, requires at least seventeen gene products to convert primary metabolites into the mature antibiotic. We have solved the crystal structures of four diverse biosynthetic enzymes from the novobiocin pathway, and used these as three-dimensional frameworks for the interpretation of functional and mechanistic data, and to speculate about how they might have evolved. The structure determinations have ranged from the routine to the challenging, necessitating a variety of different approaches.     

Construction and characterization of new cloning vectors derived from Streptomyces griseobrunneus plasmid pBT1 and containing amikacin and sulfomycin resistance genes

Three cryptic plasmids, designated pBT1 (5.6 kb), pBT2 (9.7 kb), and pBT3 (16.6 kb), were isolated from Streptomyces griseobrunneus ISP5066 and physically characterized. pBT1 and pBT2, which differ by a 4.1-kb segment, are high copy-number plasmids (40-100 copies per chromosome) that coexist with each other. pBT3 is a low copy-number plasmid. Vectors containing amikacin (or kanamycin) and sulfomycin (or thiostrepton) resistance genes from Streptomyces litmocidini ISP5164 and Streptomyces viridochromogenes subsp. sulfomycini ATCC 29776, respectively, were constructed from pBT1. One such vector, pBT37, has unique restriction sites for cloning, including BglII, XhoI, PvuII, ClaI, and SacI, with the PvuII and ClaI sites allowing clone recognition by insertional inactivation of sulfomycin resistance. Since many Streptomyces species were very sensitive to amikacin and sulfomycin, these resistance genes serve as useful selective markers. pBT37 could transform several Streptomyces strains that produce antibiotics such as tetracyclines, macrolides, beta-lactams, and aminoglycosides. This plasmid is a potentially useful vector for cloning antibiotic biosynthetic genes.     

Genome analyses of Streptomyces peucetius ATCC 27952 for the identification and comparison of cytochrome P450 complement with other Streptomyces

We have determined the genome sequence of 8.7 Mb chromosome of Streptomyces peucetius ATCC 27952, which produces clinically important anthracycline chemotherapeutic agents of the polyketide class of antibiotics, daunorubicin and doxorubicin. The cytochrome P450 (CYP) superfamily is represented by 19 sequences in the S. peucetius. Among those, 15 code for functional genes, whereas the remaining four are pseudo genes. CYPs from S. peucetius are phylogenetically close to those of Streptomyces amermitilis. Four CYPs are associated with modular PKS of avermectin and two with doxorubicin biosynthetic gene cluster. CYP252A1 is the new family found in S. peucetius, which shares 38% identity to CYP51 from Streptomyces coelicolor A3 (2). Nine CYPs from S. peucetius are found in the cluster containing various regulatory genes including rar operon, conserved in S. coelicolor A3 (2) and Streptomyces griseus. Although two ferredoxins and four ferredoxin reductases have been identified so far, only one ferredoxin reductase was found in the cluster of CYP147F1 in S. peucetius. To date, 174 CYPs have been described from 45 Streptomyces species in all searchable databases. However, only 18 CYPs are clustered with ferredoxin. The comparative study of cytochrome P450s, ferredoxins, and ferredoxin reductases should be useful for the future development and manipulation of antibiotic biosynthetic pathways.     

Biosynthesis of a natural polyketide-isoprenoid hybrid compound, furaquinocin A: identification and heterologous expression of the gene cluster

Furaquinocin (FQ) A, produced by Streptomyces sp. strain KO-3988, is a natural polyketide-isoprenoid hybrid compound that exhibits a potent antitumor activity. As a first step toward understanding the biosynthetic machinery of this unique and pharmaceutically useful compound, we have cloned an FQ A biosynthetic gene cluster by taking advantage of the fact that an isoprenoid biosynthetic gene cluster generally exists in flanking regions of the mevalonate (MV) pathway gene cluster in actinomycetes. Interestingly, Streptomyces sp. strain KO-3988 was the first example of a microorganism equipped with two distinct mevalonate pathway gene clusters. We were able to localize a 25-kb DNA region that harbored FQ A biosynthetic genes (fur genes) in both the upstream and downstream regions of one of the MV pathway gene clusters (MV2) by using heterologous expression in Streptomyces lividans TK23. This was the first example of a gene cluster responsible for the biosynthesis of a polyketide-isoprenoid hybrid compound. We have also confirmed that four genes responsible for viguiepinol [3-hydroxypimara-9(11),15-diene] biosynthesis exist in the upstream region of the other MV pathway gene cluster (MV1), which had previously been cloned from strain KO-3988. This was the first example of prokaryotic enzymes with these biosynthetic functions. By phylogenetic analysis, these two MV pathway clusters were identified as probably being independently distributed in strain KO-3988 (orthologs), rather than one cluster being generated by the duplication of the other cluster (paralogs).