Novel benzene ring biosynthesis from C(3) and C(4) primary metabolites by two enzymes

The shikimate pathway, including seven enzymatic steps for production of chorismate via shikimate from phosphoenolpyruvate and erythrose-4-phosphate, is common in various organisms for the biosynthesis of not only aromatic amino acids but also most biogenic benzene derivatives. 3-Amino-4-hydroxybenzoic acid (3,4-AHBA) is a benzene derivative serving as a precursor for several secondary metabolites produced by Streptomyces, including grixazone produced by Streptomyces griseus. Our study on the biosynthesis pathway of grixazone led to identification of the biosynthesis pathway of 3,4-AHBA from two primary metabolites. Two genes, griI and griH, within the grixazone biosynthesis gene cluster were found to be responsible for the biosynthesis of 3,4-AHBA; the two genes conferred the in vivo production of 3,4-AHBA even on Escherichia coli. In vitro analysis showed that GriI catalyzed aldol condensation between two primary metabolites, l-aspartate-4-semialdehyde and dihydroxyacetone phosphate, to form a 7-carbon product, 2-amino-4,5-dihydroxy-6-one-heptanoic acid-7-phosphate, which was subsequently converted to 3,4-AHBA by GriH. The latter reaction required Mn(2+) ion but not any cofactors involved in reduction or oxidation. This pathway is independent of the shikimate pathway, representing a novel, simple enzyme system responsible for the synthesis of a benzene ring from the C(3) and C(4) primary metabolites.     

Targeting polyketide synthase gene pool within actinomycetes: new degenerate primers

Natural products provide a unique element of molecular diversity and biological functionality and they are still indispensable for drug discovery. The polyketides, comprising a large and structurally diverse family of bioactive natural products, have been isolated from a group of mycelia-forming Gram-positive microorganisms, the actinomycetes. Relatively high amino acid sequence identity of the actinomycetes type I polyketide synthases (PKSs) was used to design three degenerate primer pairs for homology-based PCR detection of novel PKS genes, with particular interest into PKSs involved in biosynthesis of immunosuppressive-like metabolites. The stepdown PCR method, described here, enables fast insight into the PKS arsenal within actinomycetes. Designed primers and stepdown PCR were applied for the analysis of two natural isolates, Streptomyces sp. strains NP13 and MS405. Sequence analysis of chosen clones revealed the presence of two distinctive sequences in strain Streptomyces sp. NP13, but only one of these showed homology to PKS-related sequences. On analysing PCR amplicons derived from Streptomyces sp. strain MS405, three different PKS-related sequences were identified demonstrating a potential of designed primers to target PKS gene pool within single organism.     

Gene cluster responsible for validamycin biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008

A gene cluster responsible for the biosynthesis of validamycin, an aminocyclitol antibiotic widely used as a control agent for sheath blight disease of rice plants, was identified from Streptomyces hygroscopicus subsp. jinggangensis 5008 using heterologous probe acbC, a gene involved in the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone of the acarbose biosynthetic gene cluster originated from Actinoplanes sp. strain SE50/110. Deletion of a 30-kb DNA fragment from this cluster in the chromosome resulted in loss of validamycin production, confirming a direct involvement of the gene cluster in the biosynthesis of this important plant protectant. A sequenced 6-kb fragment contained valA (an acbC homologue encoding a putative cyclase) as well as two additional complete open reading frames (valB and valC, encoding a putative adenyltransferase and a kinase, respectively), which are organized as an operon. The function of ValA was genetically demonstrated to be essential for validamycin production and biochemically shown to be responsible specifically for the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone in vitro using the ValA protein heterologously overexpressed in E. coli. The information obtained should pave the way for further detailed analysis of the complete biosynthetic pathway, which would lead to a complete understanding of validamycin biosynthesis.     

Selective Isolation and Ansamycin-Targeted Screenings of Commensal Actinomycetes from the “Maytansinoids-Producing” Arboreal <Emphasis Type="Italic">Trewia nudiflora</Emphasis>

To verify the hypothesis on the involvement of commensal actinomycetes in the biosynthesis of plant maytansinoids that belong to the ansamycin family, selective isolation and targeted screenings were conducted. In total, 164 endophytic actinomycetes isolates were obtained from the roots, twigs, seeds, callus, and germ-free seedlings of “maytansinoids-producing” Trewia nudiflora Linn. by selective isolation methods. Crude extracts of the isolates were screened by antifungal bioassay against Penicillium avellaneum UC-4376, thin-layer chromatography, and liquid chromatography–mass spectrometry detection according to the bioactivity as well as structural characteristics of maytansinoids. Respectively, 25.0%, 64.8%, and 26.7% of the extracts were positive in corresponding screenings. Ten isolates showed positive results in all three different screenings. To confirm on a genetic level, 28 representative isolates from 20 morphological groups were screened by polymerase chain reaction with 3-amino-5-hydroxybenzoic acid synthase gene and carbamoyltransferase gene primers, both of which were involved in the biosynthesis of ansamycins. According to the results of the targeted screenings, two isolates Streptomyces sp. 5B and Streptomyces sp. M27m3 might have the potential of producing ansamycins, which further enhanced the hypothesis that endophyte(s) might be involved in the biosynthesis of plant maytansinoids.     

Glucose regulation of cephamycin biosynthesis in Streptomyces lactamdurans is exerted on the formation of alpha-aminoadipyl-cysteinyl-valine and deacetoxycephalosporin C synthase

Glucose exerted a concentration-dependent negative regulation on the biosynthesis of cephamycin C by Streptomyces lactamdurans. Formation of the cephamycin precursor delta(alpha-aminoadipyl)-cysteinyl-valine was greatly decreased by excess glucose. The ring-expanding enzyme deacetoxycephalosporin C synthase was strongly repressed by glucose in vivo. Isopenicillin N synthase (cyclase) and isopenicillin N epimerase were not repressed by glucose. However, the activity of isopenicillin N synthase was inhibited in vitro by glucose 6-phosphate, and the activity of deacetoxycephalosporin C synthase was inhibited by inorganic phosphate, glucose 6-phosphate, fructose 2,6-diphosphate and fructose 1,6-diphosphate. The intracellular cAMP content decreased as growth proceeded and remained lower in glucose-supplemented cells than in control cultures. cAMP did not seem to be involved in glucose control of cephamycin biosynthesis.     

A genomics-guided approach for discovering and expressing cryptic metabolic pathways

Genome analysis of actinomycetes has revealed the presence of numerous cryptic gene clusters encoding putative natural products. These loci remain dormant until appropriate chemical or physical signals induce their expression. Here we demonstrate the use of a high-throughput genome scanning method to detect and analyze gene clusters involved in natural-product biosynthesis. This method was applied to uncover biosynthetic pathways encoding enediyne antitumor antibiotics in a variety of actinomycetes. Comparative analysis of five biosynthetic loci representative of the major structural classes of enediynes reveals the presence of a conserved cassette of five genes that includes a novel family of polyketide synthase (PKS). The enediyne PKS (PKSE) is proposed to be involved in the formation of the highly reactive chromophore ring structure (or "warhead") found in all enediynes. Genome scanning analysis indicates that the enediyne warhead cassette is widely dispersed among actinomycetes. We show that selective growth conditions can induce the expression of these loci, suggesting that the range of enediyne natural products may be much greater than previously thought. This technology can be used to increase the scope and diversity of natural-product discovery.     

The AcbC protein from Actinoplanes species is a C7-cyclitol synthase related to 3-dehydroquinate synthases and is involved in the biosynthesis of the alpha-glucosidase inhibitor acarbose

The putative biosynthetic gene cluster for the alpha-glucosidase inhibitor acarbose was identified in the producer Actinoplanes sp. 50/110 by cloning a DNA segment containing the conserved gene for dTDP-D-glucose 4,6-dehydratase, acbB. The two flanking genes were acbA (dTDP-D-glucose synthase) and acbC, encoding a protein with significant similarity to 3-dehydroquinate synthases (AroB proteins). The acbC gene was overexpressed heterologously in Streptomyces lividans 66, and the product was shown to be a C7-cyclitol synthase using sedo-heptulose 7-phosphate, but not ido-heptulose 7-phosphate, as its substrate. The cyclization product, 2-epi-5-epi-valiolone ((2S,3S,4S,5R)-5-(hydroxymethyl)cyclohexanon-2,3,4,5-tetrol), is a precursor of the valienamine moiety of acarbose. A possible five-step reaction mechanism is proposed for the cyclization reaction catalyzed by AcbC based on the recent analysis of the three-dimensional structure of a eukaryotic 3-dehydroquinate synthase domain (Carpenter, E. P., Hawkins, A. R., Frost, J. W., and Brown, K. A. (1998) Nature 394, 299-302).     

Molecular evolution of aromatic polyketides and comparative sequence analysis of polyketide ketosynthase and 16S ribosomal DNA genes from various streptomyces species

A 613-bp fragment of an essential ketosynthase gene from the biosynthetic pathway of aromatic polyketide antibiotics was sequenced from 99 actinomycetes isolated from soil. Phylogenetic analysis showed that the isolates clustered into clades that correspond to the various classes of aromatic polyketides. Additionally, sequencing of a 120-bp fragment from the gamma-variable region of 16S ribosomal DNA (rDNA) and subsequent comparative sequence analysis revealed incongruity between the ketosynthase and 16S rDNA phylogenetic trees, which strongly suggests that there has been horizontal transfer of aromatic polyketide biosynthesis genes. The results show that the ketosynthase tree could be used for DNA fingerprinting of secondary metabolites and for screening interesting aromatic polyketide biosynthesis genes. Furthermore, the movement of the ketosynthase genes suggests that traditional marker molecules like 16S rDNA give misleading information about the biosynthesis potential of aromatic polyketides, and thus only molecules that are directly involved in the biosynthesis of secondary metabolites can be used to gain information about the biodiversity of antibiotic production in different actinomycetes.     

放线菌核糖体工程的发展与应用

核糖体工程(ribosome engineering)是一项利用靶点位于细菌RNA聚合酶及核糖体功能因子的抗生素诱导细菌产生抗性突变,进而提升菌株次级代谢生产潜能的技术.该方法无需依赖菌株完善的遗传操作体系,可应用于发掘几乎所有放线菌菌株中潜在的宝贵活性次级代谢产物,并广泛应用于放线菌基因组挖掘和次级代谢产物增产优化....     

A transfected m1 muscarinic acetylcholine receptor stimulates adenylate cyclase via phosphatidylinositol hydrolysis

The m1 muscarinic acetylcholine receptor gene was transfected into and stably expressed in A9 L cells. The muscarinic receptor agonist, carbachol, stimulated inositol phosphate generation, arachidonic acid release, and cAMP accumulation in these cells. Carbachol stimulated arachidonic acid and inositol phosphate release with similar potencies, while cAMP generation required a higher concentration. Studies were performed to determine if the carbachol-stimulated cAMP accumulation was due to direct coupling of the m1 muscarinic receptor to adenylate cyclase via a GTP binding protein or mediated by other second messengers. Carbachol failed to stimulate adenylate cyclase activity in A9 L cell membranes, whereas prostaglandin E2 did, suggesting indirect stimulation. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated arachidonic acid release yet inhibited cAMP accumulation in response to carbachol. PMA also inhibited inositol phosphate release in response to carbachol, suggesting that activation of phospholipase C might be involved in cAMP accumulation. PMA did not inhibit prostaglandin E2-, cholera toxin-, or forskolin-stimulated cAMP accumulation. The phospholipase A2 inhibitor eicosatetraenoic acid and the cyclooxygenase inhibitors indomethacin and naproxen had no effect on carbachol-stimulated cAMP accumulation. Carbachol-stimulated cAMP accumulation was inhibited with TMB-8, an inhibitor of intracellular calcium release, and W7, a calmodulin antagonist. These observations suggest that carbachol-stimulated cAMP accumulation does not occur through direct m1 muscarinic receptor coupling or through the release of arachidonic acid and its metabolites, but is mediated through the activation of phospholipase C. The generation of cytosolic calcium via inositol 1,4,5-trisphosphate and subsequent activation of calmodulin by m1 muscarinic receptor stimulation of phospholipase C appears to generate the accumulation of cAMP.     

Stigmatella aurantiaca Sg a15 carries genes encoding type I and type II 3-deoxy-d-arabino-heptulosonate-7-phosphate synthases: involvement of a type II synthase in aurachin biosynthesis

3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthases catalyse the first step of the shikimate pathway. Two unrelated DAHP synthase types have been described in plants and bacteria. Two type II (aroA(A2) and aroA(A5)) and one type I DAHP synthase gene (aroA001) were identified from the myxobacterium Stigmatella aurantiaca Sg a15. Inactivation of aroA(A5) leads to a mutant that is impaired in the biosynthesis of aurachins, which are electron transport inhibitors and contain an anthranilate moiety. Feeding of anthranilic acid to the mutant culture restores production of aurachins. Inactivation of aroA(A2) and aroA001 does not impair production of aurachins or other known secondary metabolites of S. aurantiaca Sg a15.     

Arabidopsis carotenoid mutants demonstrate that lutein is not essential for photosynthesis in higher plants.

Lutein, a dihydroxy beta, epsilon-carotenoid, is the predominant carotenoid in photosynthetic plant tissue and plays a critical role in light-harvesting complex assembly and function. To further understand lutein synthesis and function, we isolated four lutein-deficient mutants of Arabidopsis that define two loci, lut1 and lut2 (for lutein deficient). These loci are required for lutein biosynthesis but not for the biosynthesis of beta, beta-carotenoids. The lut1 mutations are recessive, accumulate high levels of zeinoxanthin, which is the immediate precursor of lutein, and define lut1 as a disruption in epsilon ring hydroxylation. The lut2 mutations are semidominant, and their biochemical phenotype is consistent with a disruption of epsilon ring cyclization. The lut2 locus cosegregates with the recently isolated epsilon cyclase gene, thus, providing additional evidence that the lut2 alleles are mutations in the epsilon cyclase gene. It appears likely that the epsilon cyclase is a key step in regulating lutein levels and the ratio of lutein to beta,beta-carotenoids. Surprisingly, despite the absence of lutein, neither the lut1 nor lut2 mutation causes a visible deleterious phenotype or altered chlorophyll content, but both mutants have significantly higher levels of beta, beta-carotenoids. In particular, there is a stable increase in the xanthophyll cycle pigments (violaxanthin, antheraxanthin, and zeaxanthin) in both lut1 and lut2 mutants as well as an increase in zeinoxanthin in lut1 and beta-carotene in lut2. The accumulation of specific carotenoids is discussed as it pertains to the regulation of carotenoid biosynthesis and incorporation into the photosynthetic apparatus. Presumably, particular beta, beta-carotenoids are able to compensate functionally and structurally for lutein in the photosystems of Arabidopsis.     

Ceramide 1-phosphate stimulates proliferation of C2C12 myoblasts

Recent studies have established specific cellular functions for different bioactive sphingolipids in skeletal muscle cells. Ceramide 1-phosphate (C1P) is an important bioactive sphingolipid that has been involved in cell growth and survival. However its possible role in the regulation of muscle cell homeostasis has not been so far investigated. In this study, we show that C1P stimulates myoblast proliferation, as determined by measuring the incorporation of tritiated thymidine into DNA, and progression of the myoblasts through the cell cycle. C1P induced phosphorylation of glycogen synthase kinase-3β and the product of retinoblastoma gene, and enhanced cyclin D1 protein levels. The mitogenic action of C1P also involved activation of phosphatidylinositol 3-kinase/Akt, ERK1/2 and the mammalian target of rapamycin. These effects of C1P were independent of interaction with a putative G(i)-coupled C1P receptor as pertussis toxin, which maintains G(i) protein in the inactive form, did not affect C1P-stimulated myoblast proliferation. By contrast, C1P was unable to inhibit serum starvation- or staurosporine-induced apoptosis in the myoblasts, and did not affect myogenic differentiation. Collectively, these results add up to the current knowledge on cell types targeted by C1P, which so far has been mainly confined to fibroblasts and macrophages, and extend on the mechanisms by which C1P exerts its mitogenic effects. Moreover, the biological activities of C1P described in this report establish that this phosphosphingolipid may be a relevant cue in the regulation of skeletal muscle regeneration, and that C1P-metabolizing enzymes might be important targets for developing cellular therapies for treatment of skeletal muscle degenerative diseases, or tissue injury.     

A new method for rapid identification of ansamycin compounds by inactivating KLM gene clusters in potential ansamycin-producing actinomyces

Aims: In this study, we explored the possibility of construction of a ‘universal targeting vector’ by Red/ET recombination to inactivate L gene encoding 3‐amino‐5‐hydroxybenzoic acid (AHBA)‐oxidoreductase in AHBA biosynthetic gene cluster to facilitate the detection of ansamycins production in actinomycetes. Methods and Results: Based on the conserved regions of linked AHBA synthase (K), oxidoreductase (L) and phosphatase (M) gene clusters, degenerate primers were designed and PCR was performed to detect KLM gene clusters within 33 AHBA synthase gene‐positive actinomycetes strains. Among them, 22 KLM gene cluster‐positive strains were identified. A ‘universal targeting vector’ was further constructed using the 50‐nt homologous sequences chosen from four strains internal L gene in KLM gene clusters through Red/ET recombination. The L gene from nine of the KLM gene cluster‐positive actinomycetes strains was inactivated by insertion of a kanamycin (Km) resistance marker into its internal region from the ‘universal targeting vector’. By comparison of the metabolites produced in parent strains with those in L gene‐inactivated mutants, we demonstrated the possible ansamycins production produced by these strains. One strain (4089) was proved to be a geldanamycin producer. Three strains (3‐20, 7‐32 and 8‐32) were identified as potential triene‐ansamycins producers. Another strain (3‐27) was possible to be a streptovaricin C producer. Strains 24‐100 and 4‐124 might be served as ansamitocin‐like producers. Conclusions: The results confirmed the feasibility that a ‘universal targeting vector’ could be constructed through Red/ET recombination using the conserved regions of KLM gene clusters to detect ansamycins production in actinomycetes. Significance and Impact of the Study: The ‘universal targeting vector’ provides a rapid approach in certain degree to detect the potential ansamycin producers from the 22 KLM gene cluster‐positive actinomycetes strains.     

Carboxyl-terminal domain characterization of polyene-specific P450 hydroxylase in <Emphasis Type="Italic">Pseudonocardia autotrophica</Emphasis>

A polyene compound NPP identified in Pseudonocardia autotrophica was shown to contain an aglycone identical to nystatin, but to harbor a unique disaccharide moiety that led to higher solubility and reduced hemolytic activity. Recently, it was revealed that the final step of NPP (nystatin-like polyene) biosynthesis is C10 regio-specific hydroxylation by the cytochrome P450 hydroxylase (CYP) NppL (Kim et al. [7]). Through mutation and cross-complementation, here we found that NppL preferred a polyene substrate containing a disaccharide moiety for C10 hydroxylation, while its orthologue NysL involved in nystatin biosynthesis showed no substrate preference toward mono- and disaccharide moieties, suggesting that two homologous polyene CYPs, NppL and NysL might possess a unique domain recognizing a sugar moiety. Two hybrid NppL constructs containing the C-terminal domain of NysL exhibited no substrate preference toward 10-deoxy NPP and 10-deoxy nystatin-like NysL, implying that the C-terminal domain plays a major role in differentiating the sugar moiety responsible for substrate specificity. Further C-terminal domain dissection of NppL revealed that the last fifty amino acids play a critical role in determining substrate specificity of polyene-specific hydroxylation, setting the stage for the biotechnological application of hydroxyl diversification for novel polyene biosynthesis in actinomycetes.     

Multiple hybrid polyketide synthase/non-ribosomal peptide synthetase gene clusters in the myxobacterium Stigmatella aurantiaca

Many bacterial and fungal secondary metabolites are produced by polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS). Recently, it has been discovered that these modular enzymatic systems can also closely cooperate to form natural products. The analysis of the corresponding biosynthetic machineries, in the form of hybrid systems, is of special interest for combinatorial biosynthesis, because the combination of PKS and NRPS can lead to an immense variety of structures that might be produced. During our screening for hybrid PKS/NRPS systems from myxobacteria, we scanned the genome of Stigmatella aurantiaca DW4/3-1 for the presence of gene loci that encode both the PKS and NRPS genes. In addition to the previously characterized myxothiazol system, we identified three further hybrid loci, three additional PKS and one further NRPS gene locus. These were analyzed by hybridization, physical mapping, PCR with degenerate oligonucleotides and sequencing of fragments of the gene clusters. The function of these genes was not known but it had already been speculated that one compound produced by the strain and detected via HPLC was a secondary metabolite. This was based on the observation that its production is dependent on an active copy of the phosphopantetheinyl transferase gene mtaA. We show here that one of the identified hybrid gene loci is responsible for the formation of this secondary metabolite. In agreement with the genetic data, the chemical structure resembles a cyclic polypeptide with a PKS sidechain. Our data show that S. aurantiaca has a broader genetic capacity to produce natural products than the number of compounds isolated from the strain so far suggests.