Integrated pathway mining and selection of an artificial CYP79-mediated bypass to improve benzylisoquinoline alkaloid biosynthesis.

BACKGROUND: Computational mining of useful enzymes and biosynthesis pathways is a powerful strategy for metabolic engineering. Through systematic exploration of all conceivable combinations of enzyme reactions, including both known compounds and those inferred from the chemical structures of established reactions, we can uncover previously undiscovered enzymatic processes. The application of the novel alternative pathways enables us to improve microbial bioproduction by bypassing or reinforcing metabolic bottlenecks. Benzylisoquinoline alkaloids (BIAs) are a diverse group of plant-derived compounds with important pharmaceutical properties. BIA biosynthesis has developed into a prime example of metabolic engineering and microbial bioproduction. The early bottleneck of BIA production in Escherichia coli consists of 3,4-dihydroxyphenylacetaldehyde (DHPAA) production and conversion to tetrahydropapaveroline (THP). Previous studies have selected monoamine oxidase (MAO) and DHPAA synthase (DHPAAS) to produce DHPAA from dopamine and oxygen; however, both of these enzymes produce toxic hydrogen peroxide as a byproduct. RESULTS: In the current study, in silico pathway design is applied to relieve the bottleneck of DHPAA production in the synthetic BIA pathway. Specifically, the cytochrome P450 enzyme, tyrosine N-monooxygenase (CYP79), is identified to bypass the established MAO- and DHPAAS-mediated pathways in an alternative arylacetaldoxime route to DHPAA with a peroxide-independent mechanism. The application of this pathway is proposed to result in less formation of toxic byproducts, leading to improved production of reticuline (up to 60 mg/L at the flask scale) when compared with that from the conventional MAO pathway. CONCLUSIONS: This study showed improved reticuline production using the bypass pathway predicted by the M-path computational platform. Reticuline production in E. coli exceeded that of the conventional MAO-mediated pathway. The study provides a clear example of the integration of pathway mining and enzyme design in creating artificial metabolic pathways and suggests further potential applications of this strategy in metabolic engineering.

Draft Genome Sequence of the Marine-Derived, Anticancer Compound-Producing Bacterium Streptomyces sp. Strain GMY01.

The marine Streptomyces sp. strain GMY01 was isolated from Indonesian marine sediment. Genome mining analysis revealed that GMY01 has 28 biosynthetic gene clusters, dominated by genes encoding nonribosomal peptide synthetase and polyketide synthase.

In Silico Analysis of PKS and NRPS Gene Clusters in Arisostatin- and Kosinostatin-Producers and Description of Micromonospora okii sp. nov.

Micromonospora sp. TP-A0316 and Micromonospora sp. TP-A0468 are producers of arisostatin and kosinostatin, respectively. Micromonospora sp. TP-A0316 showed a 16S rRNA gene sequence similarity of 100% to Micromonosporaoryzae CP2R9-1T whereas Micromonospora sp. TP-A0468 showed a 99.3% similarity to Micromonospora haikouensis 232617T. A phylogenetic analysis based on gyrB sequences suggested that Micromonospora sp. TP-A0316 is closely related to Micromonospora oryzae whereas Micromonospora TP-A0468 is an independent genomospecies. As Micromonospora sp. TP-A0468 showed some phenotypic differences to its closely related species, it was classified as a novel species, for which the name Micromonospora okii sp. nov. is proposed. The type strain is TP-A0468T (= NBRC 110461T). Micromonospora sp. TP-A0316 and M. okii TP-A0468T were both found to harbor 15 gene clusters for secondary metabolites such as polyketides and nonribosomal peptides in their genomes. Arisostatin-biosynthetic gene cluster (BGC) of Micromonospora sp. TP-A0316 closely resembled tetrocarcin A-BGC of Micromonospora chalcea NRRL 11289. A large type-I polyketide synthase gene cluster was present in each genome of Micromonospora sp. TP-A0316 and M. okii TP-A0468T. It was an ortholog of quinolidomicin-BGC of M. chalcea AK-AN57 and widely distributed in the genus Micromonospora.

Nitrosopumilus zosterae sp. nov., an autotrophic ammonia-oxidizing archaeon of phylum Thaumarchaeota isolated from coastal eelgrass sediments of Japan.

A novel mesophilic and aerobic ammonia-oxidizing archaeon of the phylum Thaumarchaeota, strain NM25T, was isolated from coastal eelgrass zone sediment sampled in Shimoda (Japan). The cells were rod-shaped with an S-layer cell wall. The temperature range for growth was 20-37 °C, with an optimum at 30 °C. The pH range for growth was pH 6.1-7.7, with an optimum at pH 7.1. The salinity range for growth was 5-40 %, with an optimum range of 15-32 %. Cells obtained energy from ammonia oxidation and used bicarbonate as a carbon source. Utilization of urea was not observed for energy generation and growth. Strain NM25T required a hydrogen peroxide scavenger, such as α-ketoglutarate, pyruvate or catalase, for sustained growth on ammonia. Growth of strain NM25T was inhibited by addition of low concentrations of some organic compounds and organic mixtures, including complete inhibition by glycerol, peptone and yeast extract. Phylogenetic analysis of four concatenated housekeeping genes (16S rRNA, rpoB, rpsI and atpD) and concatenated AmoA, AmoB, AmoC amino acid sequences indicated that the isolate is similar to members of the genus Nitrosopumilus. The closest relative is Nitrosopumilus ureiphilus PS0T with sequence similarities of 99.5 % for the 16S rRNA gene and 97.2 % for the amoA gene. Genome relatedness between strain NM25T and N. ureiphilus PS0T was assessed by average nucleotide identity and digital DNA-DNA hybridization, giving results of 85.4 and 40.2 %, respectively. On the basis of phenotypic, genotypic and phylogenetic data, strain NM25T represents a novel species of the genus Nitrosopumilus, for which the name sp. nov, is proposed. The type strain is NM25T (=NBRC 111181T=ATCC TSD-147T).

Vestiges of Adaptation to the Mesophilic Environment in the Genome of Tepiditoga spiralis gen. nov., sp. nov.

A novel anaerobic heterotrophic strain, designated strain sy52T, was isolated from a hydrothermal chimney at Suiyo Seamount in the Pacific Ocean. A 16S rRNA gene analysis revealed that the strain belonged to the family Petrotogaceae in the phylum Thermotogae. The strain was mesophilic with optimum growth at 48°C and the phylum primarily comprised hyperthermophiles and thermophiles. Strain sy52T possessed unique genomic characteristics, such as an extremely low G+C content and 6 copies of rRNA operons. Genomic analyses of strain sy52T revealed that amino acid usage in the predicted proteins resulted from adjustments to mesophilic environments. Genomic features also indicated independent adaptions to the mesophilic environment of strain sy52T and Mesotoga species, which belong to the mesophilic lineage in the phylum Thermotogae. Based on phenotypic and phylogenetic evidence, strain sy52T is considered to represent a novel genus and species in the family Petrotogaceae with the proposed name Tepiditoga spiralis gen. nov., sp. nov.

Draft genome sequence of Actinomadura sp. K4S16 and elucidation of the nonthmicin biosynthetic pathway.

Actinomadura sp. K4S16 (=NBRC 110471) is a producer of a novel tetronate polyether compound nonthmicin. Here, we report the draft genome sequence of this strain together with features of the organism and assembly, annotation and analysis of the genome sequence. The 9.6 Mb genome of Actinomadura sp. K4S16 encoded 9,004 putative ORFs, of which 7,701 were assigned with COG categories. The genome contained four type-I polyketide synthase (PKS) gene clusters, two type-II PKS gene clusters, and three nonribosomal peptide synthetase (NRPS) gene clusters. Among the type-I PKS gene (t1pks) clusters, a large t1pks cluster was annotated to be responsible for nonthmicin synthesis based on bioinformatic analyses. We also performed feeding experiments using labeled precursors and propose the biosynthetic pathway of nonthmicin.

Aerobic degradation of cis-dichloroethene by the marine bacterium Marinobacter salsuginis strain 5N-3.

An aerobic bacterium, designated strain 5N-3 (NBRC 113055), that degrades cis-dichloroethene (cDCE) was isolated from a sea sediment in Japan. Strain 5N-3 was able to degrade a certain amount of cDCE in the presence of pyruvate without the action of inducers. In the presence of inducers, such as phenol and benzene, the strain completely removed cDCE. By the application of 16S ribosomal RNA (16S rRNA) gene sequencing and average nucleotide identity analyses, the strain 5N-3 was identified as Marinobacter salsuginis. On the other hand, identified species of Marinobacter are not known to degrade cDCE at all. A draft genome sequence analysis of the strain 5N-3 suggested that the dmp-homologous operon (operon for phenol degradation) may be contributing to the aerobic degradation of cDCE. This is the first report on an aerobic marine bacterium that has been found to degrade cDCE.

Streptomyces lydicamycinicus sp. nov. and Its Secondary Metabolite Biosynthetic Gene Clusters for Polyketide and Nonribosomal Peptide Compounds.

(1) Background: Streptomyces sp. TP-A0598 derived from seawater produces lydicamycin and its congeners. We aimed to investigate its taxonomic status; (2) Methods: A polyphasic approach and whole genome analysis are employed; (3) Results: Strain TP-A0598 contained ll-diaminopimelic acid, glutamic acid, glycine, and alanine in its peptidoglycan. The predominant menaquinones were MK-9(H6) and MK-9(H8), and the major fatty acids were C16:0, iso-C15:0, iso-C16:0, and anteiso-C15:0. Streptomyces sp. TP-A0598 showed a 16S rDNA sequence similarity value of 99.93% (1 nucleottide difference) to Streptomyces angustmyceticus NRRL B-2347T. The digital DNA-DNA hybridisation value between Streptomyces sp. TP-A0598 and its closely related type strains was 25%-46%. Differences in phenotypic characteristics between Streptomyces sp. TP-A0598 and its phylogenetically closest relative, S. angustmyceticus NBRC 3934T, suggested strain TP-A0598 to be a novel species. Streptomyces sp. TP-A0598 and S. angustmyceticus NBRC 3934T harboured nine and 13 biosynthetic gene clusters for polyketides and nonribosomal peptides, respectively, among which only five clusters were shared between them, whereas the others are specific for each strain; and (4) Conclusions: For strain TP-A0598, the name Streptomyces lydicamycinicus sp. nov. is proposed; the type strain is TP-A0598T (=NBRC 110027T).

Classification of 'Streptomyces hyalinum' Hamada and Yokoyama as Embleya hyalina sp. nov., the second species in the genus Embleya, and emendation of the genus Embleya.

The 16S rRNA gene sequence of 'Streptomyces hyalinum' NBRC 13850T shows 99.7 % similarity to that of Embleya scabrispora DSM 41855T; however, it shows <96.1 % similarity to any other type strains, including Streptomyces spp. Phylogenetic analysis based on 16S rRNA gene sequences clearly suggests that 'S. hyalinum' belongs to the genus Embleya rather than to Streptomyces. The strain possesses ll-diaminopimelic acid in the cell wall. The major menaquinone observed is MK-9(H6), and MK-9(H4) and MK-9(H8) are minor components. The major polar lipids are diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. In this study, the whole genome of strain NBRC 13850T was sequenced, and digital DNA-DNA hybridisation between 'S. hyalinum' NBRC 13850T and E. scabrispora DSM 41855T demonstrated 31.2 % of relatedness value between the two genomes. Morphological, chemotaxonomic, biochemical and physiological data also revealed that 'S. hyalinum' can be easily differentiated from E. scabrispora (the only the valid species of the genus Embleya) and that it merits separate species status. This phenotypic and genetic evidence reveals that 'S. hyalinum' represents a novel species of the genus Embleya; the name Embleya hyalina sp. nov. is proposed for this species. The type strain is NBRC 13850T (=ATCC 29817T=MB 891-A1T). We also emended the description of the genus Embleya considering the feature of E. hyalina.

Diversity of PKS and NRPS gene clusters between Streptomyces abyssomicinicus sp. nov. and its taxonomic neighbor.

Streptomyces sp. CHI39, isolated from a rock soil sample, is a producer of abyssomicin I. The taxonomic status was clarified by a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain was closely related to Streptomyces fragilis, with similarity of 99.9%. Strain CHI39 comprised LL-diaminopimelic acid, glutamic acid, glycine, and alanine in its peptidoglycan. The predominant menaquinones were MK-9(H6), and major fatty acids were anteiso-C15:0, anteiso-C17:0, and iso-C16:0. The chemotaxonomic features matched those described for the genus Streptomyces. Genome sequencing was conducted for strain CHI39 and S. fragilis NBRC 12862T. The results of digital DNA-DNA hybridization along with differences in phenotypic characteristics between the strains suggested strain CHI39 to be a novel species, for which Streptomyces abyssomicinicus sp. nov. is proposed; the type strain is CHI39T (=NBRC 110469T). Next, we surveyed polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene clusters in genomes of S. abyssomicinicus CHI39T and S. fragilis NBRC 12862T. These strains encoded 9 and 12 clusters, respectively, among which only four clusters were shared between them while the others are specific in each strain. This suggests that strains classified to distinct species each harbor many specific secondary metabolite-biosynthetic pathways even if the strains are taxonomically close.

Gene Mutations in Ganoderma lucidum During Long-Term Preservation by Repeated Subculturing.

Subculturing is frequently used for the preservation of basidiomycetes. In this study, to assess and verify the risks of repeated subculturing on the long-term preservation of strains of culture collections, we performed single nucleotide polymorphism (SNP) analysis in genes encoding enzymes of the mevalonate pathway, 1,3-ß-glucan synthesis, lignin degradation, and the tricarboxylic acid (TCA) cycle of mycelia before and after preserving for a 4-year period by the subculturing 30 times every 45 days of Ganoderma lucidum NBRC 8346. As a result of analyzing 60 genes of the strain, SNPs were found in 18 genes, and 14 of them were found in the exon region. Nonsynonymous coding SNPs were found in two genes (atoB_2, hmgr) encoding enzymes of mevalonate pathway and five genes (lcc1_9, lcc1_11, lcc1_13, dslcc6, aa5_1_9) encoding enzymes of lignin degradation after subculturing of G. lucidum NBRC 8346.

Complete genome sequence of natural rubber-degrading, gram-negative bacterium, Rhizobacter gummiphilus strain NS21T.

Gram-negative natural rubber-degrader, Rhizobacter gummiphilus NS21T, which was isolated from soil in the botanical garden in Japan, is a newly proposed species of genus of Rhizobacter. It has been reported that the latA1 gene is involved in the natural rubber degradation in this strain. To gain novel insights into natural rubber degradation pathway, the complete genome sequence of this strain was determined. The genome of strain NS21T consists of 6,398,096 bp of circular chromosome (GenBank accession number CP015118.1) with G + C content of 69.72%. The genome contains 5687 protein-coding and 68 RNA genes. Among the predicted genes, 4810 genes were categorized as functional COGs. Homology search revealed that existence of latA1 homologous gene (latA2) in this genome. Quantitative reverse-transcription-PCR and deletion analyses indicated that natural rubber degradation of this strain requires latA2 as well as latA1.

An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis.

Certain methanogens deteriorate steel surfaces through a process called microbiologically influenced corrosion (MIC). However, the mechanisms of MIC, whereby methanogens oxidize zerovalent iron (Fe0), are largely unknown. In this study, Fe0-corroding Methanococcus maripaludis strain OS7 and its derivative (strain OS7mut1) defective in Fe0-corroding activity were isolated. Genomic analysis of these strains demonstrated that the strain OS7mut1 contained a 12-kb chromosomal deletion. The deleted region, termed "MIC island", encoded the genes for the large and small subunits of a [NiFe] hydrogenase, the TatA/TatC genes necessary for the secretion of the [NiFe] hydrogenase, and a gene for the hydrogenase maturation protease. Thus, the [NiFe] hydrogenase may be secreted outside the cytoplasmic membrane, where the [NiFe] hydrogenase can make direct contact with Fe0, and oxidize it, generating hydrogen gas: Fe0 + 2 H+ → Fe2+ + H2. Comparative analysis of extracellular and intracellular proteomes of strain OS7 supported this hypothesis. The identification of the MIC genes enables the development of molecular tools to monitor epidemiology, and to perform surveillance and risk assessment of MIC-inducing M. maripaludis.

Linfuranones B and C, Furanone-Containing Polyketides from a Plant-Associated Sphaerimonospora mesophila.

Two new furanone-containing polyketides, linfuranones B and C, were isolated from a plant-associated actinomycete of the genus Sphaerimonospora. Their structures were determined by NMR and MS spectroscopic analyses, and the absolute configurations were established by anisotropic methods and chemical degradation approaches. In silico analysis of biosynthetic genes suggested that linfuranone B is generated from linfuranone C by oxidative cleavage of the polyketide chain. Linfuranones B and C induced preadipocyte differentiation into matured adipocytes at 20-40 µM without showing cytotoxicity.

Diversity of nonribosomal peptide synthetase and polyketide synthase gene clusters among taxonomically close Streptomyces strains.

To identify the species of butyrolactol-producing Streptomyces strain TP-A0882, whole genome-sequencing of three type strains in a close taxonomic relationship was performed. In silico DNA-DNA hybridization using the genome sequences suggested that Streptomyces sp. TP-A0882 is classified as Streptomyces diastaticus subsp. ardesiacus. Strain TP-A0882, S. diastaticus subsp. ardesiacus NBRC 15402T, Streptomyces coelicoflavus NBRC 15399T, and Streptomyces rubrogriseus NBRC 15455T harbor at least 14, 14, 10, and 12 biosynthetic gene clusters (BGCs), respectively, coding for nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). All 14 gene clusters were shared by S. diastaticus subsp. ardesiacus strains TP-A0882 and NBRC 15402T, while only four gene clusters were shared by the three distinct species. Although BGCs for bacteriocin, ectoine, indole, melanine, siderophores such as deferrioxamine, terpenes such as albaflavenone, hopene, carotenoid and geosmin are shared by the three species, many BGCs for secondary metabolites such as butyrolactone, lantipeptides, oligosaccharide, some terpenes are species-specific. These results indicate the possibility that strains belonging to the same species possess the same set of secondary metabolite-biosynthetic pathways, whereas strains belonging to distinct species have species-specific pathways, in addition to some common pathways, even if the strains are taxonomically close.

Reclassification of Nocardia species based on whole genome sequence and associated phenotypic data.

Type strains of 72 validated Nocardia species were phylogenetically analyzed based on the multilocus sequence analysis (MLSA) concatenated atpD-groL1-groL2-recA-rpoA-secY-sodA-ychF. Furthermore, their similarity based on digital DNA-DNA hybridization (dDDH) was calculated. Nocardia soli, Nocardia cummidelens and Nocardia salmonicida, Nocardia nova and Nocardia elegans, Nocardia exalbida and Nocardia gamkensis, and Nocardia coubleae and Nocardia ignorata formed coherent clades, respectively. Moreover, each set showed over 70% relatedness by dDDH and shared common phenotypic characteristics. Therefore, we propose a reclassification of Nocardia soli and Nocardia cummidelens as a later heterotypic synonym of Nocardia salmonicida, Nocardia elegans as a later heterotypic synonym of Nocardia nova, Nocardia gamkensis as a later heterotypic synonym of Nocardia exalbida, and Nocardia coubleae as a later heterotypic synonym of Nocardia ignorata.

Draft genome sequence of Streptomyces hyaluromycini MB-PO13T, a hyaluromycin producer.

Streptomyces hyaluromycini MB-PO13T (=NBRC 110483T = DSM 100105T) is type strain of the species, which produces a hyaluronidase inhibitor, hyaluromycin. Here, we report the draft genome sequence of this strain together with features of the organism and generation, annotation and analysis of the genome sequence. The 11.5 Mb genome of Streptomyces hyaluromycini MB-PO13T encoded 10,098 putative ORFs, of which 5317 were assigned with COG categories. The genome harbored at least six type I PKS clusters, three type II PKS gene clusters, two type III PKS gene clusters, six NRPS gene clusters, and one hybrid PKS/NRPS gene cluster. The type II PKS gene cluster including 2-amino-3-hydroxycyclopent-2-enone synthetic genes was identified to be responsible for hyaluromycin synthesis. We propose the biosynthetic pathway based on bioinformatic analysis.

Comparative analysis of the intestinal flora in type 2 diabetes and nondiabetic mice.

A relationship between type 2 diabetes mellitus (T2DM) and intestinal flora has been suggested since development of analysis technology for intestinal flora. An animal model of T2DM is important for investigation of T2DM. Although there are some animal models of T2DM, a comparison of the intestinal flora of healthy animals with that of T2DM animals has not yet been reported. The intestinal flora of Tsumura Suzuki Obese Diabetes (TSOD) mice was compared with that of Tsumura, Suzuki, Non Obesity (TSNO) mice in the present study. The TSOD mice showed typical type 2 diabetes symptoms, which were high-fat diet-independent. The TSOD and the TSNO mouse models were derived from the same strain, ddY. In this study, we compared the intestinal flora of TSOD mice with that if TSNO mice at 5 and 12 weeks of age. We determined that that the number of operational taxonomic units (OTUs) was significantly higher in the cecum of TSOD mice than in that of TSNO mice. The intestinal flora of the cecum and that of the feces were similar between the TSNO and the TSOD strains. The dominant bacteria in the cecum and feces were of the phyla Firmicutes and Bacteroidetes. However, the content of some bacterial species varied between the two strains. The percentage of Lactobacillus spp. within the general intestinal flora was higher in TSOD mice than in TSNO mice. In contrast, the percentages of order Bacteroidales and family Lachnospiraceae were higher in TSNO mice than in TSOD mice. Some species were observed only in TSOD mice, such as genera Turicibacter and SMB53 (family Clostridiaceae), the percentage of which were 3.8% and 2.0%, respectively. Although further analysis of the metabolism of the individual bacteria in the intestinal flora is essential, genera Turicibacter and SMB53 may be important for the abnormal metabolism of type 2 diabetes.

Complete Genome Sequence of the Polychlorinated Biphenyl-Degrading Bacterium Pseudomonas putida KF715 (NBRC 110667) Isolated from Biphenyl-Contaminated Soil.

Pseudomonas putida KF715 (NBRC 110667) utilizes biphenyl as a sole source of carbon and degrades polychlorinated biphenyls (PCBs). Here, we report a complete genome sequence of the KF715 strain, which comprises a circular chromosome and four plasmids. Biphenyl catabolic genes were located on the largest plasmid, pKF715A.

Draft Genome Sequence of an Anicemycin Producer, Streptomyces sp. TP-A0648.

We report the draft genome sequence of Streptomyces sp. TP-A0648 isolated from a leaf of Aucuba japonica This strain produces a new tumor cell growth inhibitor designated anicemycin. The genome harbors at least 12 biosynthetic gene clusters for polyketides and nonribosomal peptides, suggesting the potential to produce diverse secondary metabolites.