Chanikul Chutrakul

Nonomuraea stahlianthi sp. nov., an endophytic actinomycete isolated from the stem of Stahlianthus campanulatus

A novel endophytic actinomycete, designated strain SC1-1T, was isolated from sterilized stem tissue from Stahlianthus campanulatus collected in Udon Thani province, Thailand. The isolate formed short chains of spores on aerial mycelium and presented meso-diaminopimelic acid in the cell wall peptidoglycan. Glucose, madurose, mannose, rhamnose and ribose were observed as sugars in the cells. The cell membrane phospholipids were phosphatidylethanolamine, phosphatidylmethylethanolamine, hydroxy-phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and ninhydrin-positive phosphoglycolipids. The major menaquinones were MK-9(H4) and MK-9(H2). The main cellular fatty acids were iso-C16:0, 10-methyl C17 : 0 and C17 : 1ω6c. A high G+C content (70.7 mol%) was present in the genomic DNA. The taxonomic position based on the 16S rRNA gene sequence analysis revealed that strain SC1-1T belonged to the genus Nonomuraea and shared the highest 16S rRNA gene sequence similarity value with Nonomuraea dietziae DSM 44320T (98.82 %), followed by Nonomuraea africana IFO 14745T (98.58 %), Nonomuraea jabiensis A4036T (98.43 %), Nonomuraea endophytica YIM 65601T (98.36 %), Nonomuraea purpurea 1SM4-01T (98.34 %), Nonomuraea angiospora IFO 13155T (98.29 %), Nonomuraea roseola IFO 14685T (98.23 %) and Nonomuraea recticatena IFO 14525T (98.23 %). On the basis of the DNA-DNA hybridization relatedness and including the physiological and biochemical characteristics, strain SC1-1T should be judged as a novel species of the genus Nonomuraea, for which the name Nonomuraea stahlianthi sp. nov. is proposed. The type strain is strain SC1-1T (=BCC 66361T=NBRC 110006T).

Metabolic engineering of long chain-polyunsaturated fatty acid biosynthetic pathway in oleaginous fungus for dihomo-gamma linolenic acid production

Microbial lipids are promising alternative sources of long chain-polyunsaturated fatty acids (LC-PUFAs) for food, feed, nutraceutical and pharmaceutical sectors. Dihomo-γ-linolenic acid (C20:3Δ(8,11,14); DGLA) is an important LC-PUFAs with anti-inflammatory and anti-proliferative effects. To generate a DGLA-producing strain, fatty acid reconstitution in Aspergillus oryzae was performed by metabolic engineering through co-expression of codon-optimized Pythium Δ(6)-desaturase and Δ(6)-elongase, which had high conversion rates of substrates to respective products as compared to the native enzymes. The Δ(6)-desaturated and Δ(6)-elongated products, γ-linolenic acid (C18:3Δ(6,9,12); GLA) and DGLA, were accumulated in phospholipids rather than triacylglycerol. Interestingly, the manipulation of lipid quality in the oleaginous fungus did not affect growth and lipid phenotypes. This strategy might expand to development of the oleaginous fungal strain for producing other tailor-made oils with industrial applications.

Novel elongase of Pythium sp. with high specificity on Δ 6 -18C desaturated fatty acids

We identified a novel elongase gene from a selected strain of the Oomycete, Pythium sp. BCC53698. Using a PCR approach, the cloned gene (PyElo) possessed an open reading frame (ORF) of 834 bp encoding 277 amino acid residues. A similarity search showed that it had homology with the PUFA elongases of several organisms. In addition, the signature characteristics, including four conserved motifs, a histidine-rich catalytic motif and membrane-associated feature were present in the Pythium gene. Heterologous expression in Saccharomyces cerevisiae showed that it was specific for fatty acid substrates, having a double bond at Δ(6)-position, which included γ-linolenic acid (GLA) and stearidonic acid (STA), and preferentially elongated the n3-18C PUFA. This is an elongase in Oomycete fungi, which displays very high specificity on Δ(6)-18C desaturated fatty acids. This will be a powerful tool to engineer PUFA biosynthesis in organisms of interest through the n-6 series pathway for producing value-added fatty acids.

Diacylglycerol acyltransferase 2 of Mortierella alpina with specificity on long-chain polyunsaturated fatty acids: A potential tool for reconstituting lipids with nutritional value

Based on available genome sequences and bioinformatics tools, we searched for an uncharacterized open reading frame of Mortierella alpina (MaDGAT2) using diacylglycerol acyltransferase sequence (fungal DGAT type 2B) as a query. Functional characterization of the identified native and codon-optimized M. alpina genes were then performed by heterologous expression in Saccharomyces cerevisiae strain defective in synthesis of neutral lipid (NL). Lipid analysis of the yeast tranformant carrying MaDGAT2 showed that the NL biosynthesis and lipid particle formation were restored by the gene complementation. Substrate specificity study of the fungal enzyme by fatty acid supplementation in the transformant cultures showed that it had a broad specificity on saturated and unsaturated fatty acid substrates for esterification into triacylglycerol (TAG). The n-6 polyunsaturated fatty acids (PUFAs) with 18 and 20 carbon atoms, including linoleic acid, γ-linolenic acid, dihomo γ-linolenic and arachidonic acid could be incorporated into TAG fraction in the yeast cells. Interestingly, among n-3 PUFAs tested, the MaDGAT2 enzyme preferred eicosapentaenoic acid (EPA) substrate as its highly proportional constituent found in TAG fraction. This study provides a potential genetic tool for reconstituting oils rich in long-chain PUFAs with nutritional value.

Phytohabitans kaempferiae sp. nov., an endophytic actinomycete isolated from the leaf of Kaempferia larsenii.

A novel endophytic actinomycete, designated strain KK1-3T, which formed single spores and long chains of spores (more than 10 spores) was isolated from surface-sterilized Kaempferia larsenii leaf collected from Ubon Ratchathani province, Thailand. The isolate contained l-lysine, meso-diaminopimelic acid and hydroxyl diaminopimelic acid in the cell-wall peptidoglycan. The whole-cell sugars included glucose, mannose, rhamnose, ribose, galactose and xylose. The characteristic phospholipids were phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and phosphoglycolipids. The predominant menaquinones were MK-10(H8), MK-10(H6) and MK-10(H4). The predominant cellular fatty acids were anteiso-C17 : 0 and iso-C16 : 0. The G+C content of the genomic DNA was 71 mol%. Phylogenetic analysis using 16S rRNA gene sequences revealed that strain KK1-3T should be classified as representing a member of the genus Phytohabitans. The similarity values of sequences between this strain and those of the closely related species, Phytohabitans houttuyneae K11-0057T (99.0 %), Phytohabitans suffuscus K07-0523T (98.9 %), Phytohabitans flavus K09-0627T (98.6 %) and Phytohabitans rumicisK11-0047T (98.1 %) were observed. The DNA-DNA hybridization result and some physiological and biochemical properties indicated that KK1-3T could be readily distinguished from its closest phylogenetic relatives. On the basis of these phenotypic and genotypic data, this strain represents a novel species, for which the name Phytohabitans kaempferiae sp. nov. is proposed. The type strain is strain KK1-3T (=BCC 66360T =NBRC 110005T).

Genes differentially expressed under naphthoquinone-producing conditions in the entomopathogenic fungus Ophiocordyceps unilateralis

The ant-pathogenic fungus Ophiocordyceps unilateralis BCC1869 produces six naphthoquinone (NQ) derivatives. These NQs can be found in fungal-infected ants or produced in culture. Also, the NQs have antibacterial, anticancer, and antimalarial activities and are red pigments with potential for use as natural colorants. Suppressive subtractive hybridization identified genes that were expressed under NQ-producing conditions but not under nonproducing conditions. On potato dextrose agar, the mycelia produced red pigments and secreted them into the medium and as droplets on top of the colony. High-performance liquid chromatography analysis indicated that the red pigment was predominantly erythrostominone with small amounts of its derivatives. For suppressive subtractive hybridization, the cDNA from O. unilateralis cultures on complete medium agar cultures (lacking NQs) were subtracted from those on potato dextrose agar (which produce and secrete NQs). Sixty-six unique expressed sequence tags (ESTs) were identified and include five transporter genes, two transcriptional regulator genes, and several genes in secondary metabolism and biodegradation. The transporter genes include an ATP-binding cassette transporter gene OuAtr1 and a major facilitator superfamily transporter gene OuMfs1. Expression of selected ESTs was further validated using quantitative reverse transcription PCR. Gene expression result indicates that OuAtr1 and OuMfs1 were dramatically upregulated (136- and 29-fold increase, respectively) during the NQ-producing stage compared with the NQ-nonproducing stage. Upregulation of other genes was also detected. This EST collection represents the first group of genes identified from this potential biocontrol agent and includes candidate genes for production and secretion of the red NQs. Roles of these genes could be further determined using a functional analysis.

Asanoa endophytica sp. nov., an endophytic actinomycete isolated from the rhizome of Boesenbergia rotunda

A novel Gram-stain-positive, non-motile, endophytic actinomycete, designated strain BR3-1T, which produced spore chains borne on the tips of short sporophores, was isolated from the rhizome of Boesenbergia rotunda collected from Udon Thani province, Thailand. This strain was investigated for its taxonomic position using a polyphasic approach. The strain contained 3-hydroxydiaminopimelic acid and meso-diaminopimelic acid in the cell-wall peptidoglycan. The whole-cell sugars comprised glucose, mannose, rhamnose, ribose and xylose. Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides were found as the characteristic phospholipids. The predominant menaquinones were MK-10(H8) and MK-10(H6). The major cellular fatty acids were iso-C16 : 0, iso-C15 : 0 and anteiso-C15 : 0. The G+C content of the genomic DNA was 71.4 mol%. 16S rRNA gene sequence analysis revealed that strain BR3-1T belonged to the genus Asanoa and was most closely related to Asanoa ishikariensis (99.39 %), Asanoa iriomotensis (99.31 %), Asanoa siamensis (99.17 %), Asanoa ferruginea (98.84 %) and Asanoa hainanensis (98.71 %). The DNA-DNA relatedness value between strain BR3-1T and its phylogenetically closest relatives was in the range of 15.4 % ± 1.2 to 45.8 % ± 2.6. In addition, some physiological and biochemical properties indicated that strain BR3-1T could be readily distinguished from all type strains in the genus Asanoa. Thus, strain BR3-1T should be classified as a representative of a novel species, for which the name Asanoa endophytica sp. nov. is proposed. The type strain is BR3-1T ( = BCC 66355T = NBRC 110002T).