Efficient de novo production of bioactive cordycepin by Aspergillus oryzae using a food-grade expression platform.

BACKGROUND: Cordycepin (3'-deoxyadenosine) is an important bioactive compound in medical and healthcare markets. The drawbacks of commercial cordycepin production using Cordyceps spp. include long cultivation periods and low cordycepin yields. To overcome these limitations and meet the increasing market demand, the efficient production of cordycepin by the GRAS-status Aspergillus oryzae strain using a synthetic biology approach was developed in this study. RESULTS: An engineered strain of A. oryzae capable of cordycepin production was successfully constructed by overexpressing two metabolic genes (cns1 and cns2) involved in cordycepin biosynthesis under the control of constitutive promoters. Investigation of the flexibility of carbon utilization for cordycepin production by the engineered A. oryzae strain revealed that it was able to utilize C6-, C5-, and C12-sugars as carbon sources, with glucose being the best carbon source for cordycepin production. High cordycepin productivity (564.64 ± 9.59 mg/L/d) was acquired by optimizing the submerged fermentation conditions. CONCLUSIONS: This study demonstrates a powerful production platform for bioactive cordycepin production by A. oryzae using a synthetic biology approach. An efficient and cost-effective fermentation process for cordycepin production using an engineered strain was established, offering a powerful alternative source for further upscaling.

Transcriptome-based Mining of the Constitutive Promoters for Tuning Gene Expression in Aspergillus oryzae.

Transcriptional regulation has been adopted for developing metabolic engineering tools. The regulatory promoter is a crucial genetic element for strain optimization. In this study, a gene set of Aspergillus oryzae with highly constitutive expression across different growth stages was identified through transcriptome data analysis. The candidate promoters were functionally characterized in A. oryzae by transcriptional control of ß-glucuronidase (GUS) as a reporter. The results showed that the glyceraldehyde triphosphate dehydrogenase promoter (PgpdA1) of A. oryzae with a unique structure displayed the most robust strength in constitutively controlling the expression compared to the PgpdA2 and other putative promoters tested. In addition, the ubiquitin promoter (Pubi) of A. oryzae exhibited a moderate expression strength. The deletion analysis revealed that the 5' untranslated regions of gpdA1 and ubi with the length of 1028 and 811 nucleotides, counted from the putative translation start site (ATG), respectively, could efficiently drive the GUS expression. Interestingly, both promoters could function on various carbon sources for cell growth. Glucose was the best fermentable carbon source for allocating high constitutive expressions during cell growth, and the high concentrations (6-8% glucose, w/v) did not repress their functions. It was also demonstrated that the secondary metabolite gene coding for indigoidine could express under the control of PgpdA1 or Pubi promoter. These strong and moderate promoters of A. oryzae provided beneficial options in tuning the transcriptional expression for leveraging the metabolic control towards the targeted products.

The Exploring Functional Role of Ammonium Transporters of Aspergillus oryzae in Nitrogen Metabolism: Challenges towards Cell Biomass Production.

Ammonium is a source of fermentable inorganic nitrogen essential for the growth and development of filamentous fungi. It is involved in several cellular metabolic pathways underlying nitrogen transport and assimilation. Ammonium can be transferred into the cell by an ammonium transporter. This study explored the role of ammonium transporters in nitrogen metabolism and cell biomass production in Aspergillus oryzae strain BCC 7051. Specific sequences encoding ammonium transporters (Amts) in A. oryzae were identified using genomic analysis. Four of the identified ammonium transporter genes, aoamt1-aoamt4, showed similarity in deduced amino acid sequences to the proteins in the ammonium transporter/methylammonium permease (AMT/MEP) family. Transcriptional analysis showed that the expression of aoamt2 and aoamt3 was ammonium-dependent, and was highly upregulated under ammonium-limited conditions. Their functional roles are characterized by genetic perturbations. The gene disruption and overexpression of aoamt3 indicated that the protein encoded by it was a crucial ammonium transporter associated with nitrogen metabolism and was required for filamentous growth. Compared with the wild type, the aoamt3-overexpressing strain showed superior growth performance, high biomass yield, and low glucose consumption. These results shed light on further improvements in the production of potent bioproducts by A. oryzae by manipulating the ammonium uptake capacity and nitrogen metabolism.

Potential of Aspergillus oryzae as a biosynthetic platform for indigoidine, a non-ribosomal peptide pigment with antioxidant activity.

The growing demand for natural pigments in the industrial sector is a significant driving force in the development of production processes. The production of natural blue pigments, which have wide industrial applications, using microbial systems has been gaining significant attention. In this study, we used Aspergillus oryzae as a platform cell factory to produce the blue pigment indigoidine (InK), by genetic manipulation of its non-ribosomal peptide synthetase system to overexpress the indigoidine synthetase gene (AoinK). Phenotypic analysis showed that InK production from the engineered strain was growth associated, owing to the constitutive control of gene expression. Furthermore, the initial pH, temperature, and glutamine and MgSO4 concentrations were key factors affecting InK production by the engineered strain. The pigment secretion was enhanced by addition of 1% Tween 80 solution to the culture medium. The maximum titer of total InK was 1409.22 ± 95.33 mg/L, and the maximum productivity was 265.09 ± 14.74 mg/L·d. Moreover, the recombinant InK produced by the engineered strain exhibited antioxidant activity. These results indicate that A. oryzae has the potential to be used as a fungal platform for overproduction of extracellular non-ribosomal peptide pigments.

Novel pentose-regulated promoter of Aspergillus oryzae with application in controlling heterologous gene expression.

The potent promoter and its transcriptional control make a significant contribution to strain optimization. Using transcriptome-based approach, a novel pentose-regulated promoter of the xylose reductase gene (PxyrA) of Aspergillus oryzae was identified. The promoter analysis showed that the PxyrA was tightly regulated by pentose sugars, which xylose and xylan were favorable inducers. The PxyrA function was highly efficient as compared with the maltose-inducible promoters of A. oryzae. It also exhibited the efficient transcription induction even though certain amounts of glucose and sucrose existed in the cultures. The expression control of PxyrA was dependent on xylose consumption capacity for fungal growth. The control mode of PxyrA offers a simple operation in simultaneous gene expression and cultivation optimization in Aspergilli. This study provides a prospective development of fungal production platform using cellulosic sugars by the xylose-utilizing strains for sustainable growing in circular economy.

Promoter exchange of the cryptic nonribosomal peptide synthetase gene for oligopeptide production in Aspergillus oryzae.

Oligopeptides with functional activities are of current interest in the nutraceutical and medical sectors. The development of the biosynthetic process of oligopeptides through a nonribosomal peptide synthetase (NRPS) system has become more challenging. To develop a production platform for nonribosomal peptides (NRPs), reprogramming of transcriptional regulation of the acv gene encoded ACV synthetase (ACVS) was implemented in Aspergillus oryzae using the CRISPR-Cas9 system. Awakening silent acv expression was successfully achieved by promoter substitution. Among the three exchanged promoters, AoPgpdA, AoPtef1, and PtPtoxA, the replacement of the native promoter with AoPgpdA led to the highest ACV production in A. oryzae. However, the ACV production of the AoPGpdA strain was also dependent on the medium composition, in which urea was the best nitrogen source, and a C:N ratio of 20:1 was optimal for tripeptide production. In addition to cell growth, magnesium ions are an essential element for ACV production and might participate in ACVS activity. It was also found that ACV was the growth-associated product of the engineered strain that might be a result of constitutive transcriptional control by the AoPgpdA promoter. This study offers a potential strategy for nonribosomal ACV production using the fungal system, which is applicable for redesigning bioactive oligopeptides with industrial relevance.

Significance of two intracellular triacylglycerol lipases of Aspergillus oryzae in lipid mobilization: A perspective in industrial implication for microbial lipid production.

Microbial lipid production of oleaginous strains involves in a complex cellular metabolism controlling lipid biosynthesis, accumulation and degradation. Particular storage lipid, triacylglycerol (TAG), contributes to dynamic traits of intracellular lipids and cell growth. To explore a basis of TAG degradation in the oleaginous strain of Aspergillus oryzae, the functional role of two intracellular triacylglycerol lipases, AoTgla and AoTglb, were investigated by targeted gene disruption using CRISPR/Cas9 system. Comparative lipid profiling of different cultivation stages between the control, single and double disruptant strains (ΔAotgla, ΔAotglb and ΔAotglaΔAotglb strains) showed that the inactivation of either AoTgla or AoTglb led to the increase of total lipid contents, particularly in the TAG fraction. Moreover, the prolonged lipid-accumulating stage of all disruptant strains was obtained as indicated by a reduction in specific rate of lipid turnover, in which a holding capacity in maximal lipid and TAG levels was achieved. The involvement of AoTgls in spore production of A. oryzae was also discovered. In addition to the significance in lipid physiology of the oleaginous fungi, this study provides an impact on industrial practice by overcoming the limitation in short lipid-accumulating stage of the fungal strain, which facilitate the cell harvesting step at the maximum lipid production yield.

Functional Characterization of Novel U6 RNA Polymerase III Promoters: Their Implication for CRISPR-Cas9-Mediated Gene Editing in Aspergillus oryzae.

U6 RNA polymerase III promoter (PU6), which is a key element in controlling the generation of single-guide RNA (sgRNA) for gene editing through CRISPR-Cas9 system, was investigated in this work. Using bioinformatics approach, two novel U6 ribonucleic acid (U6 RNA) sequences of Aspergillus niger were identified, showing that they had conserved motifs similar to other U6 RNAs. The putative PU6 located at the upstream sequence of A. niger U6 RNA exhibited the consensus motif, CCAATYA, and the TATA box which shared highly conserved characteristics across Aspergilli, whereas the A- and B-boxes were found at the intragenic and downstream of the structural genes, respectively. Using Aspergillus oryzae as a workhorse system, the function of A. niger PU6s for controlling the transcripts of sgRNA was verified, in which the orotidine-5'-phosphate decarboxylase (pyrG) sequence was used as a target for gene disruption through CRISPR-Cas9 system. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis of the selected pyrG auxotrophic strains showed the expression of sgRNA, indicating that the non-native promoters could efficiently drive sgRNA expression in A. oryzae. These identified promoters are useful genetic tools for precise engineering of metabolic pathways in the industrially important fungus through the empowered CRISPR-Cas9-associated gene-editing system.

Morphologically engineered strain of Aspergillus oryzae as a cell chassis for production development of functional lipids.

Cell morphology of the oleaginous fungus, Aspergillus oryzae BCC7051, was genetically engineered by disruption of non-essential genes involved in cell wall biosynthesis. Comparative phenotypic analysis of two disruptant strains defective either in α-1,3-glucan synthase 1 (ΔAoAgs1) or chitin synthase B (ΔAoChsB), and the wild type showed that the ΔAoAgs1 strain had no alterations in colonial growth and sporulation when grown on agar medium whereas the ΔAoChsB disruptant showed growth retardation and lower sporulation. However, tiny and loose pellets were found in the ΔAoAgs1 culture grown in liquid medium, where fungal pellet size was decreased by 35-50% of the wild type size. Further investigation of the ΔAoAgs1 mutant grown under stress-induced conditions, including high salt concentration, ionic strength and osmolarity, showed that its growth and development remained similar to that of the wild type. When cultivating the ΔAoAgs1 strain in a stirred-tank bioreactor, lipid production in terms of titer and productivity was significantly improved. As compared to the wild type, an increase of triacylglycerol and ergosterol contents with a proportional decrease in steryl ester content was observed in the ΔAoAgs1 strain. These results suggest that the morphologically engineered strain of A. oryzae is a robust cell chassis useful for exploitation in further production development of functional lipids with industrial significance.

Reengineering lipid biosynthetic pathways of Aspergillus oryzae for enhanced production of γ-linolenic acid and dihomo-γ-linolenic acid.

Biological significance of 18-carbon polyunsaturated fatty acids, γ-linolenic acid (GLA; C18:3 n-6) and dihomo-γ-linolenic acid (DGLA; C20:3 n-6) has gained much attention in the systematic development of optimized strains for industrial applications. In this work, a n-6 PUFAs-producing strain of Aspergillus oryzae was generated by manipulating metabolic reactions in fatty acid modification and triacylglycerol biosynthesis. The codon-optimized genes coding for Δ6-desaturase and Δ6-elongase of Pythium sp., and diacylglycerol acyltransferase 2 (mMaDGAT2) of Mortierella alpina were co-transformed in a single vector into A. oryzae BCC14614, yielding strain TD6E6-DGAT2. Comparative phenotypic analysis showed that a 70% increase of lipid titer was found in the engineered strain, which was a result of a significant increase in triacylglycerol (TAG) content (52.0 ±â€¯1.8% of total lipids), and corresponded to the increased size of lipid particles observed in the fungal cells. Interestingly, the proportions of GLA and DGLA in neutral lipids of the engineered strain were similar, with the highest titers obtained in the high C:N culture (29:0; 6% glucose) during the lipid-accumulating stage of growth. Time-course expression analysis of the engineered strain revealed transcriptional control of TAG biosynthesis through a co-operation between the native DGAT2 of A. oryzae and the transformed mMaDGAT2.

Jiangella endophytica sp. nov., an endophytic actinomycete isolated from the rhizome of Kaempferia elegans.

An endophytic actinobacterium, designated strain KE2-3T, was isolated from surface-sterilised rhizome of Kaempferia elegans. The polyphasic approach was used for evaluating the taxonomic position of this strain. The taxonomic affiliation of this strain at genus level could be confirmed by its chemotaxonomic characteristic, i.e. the presence of ll-diaminopimelic acid in the cell peptidoglycan, MK-9(H4) as the major menaquinone, iso-C16 : 0, anteiso-C15 : 0, iso-C14 : 0 and iso-C15 : 0 as the predominant fatty acids in cells, and the presence of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside in its membranes. Based on 16S rRNA gene sequence analysis, strain KE2-3T was identified as a member of the genus Jiangella and showed the highest similarities to Jiangella muralis DSM 45357T (99.3 %) followed by Jiangella albaDSM 45237T (99.2 %), Jiangella alkaliphilia DSM 45079T (99.0 %), Jiangella gansuensisDSM 44835T (98.8 %) and Jiangella mangrovi3SM4-07T (98.6 %). However, the draft genome sequence of strain KE2-3T exhibited low average nucleotide identity values to the reference strains (85.5-90.2 %), which were well below the 95-96 % species circumscription threshold. The DNA G+C content of genomic DNA was 72.3 mol%. With the differences of physiological, biochemical and genotypic data, strain KE2-3T could be discriminated from its closest neighbour. Thus, strain KE2-3T should be recognised as a novel species of genus Jiangella, for which the name Jiangellaendophytica sp. nov. is proposed. The type strain is KE2-3T (=BCC 66359T=NBRC 110004T).

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.

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).

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).

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).

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.

The codon-optimized Δ(6)-desaturase gene of Pythium sp. as an empowering tool for engineering n3/n6 polyunsaturated fatty acid biosynthesis.

BACKGROUND: The ∆(6)-desaturase gene, encoding a key enzyme in the biosynthesis of polyunsaturated fatty acids, has potential in pharmaceutical and nutraceutical applications. RESULTS: The ∆(6)-desaturase gene has been isolated from a selected strain of Oomycetes, Pythium sp. BCC53698. The cloned gene (PyDes6) contained an open reading frame (ORF) of 1401 bp encoding 466 amino acid residues. The deduced amino acid sequence shared a high similarity to those of other ∆(6)-desaturases that contained the signature features of a membrane-bound ∆(6)-desaturase, including a cytochrome b 5 and three histidine-rich motifs and membrane-spanning regions. Heterologous expression in Saccharomyces cerevisiae showed that monoene, diene and triene fatty acids having ∆(9)-double bond were substrates for PyDes6. No distinct preference between the n-3 and n-6 polyunsaturated fatty acyl substrates was found. The ∆(6)-desaturated products were markedly increased by codon optimization of PyDes6. CONCLUSION: The codon-optimized ∆(6)-desaturase gene generated in this study is a promising tool for further reconstitution of the fatty acid profile, in a host system of choice, for the production of economically important fatty acids, particularly the n-3 and n-6 polyunsaturated fatty acids.

Tenellin acts as an iron chelator to prevent iron-generated reactive oxygen species toxicity in the entomopathogenic fungus Beauveria bassiana.

Iron is an essential element for life. However, the iron overload can be toxic. Here, we investigated the significant increase of tenellin and iron-tenellin complex production in ferricrocin-deficient mutants of Beauveria bassiana. Our chemical analysis indicated that the ferricrocin-deficient mutants T1, T3 and T5 nearly abolished ferricrocin production. In turn, these mutants had significant accumulation of iron-tenellin complex in their mycelia at 247-289 mg g(-1) cell dry weight under iron-replete condition. Both tenellin and iron-tenellin complex were not detected in the wild-type under such condition. Mass analysis of the mutants' crude extracts demonstrated that tenellin formed a 3:1 complex with iron in the absence of ferricrocin. The unexpected link between ferricrocin and tenellin biosynthesis in ferricrocin-deficient mutants could be a survival strategy during iron-mediated oxidative stress.

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.

The potential of a fluorescent-based approach for bioassay of antifungal agents against chili anthracnose disease in Thailand.

Severe chili anthracnose disease in Thailand is caused by Colletotrichum gloeosporioides and C. capsici. To discover anti-anthracnose substances we developed an efficient dual-fluorescent labeling bioassay based on a microdilution approach. Indicator strains used in the assay were constructed by integrating synthetic green fluorescent protein (sGFP) and Discosoma sp. red fluorescent protein (DsRedExp) genes into the genomes of C. gloeosporioides or C. capsici respectively. Survival of co-spore cultures in the presence of inhibitors was determined by the expression levels of these fluorescent proteins. This developed assay has high potential for utilization in the investigation of selective inhibition activity to either one of the pathogens as well as the broad-range inhibitory effect against both pathogens. The value of using the dual-fluorescent assay is rapid, reliable, and consistent identification of anti-anthracnose agents. Most of all, the assay enables the identification of specific inhibitors under the co-cultivation condition.