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Resilience at the individual, community, government and global food system levels must be built in such a way that the economic, social and environmental bases to generate food security and nutrition for current and future generations are not compromised anywhere in the world. We present opportunities for diversification of production, diversification at the household level, and diversification through the global, regional and local trading systems for resilient food systems. There are a number of trade-offs which must be navigated as we strive to achieve greater food system resilience. These include the need to deliver short-term humanitarian aid without jeopardizing long run development, mitigation of rising global temperatures even as the food system adapts to the inevitable changes in the earth’s climate, taking advantage of the benefits of globalization while avoiding the downsides, and encouraging agricultural production and boosting rural incomes while also protecting the environment. Much will be gained for resilience building by highlighting successful solutions and facilitating the exchange of tools, data, information and knowledge and capacity.

This chapter focusses on the role of science, technology and innovation (STI) in transforming the food systems of Asia and the Pacific to achieve long-term environmentally sustainable food and nutritional security (FNS). A “whole of systems” approach is required to address the issues, and that work is urgently needed to define ‘healthy’ diets for different regions, societies and cultures. Emphasis should shift from the provision of calories to the supply of balanced patterns of all essential nutrients, and the ‘holistic’ properties of foods should be recognised. The chapter identifies countries and regions, within Asia, considered to be at particularly high risk for future food insecurity. Systems analysis should be applied across the agricultural and food sectors of these countries to identify the actual technical and other impediments to FNS. It is envisaged that the results from such an analysis would be used to formulate a ‘blueprint’ for agricultural and food STI in Asia. Overarching recommendations are the establishment of a trans-national funding mechanism for the entire region, focussing on targeted interdisciplinary STI, and the establishment of regional centres of excellence for research, education and extension, focussing on the identified key areas of opportunity. It was concluded that there is an urgent need for investment and action.

The putative ferricrocin synthetase gene ferS in the fungal entomopathogen Beauveria bassiana BCC 2660 was identified and characterized. The 14,445-bp ferS encodes a multimodular nonribosomal siderophore synthetase tightly clustered with Fusarium graminearum ferricrocin synthetase. Functional analysis of this gene was performed by disruption with the bar cassette. ΔferS mutants were verified by Southern and PCR analyses. HPLC and TLC analyses of crude extracts indicated that biosynthesis of ferricrocin was abolished in ΔferS. Insect bioassays surprisingly indicated that ΔferS killed the Spodoptera exigua larvae faster (LT50 59 h) than wild type (66 h). Growth and developmental assays of the mutant and wild type demonstrated that ΔferS had a significant increase in germination under iron depletion and radial growth and a decrease in conidiation. Mitotracker staining showed that the mitochondrial activity was enriched in ΔferS under both iron excess and iron depletion. Comparative transcriptomes between wild type and ΔferS indicated that the mutant was increased in the expression of eight cytochrome P450 genes and those in iron homeostasis, ferroptosis, oxidative stress response, ergosterol biosynthesis, and TCA cycle, compared to wild type. Our data suggested that ΔferS sensed the iron excess and the oxidative stress and, in turn, was up-regulated in the antioxidant-related genes and those in ergosterol biosynthesis and TCA cycle. These increased biological pathways help ΔferS grow and germinate faster than the wild type and caused higher insect mortality than the wild type in the early phase of infection.

Analysis of metabolic flux was used for system level assessment of carbon partitioning in Kasetsart 50 (KU50) and Hanatee (HN) cassava cultivars to understand the metabolic routes for their distinct phenotypes. First, the constraint-based metabolic model of cassava storage roots, rMeCBM, was developed based on the carbon assimilation pathway of cassava. Following the subcellular compartmentalization and curation to ensure full network connectivity and reflect the complexity of eukaryotic cells, cultivar specific data on sucrose uptake and biomass synthesis were input, and rMeCBM model was used to simulate storage root growth in KU50 and HN. Results showed that rMeCBM-KU50 and rMeCBM-HN models well imitated the storage root growth. The flux-sum analysis revealed that both cultivars utilized different metabolic precursors to produce energy in plastid. More carbon flux was invested in the syntheses of carbohydrates and amino acids in KU50 than in HN. Also, KU50 utilized less flux for respiration and less energy to synthesize one gram of dry storage root. These results may disclose metabolic potential of KU50 underlying its higher storage root and starch yield over HN. Moreover, sensitivity analysis indicated the robustness of rMeCBM model. The knowledge gained might be useful for identifying engineering targets for cassava yield improvement.

Entomopathogenic fungi utilize specific secondary metabolites to defend against insect immunity, thereby enabling colonization of their specific hosts. We are particularly interested in the polyketide synthesis gene pks15, which is involved in metabolite production, and its role in fungal virulence. Targeted disruption of pks15 followed by genetic complementation with a functional copy of the gene would allow for functional characterization of this secondary metabolite biosynthesis gene. Using a Beauveria bassiana ∆pks15 mutant previously disrupted by a bialophos-resistance (bar) cassette, we report here an in-cis complementation at bar cassette using CRISPR/Cas9 gene editing. A bar-specific short guide RNA was used to target and cause a double-strand break in bar, and a donor DNA carrying a wild-type copy of pks15 was co-transformed with the guide RNA. Isolate G6 of ∆pks15 complemented with pks15 was obtained and verified by PCR, Southern analyses and DNA sequencing. Compared to ∆pks15 which showed a marked reduction in sporulation and insect virulence, the complementation in G6 restored with insect virulence, sporulation and conidial germination to wild-type levels. Atomic force and scanning electron microscopy revealed that G6 and wild-type conidial wall surfaces possessed the characteristic rodlet bundles and rough surface while ∆pks15 walls lacked the bundles and were relatively smoother. Conidia of ∆pks15 were larger and more elongated than that of G6 and the wild type, indicating changes in their cell wall organization. Our data indicate that PKS15 and its metabolite are likely not only important for fungal virulence and asexual reproduction, but also cell wall formation.

The reducing clade IIb polyketide synthase gene, pks14, is preserved throughout the evolution of entomopathogenic fungi. We examined the functions of pks14 in Beauveria bassiana using targeted gene disruption, and pks14 disruption was verified by Southern blot and PCR analyses. The radial growth, cell dry weight and conidial germination of Δpks14 were comparable to that of the wild type. Our sequence and gene expression analyses of the pks14 biosynthetic cluster demonstrated: (i) cotranscription and constitutive expression of nearly all the genes of the aforementioned cluster including the C2H2 zinc finger transcription regulator gene, but not pks14 and the cytochrome P450 gene; (ii) expression of the pks14 gene in the insect-containing culture condition only; and (iii) a KAR9-like gene in direct proximity with pks14 is the only gene showing co-regulation. The Δpks14-infected Spodoptera exigua larvae survived significantly longer than those infected by the wild type, indicating a marked reduction in the virulence of Δpks14 against the insect. LT50 of Δpks14 was increased by 1.55 days. Hyphal body formation was decreased in the hemolymph of insects infected by Δpks14 as compared with those inoculated by the wild type. Our results suggest that PKS14-catalyzed polyketide enhances virulence and pathogenicity of B. bassiana on insects.

Arthrospira (Spirulina) platensis is a well-known commercial cyanobacterium that is used as a food and in feed supplements. In this study, we examined the physiological changes and whole-genome expression in A. platensis C1 exposed to high temperature. We found that photosynthetic activity was significantly decreased after the temperature was shifted from 35°C to 42°C for 2 h. A reduction in biomass production and protein content, concomitant with the accumulation of carbohydrate content, was observed after prolonged exposure to high temperatures for 24 h. Moreover, the results of the expression profiling in response to high temperature at the designated time points (8 h) revealed two distinct phases of the responses. The first was the immediate response phase, in which the transcript levels of genes involved in different mechanisms, including genes for heat shock proteins; genes involved in signal transduction and carbon and nitrogen metabolism; and genes encoding inorganic ion transporters for magnesium, nitrite and nitrate, were either transiently induced or repressed by the high temperature. In the second phase, the long-term response phase, both the induction and repression of the expression of genes with important roles in translation and photosynthesis were observed. Taken together, the results of our physiological and transcriptional studies suggest that dynamic changes in the transcriptional profiles of these thermal-responsive genes might play a role in maintaining cell homeostasis under high temperatures, as reflected in the growth and biochemical composition, particularly the protein and carbohydrate content, of A. platensis C1.

Histopathogenesis of living insects of Myzus persicae Sulzer (Hemiptera: Aphididae) and Phenacoccus manihoti Matile‐Ferrero (Hemiptera: Pseudococcidae) by Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) was monitored from penetration through insect death. Important events in aphids included fungal penetration of the integument of the less-resistant leg intersegmental membrane and invasion of natural openings, formation of hyphal bodies in live aphids by three days post-inoculation (PI), and extensive hyphal colonization of the two leg segments closest to the insect body at death of the aphids. Confocal microscopy of green fluorescent protein-labeled B. bassiana in live mealybugs indicated the fungus penetrated the host through the legs and mouthparts. The fungus was scarce in live mealybugs at 1–5 days PI, formed hyphal bodies by six days PI, and growth was limited to parts of dead hosts at 6–7 days PI. In dead mealybugs, hyphal bodies were near solid tissue. Blastospores were in the hemolymph.

The development of a reliable genetic transformation system for Arthrospira platensis has been a long-term goal, mainly for those trying either to improve its performance in large-scale cultivation systems or to enhance its value as food and feed additives. However, so far, most of the attempts to develop such a transformation system have had limited success. In this study, an efficient and stable transformation system for A. platensis C1 was successfully developed. Based on electroporation and transposon techniques, exogenous DNA could be transferred to and stably maintained in the A. platensis C1 genome. Most strains of Arthrospira possess strong restriction barriers, hampering the development of a gene transfer system for this group of cyanobacteria. By using a type I restriction inhibitor and liposomes to protect the DNA from nuclease digestion, the transformation efficiency was significantly improved. The transformants were able to grow on a selective medium for more than eight passages, and the transformed DNA could be detected from the stable transformants. We propose that the intrinsic endonuclease enzymes, particularly the type I restriction enzyme, in A. platensis C1 play an important role in the transformation efficiency of this industrial important cyanobacterium.

The effect of oxygen availability on the molecular mechanisms of fatty acid biosynthesis was investigated in Mucor rouxii, a Mucorale fungus capable of producing gamma-linolenic acid through perturbation of the gaseous environment. Shifting of the M. rouxii culture from anaerobic to aerobic conditions resulted in an increase of the biomass and total fatty acid content of the M. rouxii culture. In addition, the levels of unsaturated fatty acids were enhanced accompanied by a decrease in the levels of medium- and long-chain saturated fatty acids. These results correspond to the levels of expressions of the Delta(9)-, Delta(12)- and Delta(6)-desaturases genes, all of which were coordinately up-regulated after the shift. The transcriptional response observed was rapid and transient, with the maximal mRNA levels detected between 0.5 h and 1.0 h after the shift. Together, our findings indicate that the anaerobic M. rouxii culture acclimatised to oxygen exposure by modulating fatty acid composition that was transcriptionally co-regulated by Delta(9)-, Delta(12)- and Delta(6)-desaturase genes.

Polyketides draw much attention because of their potential use in pharmaceutical and biotechnological applications. This study identifies an abundant pool of polyketide synthase (PKS) genes from local isolates of tropical fungi found in Thailand in three different ecological niches: insect pathogens, marine inhabitants, and lichen mutualists. We detected 149 PKS genes from 48 fungi using PCR with PKS-specific degenerate primers. We identified and classified 283 additional PKS genes from 13 fungal genomes. Phylogenetic analysis of all these PKS sequences the comprising ketosynthase (KS) conserved region and the KS-acyltransferase interdomain region yielded results very similar to those for phylogenies of the KS domain and suggested a number of remarkable points. (i) Twelve PKS genes amplified from 12 different insect-pathogenic fungi form a tight cluster, although along with two PKS genes extracted from genomes of Aspergillus niger and Aspergillus terreus , in reducing clade III. Some of these insect-specific fungal PKSs are nearly identical. (ii) We identified 38 new PKS-nonribosomal peptide synthetase hybrid genes in reducing clade II. (iii) Four distinct clades were discovered with more than 75% bootstrap support. We propose to designate the novel clade D1 with 100% bootstrap support “reducing clade V.” The newly cloned PKS genes from these tropical fungi should provide useful and diverse genetic resources for future research on the characterization of polyketide compounds synthesized by these enzymes.

The present study addresses the differential expression of Spirulina platensis proteins detected during cold-induced stress, analyzed at the subcellular level. In performing differential expression analysis, the results revealed upregulated proteins in every subcellular fraction, including two-component response systems, DNA repair, molecular chaperones, stress-induced proteins and proteins involved in other biological processes such as secretion systems and nitrogen assimilation. The chlorophyll biosynthetic proteins, protochlorophyllide oxidoreductase and ChlI, had unique expression patterns as detected in the thylakoid membrane; the levels of these proteins immediately decreased during the first 45 min of low-temperature exposure. In contrast, their expression levels significantly increased after low-temperature exposure, indicating the relevance of the chlorophyll biosynthesis in Spirulina in response to low-temperature stress in the light condition. In addition, this is the first report in which genome-based protein identification in S. platensis by peptide mass fingerprinting was performed using the database derived from the unpublished Spirulina genome sequence.

Two yeast strains of the genus Candida were isolated from wild flowers collected in Khao Yai National Park, Nakhonratchasima Province, Thailand. Based on morphological, physiological and chemotaxonomic characteristics and sequence analysis of the D1/D2 domain of the 26S rRNA gene, strains BCC 7722(T) (=NBRC 102563(T)=CBS 10611(T)) and BCC 7729(T) (=NBRC 102565(T)=CBS 10839(T)) were found to represent two distinct novel Candida species, for which the names Candida ratchasimensis sp. nov. and Candida khaoyaiensis sp. nov. are proposed, respectively. In the phylogenetic tree constructed according to the neighbour-joining method based on sequences of the D1/D2 domain of the 26S rRNA gene, strains BCC 7722(T) (GenBank accession no. AY228492) and BCC 7729(T) (accession no. DQ400367) constituted a cluster with Candida cellae that was connected with a clade with Starmerella meliponinorum and Candida etschellsii. Within the D1/D2 domain, C. ratchasimensis and C. khaoyaiensis differ from C. cellae in 25 nucleotide substitutions with five gaps and 29 nucleotide substitutions with one gap, respectively.

Asaia lannaensis sp. nov. was described for two strains isolated from flowers of the spider lily collected in Chiang Mai, Thailand. The isolates produced acetic acid from ethanol on ethanol/calcium carbonate agar, differing from the type strains of Asaia bogorensis, Asaia siamensis, and Asaia krungthepensis, but did not grow in the presence of 0.35% acetic acid (v/v). The new species is the fourth of the genus Asaia, the family Acetobacteraceae.

Strain NBRC 12467 T was examined genetically, phylogenetically, phenotypically, and chemotaxonomically. The DNA G+C content of the strain was 59.5 mol%. The strain represented low levels of DNA-DNA hybridization of 49-9% to the type strains of eight Gluconobacter species. The strain formed a cluster along with the type strains of G. albidus and G. kondonii in phylogenetic trees based on 16S rRNA gene sequences. In a phylogenetic tree based on 165-23S rRNA gene ITS sequences, however, the strain formed an independent cluster from the type strains of the eight Gluconobacter species. Such phylogenetic relationships were supported by the calculated pair-wise 16S rRNA gene and 16S-23S rRNA gene ITS sequence similarities. The strain was distinguished from the type strains of the eight Gluconobacter species by 16S-23S rRNA gene ITS restriction analysis using five restriction endonucleases. The strain produced a water-soluble brown pigment and 2,5-diketo-D-gluconate from D-glucose, differing from the type strains of the eight Gluconobacter species, and acid from meso-erythritol very weakly, differing from the type strains of the remaining seven Gluconobacter species except for the type strain of G. roseus, but not from maltose, differing from the type strain of G. oxydans, and had Q-10. For the strain, which was once classified as G. oxydans subsp. sphaericus, Gluconobacter sphaericus (Ameyama 1975) comb. nov. is proposed. The type strain is NBRC 12467 T , which is also deposited as BCC 14448 T .

119‒125 (2008) * Address reprint requests to: Dr. Yuzo Yamada, BIOTEC Cul-ture Collection, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Thailand Science Park, Phaholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand. E-mail: yamada333@kch.biglobe.ne.jp

Nonribosomal peptides, synthesized by nonribosomal peptide synthetases (NRPS), are an important group of diverse bioactive fungal metabolites. Xylaria sp. BCC1067, which is known to produce a variety of biologically active metabolites, was studied for gene encoding NRPS by two different PCR-based methods and seven different NRPS fragments were obtained. In addition, screening a genomic library with an amplified NRPS fragment as a probe identified a putative NRPS gene named XyNRPSA. The functionality of XyNRPSA for the production of a corresponding metabolite was probed by gene insertion inactivation. Comparing the disrupting metabolite profile with that of the wild type led to the identification of a speculated metabolite. The crude extract of Xylaria sp. BCC1067 also exhibits antifungal activity against the human pathogens Candida albicans and Trichophyton mentagrophytes. However, the evaluation of biological activity of the XyNRPSA product suggests that it is neither a compound with antifungal activity nor a siderophore. In the vicinity of XyNRPSA, a second gene (named XyPtB) was identified. Its localization and homology to orfB of the ergot alkaloid biosynthetic gene cluster suggests that XyPtB may be involved in XyNRPSA product biosynthesis.

Forty-four Thai isolates phenotypically assigned to the genus Gluconobacter were examined for 16S-23S rDNA ITS restriction analysis by MboII and SduI (=Bsp1286I) digestions. The Thai isolates tested were divided into seven groups: Group I for fourteen isolates, Group IX for one isolate, Group X for two isolates, Group V-2 for four isolates, Group XI for three isolates, Group IV for one isolate, and Group III for nineteen isolates. There were no isolates of either Group II or Group V-1 that were identified as G. cerinus. The isolates of Group III, Group IV, and Group XI were subjected to an additional 16S-23S rDNA ITS restriction analysis by AvaII, TaqI, BsoBI, and BstNI digestions. The isolates of Group III were divided into three groups and two subgroups: Group III-2 for five isolates, Group III-6 for two isolates, and Group III-4, which was divided into two subgroups, Subgroup III-4a for four isolates and Subgroup III-4b for eight isolates. The fourteen isolates of Group I were identified as G. oxydans, and the two isolates of Group X were temporarily identified as G. oxydans. The five isolates of Group III-2 and the one isolate of Group IV were identified as G. frateurii. The remaining twenty-two isolates of Group V-2, Group III-4, Group III-6, Group IX, and Group XI were not identified but are candidates for several new species.

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.

To understand the relationship between fatty acid metabolism and the growth morphology of Mucor rouxii, fatty acid profiling was studied comparatively in cells grown under conditions which included different atmospheric conditions or the addition of phenethyl alcohol (PEA). The significant difference in fatty acid profiles from M. rouxii grown under aerobic or anaerobic conditions was not found to be directly related to morphological growth. Oxygen limitation, which induced the formation of pure multipolar budding yeasts, led to a decrease in long-chain fatty acids-- particularly unsaturated fatty acids-- and an increase in medium-chain saturated fatty acids, a finding which contrasted with the aerobic cultures, including mycelia and PEA-induced bipolar budding cells. High levels of C18 : 1Delta(9) were found in aerobic yeast cultures with additional PEA when compared to that in the aerobically grown mycelia. The identification of unusual fatty acids in Mucor in response to alcoholic and hypoxic stresses - including odd-numbered fatty acids and 7-hydroxy dodecanoic acid (7-OH C12 : 0) in addition to the more common fatty acids - implied that an important role existed for these unusual fatty acids.

Spirulina-acyl-lipid desaturases are integral membrane proteins found in thylakoid and plasma membranes. These enzymes catalyze the fatty acid desaturation process of Spirulina to yield gamma-linolenic acid (GLA) as the final desaturation product. It has been reported that the cyanobacterial desaturases use ferredoxin as an electron donor, whereas the acyl-lipid desaturase in plant cytoplasm and the acyl-CoA desaturase of animals and fungi use cytochrome b (5). The low level of ferredoxin present in Escherichia coli cells leads to an inability to synthesize GLA when the cells are transformed with the Spirulina-(6) desaturase, desD, and grown in the presence of the reaction substrate, linoleic acid. In this study, Spirulina-(6) desaturase, encoded by the desD gene, was N-terminally fused and co-expressed with the cytochrome b (5) domain from Mucor rouxii. The product, GLA, made heterologously in E. coli and Saccharomyces cerevisiae, was then detected and analyzed. The results revealed the production of GLA by Spirulina-(6) desaturase fused or co-expressed with cytochrome b (5) in E. coli cells, in which GLA production by this gene cannot occur in the absence of cytochrome b (5). Moreover, the GLA production ability in the E. coli host cells was lost after the single substitution mutation was introduced to H52 in the HPGG motif of the cytochrome b (5) domain. These results revealed the complementation of the ferredoxin requirement by the fusion or co-expression of the fungal-cytochrome b (5) domain in the desaturation process of Spirulina-(6) desaturase. Furthermore, the free form of cytochrome b (5) domain can also enhance GLA production by the Spirulina-desD gene in yeast cells.

Fungal type I polyketide (PK) compounds are highly valuable for medical treatment and extremely diverse in structure, partly because of the enzymatic activities of reducing domains in polyketide synthases (PKSs). We have cloned several PKS genes from the fungus Xylaria sp. BCC 1067, which produces two polyketides: depudecin (reduced PK) and 19,20-epoxycytochalasin Q (PK-nonribosomal peptide (NRP) hybrid). Two new degenerate primer sets, KA-series and XKS, were designed to amplify reducing PKS and PKS-NRP synthetase hybrid genes, respectively. Five putative PKS genes were amplified in Xylaria using KA-series primers and two more with the XKS primers. All seven are predicted to encode proteins homologous to highly reduced (HR)-type PKSs. Previously designed primers in LC-, KS-, and MT-series identified four additional PKS gene fragments. Selected PKS fragments were used as probes to identify PKS genes from the genomic library of this fungus. Full-length sequences for five PKS genes were obtained: pks12, pks3, pksKA1, pksMT, and pksX1. They are structurally diverse with 1-9 putative introns and products ranging from 2162 to 3654 amino acids in length. The finding of 11 distinct PKS genes solely by means of PCR cloning supports that PKS genes are highly diverse in fungi. It also indicates that our KA-series primers can serve as powerful tools to reveal the genetic potential of fungi in production of multiple types of HR PKs, which the conventional compound screening could underestimate.

In previous studies, we characterized five histidine kinases (Hiks) and the cognate response regulators (Rres) that control the expression of approximately 70% of the hyperosmotic stress-inducible genes in the cyanobacterium Synechocystis sp. PCC 6803. In the present study, we screened a gene knock-out library of Rres by RNA slot-blot hybridization and with a genome-wide DNA microarray and identified three Hik-Rre systems, namely, Hik33-Rre31, Hik10-Rre3, and Hik16-Hik41-Rre17, as well as another system that included Rre1, that were involved in perception of salt stress and transduction of the signal. We found that these Hik-Rre systems were identical to those that were involved in perception and transduction of the hyperosmotic stress signal. We compared the induction factors of the salt stress- and hyperosmotic stress-inducible genes that are located downstream of each system and found that these genes responded to the two kinds of stress to different respective extents. In addition, the Hik33-Rre31 system regulated the expression of genes that were specifically induced by hyperosmotic stress, whereas the system that included Rre1 regulated the expression of one or two genes that were specifically induced either by salt stress or by hyperosmotic stress. Our observations suggest that the perception of salt and hyperosmotic stress by the Hik-Rre systems is complex and that salt stress and hyperosmotic stress are perceived as distinct signals by the Hik-Rre systems.

Production of red pigments (naphthoquinones) by the insect pathogenic fungus Cordyceps unilateralis BCC 1869 was investigated in this study. Cultivation conditions, including temperature, intitial pH of medium, and aeration, were optimised to improve the yield of total naphthoquinones in shake-flask culture of C. unilateralis. The highest yield of total naphthoquinones (3 g L-1) was obtained from a 28-day culture grown in potato dextrose broth with an initial pH of 7.0, at 28 degrees C with shaking-induced aeration at 200 rpm. An extraction process for isolation of the targeted naphthoquinone, 3,5,8-trihydroxy-6-methoxy-2-(5-oxohexa-1,3-dienyl)-1,4-naphthoquinone (3,5,8-TMON), from a culture of C. unilateralis, was also developed. The yield of 3,5,8-TMON obtained was about 1.2 g L-1 or 40% of total naphthoquinones. The stability of 3,5,8-TMON was very high, even upon exposure to strong sunlight (70,000 lx), high temperature up to 200 degrees C, and acid and alkali solutions at concentrations of 0.1 M.

Three bacterial strains were isolated from flowers collected in Bangkok, Thailand, by an enrichment-culture approach for acetic acid bacteria. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates were located in the lineage of the genus Asaia but constituted a cluster separate from the type strains of Asaia bogorensis and Asaia siamensis. The DNA base composition of the isolates was 60·2–60·5 mol% G+C, with a range of 0·3 mol%. The isolates constituted a taxon separate from Asaia bogorensis and Asaia siamensis on the basis of DNA–DNA relatedness. The isolates had morphological, physiological, biochemical and chemotaxonomic characteristics similar to those of the type strains of Asaia bogorensis and Asaia siamensis, but the isolates grew on maltose. The major ubiquinone was Q10. On the basis of the results obtained, the name Asaia krungthepensis sp. nov. is proposed for the isolates. The type strain is isolate AA08T (=BCC 12978T=TISTR 1524T=NBRC 100057T=NRIC 0535T), which had a DNA G+C content of 60·3 mol% and was isolated from a heliconia flower (‘paksaasawan’ in Thai; Heliconia sp.) collected in Bangkok, Thailand.

Genetic and biochemical approaches reveal the existence of a gamma-linolenic acid biosynthetic pathway in Mucor rouxii. By treatment with ultraviolet light, combined with low temperature cultivation and filtration enrichment, a mutant defective in polyunsaturated fatty acid synthesis was isolated. Genetic analysis and fatty acid supplementation indicate that the defect occurred in the Delta(12)-desaturation resulting in the absence of cis-linoleic acid and gamma-linolenic acid and in the accumulation of monounsaturated fatty acids. In addition, an unusual fatty acid, trans-linoleic acid (9c,12t-C18:2), which has not been reported previously in this fungus, was found to increase in the mutant. The information gained from the mutant was used to develop the hypothetical pathway of fatty acid desaturation in M. rouxii.

In cyanobacteria, the elevation of unsaturated fatty acid levels in membrane lipids has been shown to play a major role in the response to temperature change. The cyanobacterium Spirulina platensis strain C1 contains three desaturases--delta9, delta12 and delta6 desaturases--which are encoded by desC, desA and desD, respectively. In light of the above, a study was conducted of the regulation of desaturase gene expression in response to temperature change in S. platensis strain C1. The two lipid membranes, thylakoid and plasma, were separated, while the expressions of the desaturase genes to the downward shift of growth temperature were studied in the translation level by Western blot analysis. The results revealed that the expression of delta9 desaturase is independent of temperature. In the case of delta12 desaturase, two forms of the enzyme were found, at 45 and 40 kDa. In terms of correlation with the results in the transcription level, it is more likely that the 45 kDa-delta12 desaturase and the 40 kDa-delta12 desaturase are translated from 1.7 kb and 1.5 kb mRNA, respectively. Taken together, the results indicate that the expression of the 40 kDa-delta12 desaturase is temperature independent, whereas, the 45 kDa protein form demonstrates a response to the immediate temperature reduction. Furthermore, the activity of delta6 desaturase in the two lipid membranes is possibly regulated by temperature reduction. However, alteration in the level of gamma-linolenic acid, the product synthesized by delta6 desaturase, was observed in the plasma membrane prior to the thylakoid membrane.

Aigialomycins A-E (2-6), new 14-membered resorcylic macrolides, were isolated together with a known hypothemycin (1) from the mangrove fungus, Aigialus parvus BCC 5311. Structures of these compounds, including absolute configuration, were elucidated by spectroscopic methods, chemical conversions, and X-ray crystallographic analysis. Hypothemycin and aigialomycin D (5) exhibited in vitro antimalarial activity with IC(50) values of 2.2 and 6.6 microg/mL, respectively, while other analogues were inactive. Cytotoxicities of these compounds were also evaluated.

Bioassay-guided fractionation of the extracts from the insect pathogenic fungus Cordyceps nipponica BCC 1389 led to the isolation of N-hydroxy- and N-methoxy-2-pyridones, cordypyridones A-D (1-4). Structures of these compounds, including absolute configuration, were determined by spectroscopic methods, chemical conversions and single-crystal X-ray diffraction analyses. Codypyridones A and B, atropisomers of each other, exhibited potent in vitro antimalarial activity with IC(50) values of 0.066 and 0.037 microg/mL, respectively, while their cytotoxicity was much weaker.

Department of Chemistry, Faculty of Science,Mahidol University, Bangkok 10400, Thailand;and National Center for Genetic Engineering andBiotechnology (BIOTEC), National Science andTechnology Development Agency (NSTDA), 73/1,Rama VI Rd., Rajdhevee, Bangkok 10400, Thailandprasat@biotec.or.th.Received March 23, 2000

The reduction current of H2O2 which was generated by enzymatic reaction of glucose was measured at a microparticle-loaded polypyrrole film when glucose oxidase was in the solution or when it was immobilized in the film. This technique has the advantage of enabling detection at a low potential (0.0 V versus SCE). The current was measured at various thicknesses of the film, various concentrations of glucose and various potentials. The results were analyzed by use of previously derived diffusion-kinetic equations. It was found that the electrochemical reduction of H2O2 was the rate-determining step and that the reaction occurred at the interface between the film and the solution. Keywords: Enzyme, Amperometry, Microparticles, Polypyrrole, Hydrogen peroxide, Mass transfer model

The alteration of the degree of unsaturated fatty acids in membrane lipids has been shown to be a key mechanism in the tolerance to temperature stress of living organisms. The step that most influences the physiology of membranes has been proposed to be the amount of di-unsaturated fatty acids in membrane lipids. In this study, we found that the desaturation of fatty acid to yield the di-unsaturated fatty acid 18:2(9,12), in Spirulina platensis strain C1, was not regulated by temperature. As shown by the fatty acid composition and gene expression patterns, the levels of 18:1(9) and 18:2(9,12) remained almost constant either when the cells were grown at 35 degrees C (normal growth temperature) or 22 and 40 degrees C. The expression of desC (Delta9) and desA (Delta12) genes, which are responsible for the introduction of first and second double bonds into fatty acids, respectively, was not affected by the temperature shift from 35 to 22 degrees C or to 40 degrees C. Only the expression and mRNA stability of the desD gene (Delta6) that is responsible for the introduction of a third double bond into fatty acids were enhanced by a temperature shift from 35 to 22 degrees C, but not the shift from 35 to 40 degrees C. The increase in the level of desD mRNA elevated the desaturation of fatty acid from 18:2(9,12) to 18:3(6,9,12) at 22 degrees C. However, the increased level of 18:3(6,9,12) was observed after 36 h of incubation at 22 degrees C, indicating a slow response to temperature of fatty acid desaturation in this cyanobacterium. These findings suggest that the desaturation of fatty acids might not be a key mechanism in the response to the temperature change of S. platensis strain C1.

Metabolically-active mycelia of Penicillium sp. PT1 took up Zn2+ in a biphasic mode, involving an initial energy-dependent binding of Zn2+ to the cell surface, followed by a slower intracellular accumulation. The independent binding probably involved a simple ion exchange, as indicated by the pH decrease during the initial adsorption from 4.55 to 3.28. Intracellular accumulation probably involved polyphosphate precipitation as suggested by transmission electron microscopy

Background Starch serves as a temporal storage of carbohydrates in plant leaves during day/night cycles. To study transcriptional regulatory modules of this dynamic metabolic process, we conducted gene regulation network analysis based on small-sample inference of graphical Gaussian model (GGM). Results Time-series significant analysis was applied for Arabidopsis leaf transcriptome data to obtain a set of genes that are highly regulated under a diurnal cycle. A total of 1,480 diurnally regulated genes included 21 starch metabolic enzymes, 6 clock-associated genes, and 106 transcription factors (TF). A starch-clock-TF gene regulation network comprising 117 nodes and 266 edges was constructed by GGM from these 133 significant genes that are potentially related to the diurnal control of starch metabolism. From this network, we found that β-amylase 3 (b-amy3: At4g17090), which participates in starch degradation in chloroplast, is the most frequently connected gene (a hub gene). The robustness of gene-to-gene regulatory network was further analyzed by TF binding site prediction and by evaluating global co-expression of TFs and target starch metabolic enzymes. As a result, two TFs, indeterminate domain 5 (AtIDD5: At2g02070) and constans-like (COL: At2g21320), were identified as positive regulators of starch synthase 4 (SS4: At4g18240). The inference model of AtIDD5-dependent positive regulation of SS4 gene expression was experimentally supported by decreased SS4 mRNA accumulation in Atidd5 mutant plants during the light period of both short and long day conditions. COL was also shown to positively control SS4 mRNA accumulation. Furthermore, the knockout of AtIDD5 and COL led to deformation of chloroplast and its contained starch granules. This deformity also affected the number of starch granules per chloroplast, which increased significantly in both knockout mutant lines. Conclusions In this study, we utilized a systematic approach of microarray analysis to discover the transcriptional regulatory network of starch metabolism in Arabidopsis leaves. With this inference method, the starch regulatory network of Arabidopsis was found to be strongly associated with clock genes and TFs, of which AtIDD5 and COL were evidenced to control SS4 gene expression and starch granule formation in chloroplasts.

Isolates AH11(T) and AH13(T) were isolated from flowers of lantana and candle bush respectively collected in Thailand. In phylogenetic trees based on 16S rRNA gene sequences, the two isolates formed an independent cluster, which was then connected to the type strain of Saccharibacter floricola. The calculated pair-wise 16S rRNA gene sequence similarities of isolate AH11(T) were 95.7-92.3% to the type strains of the type species of the 12 genera of acetic acid bacteria. The DNA base composition was from 51.2 to 56.8 mol % G+C, with a range of 5.6 mol %. When isolate AH11(T) was labeled, DNA-DNA similarities were 100, 12, 4, 5, and 4% respectively to isolates AH11(T) and AH13(T) and the type strains of Saccharibacter floricola, Gluconobacter oxydans, and Acetobacter aceti. The two isolates were non-motile and did not oxidize either acetate or lactate. No growth was found in the presence of 0.35% acetic acid w/v. The two isolates were not osmophilic but osmotolerant, produced 2,5-diketo-D-gluconate from D-glucose, and did not oxidize lactate, thus differing from strains of Saccharibacter floricola, which showed weak lactate oxidation. The two isolates contained unsaturated C(18:1)ω7c fatty acid as the major fatty acid, and were unique in the presence of a considerable amount of straight-chain C(18:1)2OH fatty acid. Q-10 was present as the major isoprenoid quinone. Neokomagataea gen. nov. was proposed with the two species, Neokomagataea thailandica sp. nov. for isolate AH11(T) (=BCC 25710(T)=NBRC 106555(T)), which has 56.8 mol % G+C, and Neokomagataea tanensis sp. nov. for isolate AH13(T) (=BCC 25711(T)=NBRC 106556(T)), which has 51.2 mol % G+C.

The effect of oxygen availability on the molecular mechanisms of fatty acid biosynthesis was investigated in Mucor rouxii, a Mucorale fungus capable of producing gamma-linolenic acid through perturbation of the gaseous environment. Shifting of the M. rouxii culture from anaerobic to aerobic conditions resulted in an increase of the biomass and total fatty acid content of the M. rouxii culture. In addition, the levels of unsaturated fatty acids were enhanced accompanied by a decrease in the levels of medium- and long-chain saturated fatty acids. These results correspond to the levels of expressions of the Delta(9)-, Delta(12)- and Delta(6)-desaturases genes, all of which were coordinately up-regulated after the shift. The transcriptional response observed was rapid and transient, with the maximal mRNA levels detected between 0.5 h and 1.0 h after the shift. Together, our findings indicate that the anaerobic M. rouxii culture acclimatised to oxygen exposure by modulating fatty acid composition that was transcriptionally co-regulated by Delta(9)-, Delta(12)- and Delta(6)-desaturase genes.

Potential of methane production and trophic microbial activities at rhizospheric soil during rice cv. Supanbunri 1 cultivation were determined by laboratory anaerobic diluents vials. The methane production was higher from rhizospheric than non-rhizospheric soil, with the noticeable peaks during reproductive phase (RP) than vegetative phase (VP). Glucose, ethanol and acetate were the dominant available substrates found in rhizospheric soil during methane production at both phases. The predominance activities of trophic microbial consortium in methanogenesis, namely fermentative bacteria (FB), acetogenic bacteria (AGB), acetate utilizing bacteria (AB) and acetoclastic methanogens (AM) were also determined. At RP, these microbial groups were enhanced in the higher of methane production than VP. This correlates with our finding that methane production was greater at the rhizospheric soil with the noticeable peaks during RP (1,150 +/- 60 nmol g dw(-1) d(-1)) compared with VP (510 +/- 30 nmol g dw(-1) d(-1)). The high number of AM showed the abundant (1.1x10(4) cell g dw(-1)) with its high activity at RP, compared to the less activity with AM number at VP (9.8x10(2) cell g dw(-1)). Levels of AM are low in the total microbial population, being less than 1% of AB. These evidences revealed that the microbial consortium of these two phases were different.

Two isolates, AC04(T) and AC05, were isolated from the flowers of red ginger collected in Chiang Mai, Thailand. In phylogenetic trees based on 16S rRNA gene sequences, the two isolates were included within a lineage comprised of the genera Acidomonas, Gluconacetobacter, Asaia, Kozakia, Swaminathania, Neoasaia, Granulibacter, and Tanticharoenia, and they formed an independent cluster along with the type strain of Tanticharoenia sakaeratensis. The calculated pair-wise sequence similarities of isolate AC04(T) were 97.8-92.5% to the type strains of the type species of the 11 genera of acetic acid bacteria. The DNA base composition was 66.0-66.1 mol % G+C with a range of 0.1 mol %. A single-stranded, labeled DNA from isolate AC04(T) presented levels of DNA-DNA hybridization of 100, 85, 4, and 3% respectively to DNAs from isolates AC04(T) and AC05 and the type strains of Tanticharoenia sakaeratensis and Gluconacetobacter liquefaciens. The two isolates were unique morphologically in polar flagellation and physiologically in intense acetate oxidation to carbon dioxide and water and weak lactate oxidation. The intensity in acetate oxidation almost equaled that of the type strain of Acetobacter aceti. The two isolates had Q-10. Isolate AC04(T) was discriminated from the type strains of the type species of the 11 genera by 16S rRNA gene restriction analysis using restriction endonucleases TaqI and Hin6I. The unique phylogenetic, genetic, morphological, physiological, and biochemical characteristics obtained indicate that the two isolates can be classified into a separate genus, and Ameyamaea chiangmaiensis gen. nov., sp. nov. is proposed. The type strain is isolate AC04(T) (=BCC 15744(T), =NBRC 103196(T)), which has a DNA G+C content of 66.0 mol %.