Detection of nonribosomal peptide synthetase genes in Xylaria sp. BCC1067 and cloning of XyNRPSA

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.     

Bilateral eyestalk ablation of the blue swimmer crab, Portunus pelagicus, produces hypertrophy of the androgenic gland and an increase of cells producing insulin-like androgenic gland hormone

The androgenic glands (AG) of male decapod crustaceans produce insulin-like androgenic gland (IAG) hormone that controls male sex differentiation, growth and behavior. Functions of the AG are inhibited by gonad-inhibiting hormone originating from X-organ-sinus gland complex in the eyestalk. The AG, and its interaction with the eyestalk, had not been studied in the blue swimmer crab, Portunus pelagicus, so we investigated the AG structure, and then changes of the AG and IAG-producing cells following eyestalk ablation. The AG of P. pelagicus is a small endrocrine organ ensheathed in a connective tissue and attached to the distal part of spermatic duct and ejaculatory bulb. The gland is composed of several lobules, each containing two major cell types. Type I cells are located near the periphery of each lobule, and distinguished as small globular cells of 5-7 μm in diameter, with nuclei containing mostly heterochromatin. Type II cells are 13-15 μm in diameter, with nuclei containing mostly euchromatin and prominent nucleoli. Both cell types were immunoreactive with anti-IAG. Following bilateral eyestalk ablation, the AG underwent hypertrophy, and at day 8 had increased approximately 3-fold in size. The percentage of type I cells had increased more than twice compared with controls, while type II cells showed a corresponding decrease.     

Role of light and photosynthesis on the acclimation process of the cyanobacteriumSpirulina platensis to salinity stress

The response ofSpirulina platensis cells to salinity stress was studied. Once adapted to the higher osmoticum, photosynthetic parameters such as the maximum rate of photosynthesis under saturating irradiance (P_max) and the initial slope of the P-I curve () are reduced by 15% and 25% in 0.5 M NaCl grown cells, respectively. Salt-adapted cells have a modified biochemical composition; reduced protein and chlorophyll content, and an increased level of carbohydrates. The reduction in the photosynthetic capacity of the salt-adaptedSpirulina cells reflects a lower ability to utilize light energy and results in an increase in the susceptibility of the stressed cells to photoinhibition. This conclusion is supported by the finding that cultures exposed to salt stress show not only a decrease in growth rate (), but lose the ability to respond to increased irradiance with an increase in growth. The use of variable fluorescence as a fast and reliable measurement to follow the changes in PSII of salt-stressesSpirulina cells enables following the early events of salinity shock. It indicates that as soon as the cells are exposed to salt, a protection mechanism is induced. This mechanism does not require any protein synthesis and may take place even in the dark, though at somewhat reduced effectiveness. The significance of the result in providing a better understanding of the interaction between two environmental stresses — light and salinity — and their application in the outdoor mass cultivation ofSpirulina are discussed.Author for correspondence     

Different effects of the combined stress of nitrogen depletion and high temperature than an individual stress on the synthesis of biochemical compounds in <Emphasis Type="Italic">Arthrospira platensis</Emphasis> C1 (PCC 9438)

In nature, several abiotic stresses occur simultaneously, leading to retarded growth and biochemical changes in microalgae, including the commercial cyanobacterium, Arthrospira platensis. To gain more understanding of stress response, we investigated the integrative effects of nitrogen depletion and high temperature stress on physiological changes of A. platensis C1. The results revealed that photosynthetic activities of the stressed cells were markedly reduced by more than a half in comparison to the non-stressed cells. Moreover, a reduction of biomass was observed within 24 h after prolonged exposure to combined stress of nitrogen depletion and high temperature. The total protein contents, including phycocyanin (PC), in the stressed cells, decreased rapidly within 8 h of incubation. This finding was concomitant with the increase in carbohydrate content. However, the accumulation of carbohydrates in the nitrogen depletion-treated cells was greater than that in the cells under the combined stress. Furthermore, the levels of polysaccharides increased only under long-term incubation under nitrogen depletion but not under the combined stress. In addition, the combination of nitrogen depletion and high temperature stress resulted in an increase in the proportion of linoleic acid but a decrease in γ-linolenic acid within 24 h. These results suggest that the response of A. platensis to the combined stress was different from the responses of cells to individual stress. The PC degradation, the increased carbohydrates, and the alteration in fatty acids profiles were required for physiological response to combined nitrogen depletion and high temperature stress of A. platensis C1.     

Bullera koratensis sp. nov. and Bullera lagerstroemiae sp. nov., two new ballistoconidium-forming yeast species in the Trichosporonales clade isolated from plant leaves in Thailand

Seven strains of ballistoconidiogenous yeasts that contain xylose, form Q-10 ubiquinone, propagate by budding and don't produce stalk conidia were isolated from plant leaves collected in Thailand and were found to represent two new species. The taxonomic properties of the two species coincided with the genus Bullera so they are described as Bullera koratensis sp. nov. and Bullera lagerstroemiae sp. nov. In phylogenetic trees based on the nucleotide sequences of 18S ribosomal DNA and the D1/D2 domain of 26S rDNA, these two species are located in the Trichosporonales clade (Cryptococcus humicola-Trichosporon lineage).     

Comparative fatty acid profiling of Mucor rouxii under different stress conditions

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.     

Characterization of gamma-glutamyl hydrolase produced by Bacillus sp. isolated from Thai Thua-nao

Gamma-glutamyl hydrolase with a molecular mass of 28 kDa was purified from the culture broth of Bacillus sp. isolated from Thai Thua-nao, a natto-like fermented soybean food. The purified enzyme hydrolyzed chemically synthesized oligo-gamma-L-glutamates but not oligo-gamma-D-glutamates and degraded gamma-polyglutamic acid to a hydrolyzed product of only about 20 kDa (with D- and L-glutamic acid in a ratio of 70:30), suggesting that the enzyme is a gamma-glutamyl hydrolase that cleaves the gamma-glutamyl linkage between L- and L-glutamic acid of gamma-polyglutamic acid.     

Insect-Specific Polyketide Synthases (PKSs), Potential PKS-Nonribosomal Peptide Synthetase Hybrids,and Novel PKS Clades in Tropical Fungi

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.One hallmark of tropical countries is the tremendous availability and diversity of natural resources. Tropical forests, freshwater reservoirs, and seas are home to an uncountable number of species, ranging from microorganisms (e.g., bacteria, fungi, and protozoa) to invertebrates to vertebrates to plants. Thailand is no exception. The country has a large collection of fungi found in different niches and habitats in its ecosystems. Interesting groups include fungi that are associated with insects, those that inhabit the sea, and those that are in lichen complexes; these are referred to here as insect fungi, marine fungi, and lichenized fungi, respectively. The first group is of particular interest because it represents a remarkable relationship (in this case, pathogenesis) between the fungi and their insect hosts. These entomopathogenic fungi were isolated from the dead insect bodies in different stages (e.g., larvae, pupae, nymphs, or adults). The marine fungi used in this study were mostly isolated from the living or dead plant parts floating at the seashore, whereas the lichen mutualistic fungi were isolated from lichen complexes on the bark of trees in tropical forests in Thailand. All these fungal isolates were deposited in National Center for Genetic Engineering and Biotechnology (BIOTEC) Culture Collection (BCC). The BCC has one of the richest collections (approximately 400 species and 5,000 isolates) of insect fungi in the world (19).Secondary metabolites may play an important role in organisms that synthesize them, for example, in spore development (7), protection, or host virulence (5). Polyketides (PKs) are natural secondary metabolite compounds derived from the condensation of acyl coenzyme A subunits in a head-to-tail manner, and they have a tremendous diversity in structure (33). Structural diversification of the PKs includes a variation in the number of subunits, types of subunits, degree of chemical reduction of the β-keto thioester, extent of stereochemistry of the α-keto group at each condensation, and subsequent processing (e.g., cyclicization) (25, 28, 33). The high therapeutic and economic value of PK compounds has attracted the interest of drug companies and government research agencies. Some PKs are commercially available for medical treatments, such as grahamimycin and patulin (antibiotics), lovastatin and compactin (cholesterol-lowering agents), griseofulvin (an antibiotic/antifungal agent), and monocerin (an antifungal agent).Enzymes that synthesize the PKs are called PK synthases (PKSs). PKSs are multifunctional enzymes that are composed of three principal domains: ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP). Fungal PKSs are type I, multifunctional large enzymes and use an iterative strategy to synthesize PKs. They can be divided into two groups, nonreducing (NR) and reducing (4), and further subdivided into NR subclades I, II, and III and reducing subclades I, II, III, and IV (26). NR PKSs include those synthesizing pigments or aflatoxin. Reducing PKSs are involved in the synthesis of PK compounds with various chemical reductions in structure. Apart from the three major domains (KS, AT, and ACP) present in all PKSs, reducing PKSs contain three additional domains, i.e., dehydratase, enoyl reductase, and ketoreductase, which are involved in the reduction of the keto group to various stages (i.e., alcohol, unsaturated thiolester, and full saturation, respectively), therefore enhancing diversity of the PK structure.Kroken et al. (26) studied putative amino acid sequences of the PKS genes previously characterized in fungi and the PKS genes discovered from the genome sequencing projects for eight fungal species in the Ascomycota. PKS genes were found only in the genomes of the Pezizomycotina and not in the sequenced genomes of either Ascomycota in the Taphrinomycota or Saccharomycotina or Basidiomycota in the Hymenomycetes. Thus, we focused our search on the fungi in this subphylum. We aimed to mine valuable PKS genes from this fungal resource. One of the main objectives is to find novel secondary metabolites useful for medical or agricultural applications. One highly regarded example is the “vegetable caterpillar,” where the fungus Cordyceps sinensis grows on Hepialidae caterpillars. The fungus has long been used in traditional Chinese medicine. Extracts of Cordyceps sinensis were reported to have a variety of therapeutic effects, for example, antitumor (6), antioxidant (42), and antiaging (24) activities. The C. sinensis-Hepialidae pair is also called the “body snatcher”. This name comes from the fact that the fungus infects and consumes the insect tissue and fills up the insect cavity with its mycelia. Thus, another objective is to find metabolites involved in interaction between fungal pathogens and their insect hosts. Insect pests pose tremendous losses to humans in regard to health issues (vectors of diseases) and economic issues (crop plant losses by insect pathogens and building structure damage by termites). Little was known regarding the roles of PKs in producing fungi on their interaction with insect host. Better understanding of this relationship might have implications for insect control.We conducted our PKS screening using PCR with the degenerate KA series primers (2). In addition to our preliminary PKS screening with these primers in a few fungi (2), the KA series primers were used to clone the reducing PKS gene for radicicol biosynthesis from the fungus Pochonia chlamydosporia, and later its whole biosynthetic cluster was revealed (37). Here, the method and the primers were also proven to be successful in finding rich resources of hidden metabolic pathways for PK biosynthesis from 48 fungi that were isolated in Thailand and, particularly, have no genome sequences determined. In addition, more than 200 PKS genes were identified from our genome analysis of 13 filamentous fungi.     

Production of alkaline protease by a genetically engineered Aspergillus oryzae U1521

The production of alkaline protease of Aspergillus oryzae U1521 was examined in liquid culture. In a culture of defatted soybean only, it gave satisfactory enzyme yields at 584,000 U/g defatted soybean. When various carbohydrates were supplemented, enzyme production was significantly increased. An increase in production by lactose was the most marked. Enrichment with casitone or casein increased productivity, but not cornsteep solid. Media formulation (g/L) of defatted soybean 10, lactose 5, casitone 1, and KH(2)PO(4) 5 enhanced alkaline protease production by A. oryzae U1521 to a maximum of 1,410,000 U/g defatted soybean. Scaling-up experiments indicated the flask-scale results could be reproduced at 40 g of substrate in 5-L fermenter. The enzyme activity was maximum between pH 8-9 and at a temperature of 45 degrees C.