Determination of the effect of polyamines on an oil-degrading strain of Yarrowia lipolytica using an odc minus mutant.

Yarrowia lipolytica is an ascomycetous dimorphic yeast with immense potential for industrial applications, including bioremediation of crude oil-contaminated environments. It has been shown that a dimorphic marine isolate of Y. lipolytica (var. indica) has significant capacity to degrade fatty acids and alkanes, when in its yeast morphology. It has also been demonstrated that polyamines play an important role in the yeast-to-mycelium transition of different strains of Y. lipolytica that are unable to utilize those carbon sources. To determine the role of polyamines on their capacity to utilize oils and hydrocarbons, on the dimorphic transition, and also on other characteristics of the var. indica strain of Y. lipolytica, we proceeded to obtain ornithine decarboxylase minus (odc-) mutants. These mutants behaved as yeasts independently of the concentrations of putrescine added. Further, they conserved the oil-degrading capacity of the parent strain. The odc- mutant can thus be used in fatty acid degradation, and oil spill remediation with distinct advantages.

Fungal cell membrane-promising drug target for antifungal therapy.

Increase in invasive fungal infections over the past few years especially in immunocompromised patients prompted the search for new antifungal agents with improved efficacy. Current antifungal armoury includes very few effective drugs like Amphotericin B; new generation azoles, including voriconazole and posaconazole; echinocandins like caspofungin and micafungin to name a few. Azole class of antifungals which target the fungal cell membrane are the first choice of treatment for many years because of their effectiveness. As the fungal cell membrane is predominantly made up of sterols, glycerophospholipids and sphingolipids, the role of lipids in pathogenesis and target identification for improved therapeutics were largely pursued by researchers during the last few years. Present review focuses on cell membrane as an antifungal target with emphasis on membrane biogenesis, structure and function of cell membrane, cell membrane inhibitors, screening assays, recent advances and future prospects.

Possible mechanism of antifungal phenazine-1-carboxamide from Pseudomonas sp. against dimorphic fungi Benjaminiella poitrasii and human pathogen Candida albicans.

AIM: Investigation of antifungal mechanism of phenazine 1-carboxamide (PC) produced by a Pseudomonas strain MCC2142. METHODS AND RESULTS: An antifungal metabolite produced by a Pseudomonas was purified and identified as PC. Human pathogenic fungi such as Candida albicans, Candida glabrata, Cryptococcus neoformans, Fusarium oxysporum, Aspergillus fumigatus and Aspergillus niger were found to be inhibited by PC (MIC90 32-64 µg ml(-1)). Addition of PC (20 µg ml(-1)) during yeast (Y)-hypha (H) transitions inhibited germ tube formation by >90% and >99% in C. albicans National Collection of Industrial Microorganisms (NCIM) 3471 and nonpathogenic model Benjaminiella poitrasii, respectively. After exposure to PC (20 µg ml(-1)), 75-80% yeast cells of B. poitrasii and C. albicans NCIM 3471 showed rhodamine 123 fluorescence indicating high intracellular reactive oxygen species (ROS) production. ROS further led to hyperpolarization of mitochondrial membrane, subsequently induction of apoptosis as evident by externalization of phosphatidylserine, DNA fragmentation, chromatin condensation and finally death in B. poitrasii. In C. albicans NCIM 3471, PC (20 µg ml(-1)) induced apoptosis. CONCLUSIONS: The antifungal effect of PC in B. poitrasii and C. albicans may be due to ROS-mediated apoptotic death. SIGNIFICANCE AND IMPACT OF THE STUDY: Inhibition of Y-H transition of B. poitrasii and C. albicans by PC indicates that it may prove useful in the control of dimorphic human pathogens.

Dimorphism and hydrocarbon metabolism in Yarrowia lipolytica var. indica.

Yarrowia lipolytica is able to metabolize high Mr hydrophobic natural compounds such as fatty acids and hydrocarbons. Characteristically, strains of Y. lipolytica can grow as populations with variable proportions of yeast and filamentous forms. In the present study, we describe the dimorphic characteristics of a variant designated as Y. lipolytica var. indica isolated from petroleum contaminated sea water and the effect of cell morphology on hydrocarbon metabolism. The variant behaved as a yeast monomorphic strain, under conditions at which terrestrial Y. lipolytica strain W29 and its derived strains, grow as almost uniform populations of mycelial cells. Using organic nitrogen sources and N-acetylglucosamine as carbon source, var. indica was able to form mycelial cells, the proportion of which increased when incubated under semi-anaerobic conditions. The cell surface characteristics of var. indica and W29 were found to be different with respect to contact angle and percent hydrophobicity. For instance, percent hydrophobicity of var. indica was 89.93 ± 1.95 while that of W29 was 70.78 ± 1.1. Furthermore, while all tested strains metabolize hydrocarbons, only var. indica was able to use it as a carbon source. Yeast cells of var. indica metabolized hexadecane with higher efficiency than the mycelial form, whereas the mycelial form of the terrestrial strain metabolized the hydrocarbon more efficiently, as occurred with the mycelial monomorphic mutant AC11, compared to the yeast monomorphic mutant AC1.

A biochemical correlate of dimorphism in a zygomycete Benjaminiella poitrasii: characterization of purified NAD-dependent glutamate dehydrogenase, a target for antifungal agents.

The fungal organisms, especially pathogens, change their vegetative (Y, unicellular yeast and H, hypha) morphology reversibly for survival and proliferation in the host environment. NAD-dependent glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2) from a non-pathogenic dimorphic zygomycete Benjaminiella poitrasii was previously reported to be an important biochemical correlate of the transition process. The enzyme was purified to homogeneity and characterized. It is a 371 kDa native molecular weight protein made up of four identical subunits. Kinetic studies showed that unlike other NAD-GDHs, it may act as an anabolic enzyme and has more affinity towards 2-oxoglutarate than L-glutamate. Chemical modifications revealed the involvement of single histidine and lysine residues in the catalytic activity of the enzyme. The phosphorylation and dephosphorylation study showed that the NAD-GDH is present in active phosphorylated form in hyphal cells of B. poitrasii. Two of the 1,2,3 triazole linked ß-lactam-bile acid conjugates synthesized in the laboratory (B18, B20) were found to be potent inhibitors of purified NAD-GDH which also significantly affected Y-H transition in B. poitrasii. Furthermore, the compound B20 inhibited germ tube formation during Y-H transition in Candida albicans strains and Yarrowia lipolytica. The possible use of NAD-GDH as a target for antifungal agents is discussed.

Herbicidal bio-assay of isocladosporin enantiomers and determination of its plausible absolute configuration.

A fungal metabolite, isocladosporin was isolated from natural fungus, Cladosporium cladosporioides in the mid of 90s. Due to the lack of optical rotation of isolated natural product sample, the absolute configuration of the natural product remained undetermined for more than two decades. Herein, we demonstrated an SAR study of enantiomers of isocladosporin in herbicidal bio-assay against wheat coleoptile. Using this study as a comparative tool we further proposed the plausible absolute configuration of natural isocladosporin for the first time. The assigned configuration was also supported through biogenetic precursors.

Can fungi compete with marine sources for chitosan production?

Chitosan, a ß-1,4-linked glucosamine polymer is formed by deacetylation of chitin. It has a wide range of applications from agriculture to human health care products. Chitosan is commercially produced from shellfish, shrimp waste, crab and lobster processing using strong alkalis at high temperatures for long time periods. The production of chitin and chitosan from fungal sources has gained increased attention in recent years due to potential advantages in terms of homogenous polymer length, high degree of deacetylation and solubility over the current marine source. Zygomycetous fungi such as Absidia coerulea, Benjaminiella poitrasii, Cunninghamella elegans, Gongrenella butleri, Mucor rouxii, Mucor racemosus and Rhizopus oryzae have been studied extensively. Isolation of chitosan are reported from few edible basidiomycetous fungi like Agaricus bisporus, Lentinula edodes and Pleurotus sajor-caju. Other organisms from mycotech industries explored for chitosan production are Aspergillus niger, Penicillium chrysogenum, Saccharomyces cerevisiae and other wine yeasts. Number of aspects such as value addition to the existing applications of fungi, utilization of waste from agriculture sector, and issues and challenges for the production of fungal chitosan to compete with existing sources, metabolic engineering and novel applications have been discussed to adjudge the potential of fungal sources for commercial chitosan production.

Regulation of chitin synthase activity in the dimorphic fungus Benjaminiella poitrasii by external osmotic pressure.

The effects of changes in external osmotic pressure on chitin synthase activity of a dimorphic fungus, Benjaminiella poitrasii, have been investigated. Mycelial and yeast cells incubated in medium of low osmolality (distilled water, 0 mOsm) for 10 min had 2-3-fold higher specific activities of native chitin synthase in mixed membrane preparations than cells that had been subjected to a high osmolality medium (1.2 M sorbitol in distilled water, 1612 mOsm). Cells suspended in media of different osmolalities for 10 min were also affected in the extent of germ tube formation. Germ tube formation was highest in cells incubated in low osmolality medium. The addition of protein phosphatase inhibitors (cyclosporin A, 1.2 micrograms/ml; cantharidin, 20 microM) abolished the effect of hypo-osmotic stress on chitin synthase activation of yeast mixed membrane preparations. The presence of protein kinase inhibitors (genistein, 40 micrograms/ml; H-7, 100 microM) and a Ca2+ channel blocker (verapamil, 50 microM) reduced chitin synthase activity to 50-60% of that observed in cells under hypo-osmotic shock. These inhibitors also inhibited germ tube formation. This suggests that chitin synthase activity and yeast hyphal morphogenesis are both subject to regulation by osmotic pressure, phosphorylation and calcium.

Solid-state CP/MASS 13C-NMR spectroscopy: a sensitive method to monitor enzymatic hydrolysis of chitin.

The time-course hydrolysis of colloidal chitin by the chitinase complex isolated from Myrothecium verrucaria was monitored using solution and solid-state 13C-NMR spectroscopy. The solution NMR studies showed the presence of N-acetylglucosamine (GlcNAc) as the sole product of hydrolysis. Solid-state 13C CP/MASS studies, on the other hand, indicated the presence of high molecular weight oligomers as well as GlcNAc. The linewidth of the C1 carbon of the oligomers obtained after hydrolysis is found to be less than that of the unhydrolyzed sample. The linewidths calculated from the spin-spin relaxation times (T2) of colloidal chitin and its products of hydrolysis were in the restricted range of 40-50 Hz, compared with the observed linewidths of 143-123 Hz. Peak area measurement on monomer to polymer/oligomer indicated an initial slow formation of the monomer, GlcNAc. From the NMR data, the involvement of endo-enzymes in the initial phase of hydrolysis is suggested.

Ethanol production from cellulose by coupled saccharification/fermentation using Saccharomyces cerevisiae and cellulase complex from Sclerotium rolfsii UV-8 mutant.

Using cellulase/hemicellulase complex of Sclerotium rolfsii UV-8 mutant and Saccharomyces cerevisiae for fermentation, the coupled saccharification/fermentation (CSF) of 15% AT-rice straw was carried out at 40 degrees C, pH 4.5 for the first 24 h and further incubation was performed at 30 degrees C for 72 h. Increasing the amount of cellulase activity from 3-12 IU FPA/g of substrate resulted in increased yields of ethanol from 1.5-3.6% in 96 h. It has been observed that the coupled system was advantageous over the two stage (separate hydrolysis/fermentation) system as it produced higher amounts of ethanol from cellulose (3.6% as compared to 2.3% ethanol from rice straw).

Factors affecting stability of Sclerotium rolfsii UV-8 mutant cellulase complex under saccharification conditions.

Enzyme stability studies in case of Sclerotium rolfsii UV-8 mutant have been investigated under the conditions used for saccharification of cellulose (50 degrees C, pH 4.5, 48 h). Avicelase (measure of exoenzymes) and xylanase were found to be less stable than CMCase (endoglucanase) and beta-glucosidase. Merthiolate (and other Hg compounds) added as a biocide, inactivated avicelase and xylanase about 60-70%. Of the antibiotics tested, tetracycline, chloramphenicol, and streptomycin sulfate were found suitable as an additive in cellulose hydrolysis system. The optimum hydrolysis of alkali-treated (AT)-rice straw, AT-bagasse, Solka Floc SW40, and Avicel P.H.101 was observed under shaking conditions at pH 4.5, 50 degrees C in CO2 atmosphere. It is suggested, all the studied parameters could be used for the evaluation of mutant strains.

Dimorphism in Benjaminiella poitrasii: involvement of intracellular endochitinase and N-acetylglucosaminidase activities in the yeast-mycelium transition.

The chitinase and N-acetylglucosaminidase activities in cell-wall-bound and free fractions in the dimorphic fungus Benjaminiella poitrasii were studied as a function of morphological (unicellular yeast-mycelium) transition. The specific activities of chitinases of cell-wall-free, particularly in the membrane fraction, were significantly different in the yeast and mycelial forms. During the yeast-mycelium transition, the N-acetylglucosaminidase activity isolated in a membrane preparation increased steadily. The activity of the yeast cells (0.83 +/- 0.17 nkat/mg protein) increased 17-fold to 14.2 +/- 1.7 nkat/mg protein in 1-d-old mycelial cells. The endochitinase activity increased 12-fold between 6 and 12 h and thereafter practically remained unchanged up to 24 h. A reverse trend in the chitinolytic activities was observed during the mycelium-yeast transition. Isoelectrofocussing (pH range 3.5-10) of mixed membrane fraction free of particulate fraction of parent and morphological (Y-5, yeast-form) mutant cells separated endochitinase and N-acetylglucosaminidase activity into two pH ranges, viz. 4.3-5.7 and 6.1-7.7, respectively. The predominant N-acetylglucosaminidase activity observed at pH 6.9 and 7.1 for the parent strain membrane fraction was undetected in the mutant preparation. The results suggested that the membrane-bound (either tightly or loosely) chitinolytic enzymes, particularly, N-acetylglucosaminidase, significantly contributed to the morphological changes in B. poitrasii.

Chitinolytic enzymes: an appraisal as a product of commercial potential.

Chitin, its deacetylated form, chitosan and chitinolytic enzymes viz. endo-chitinase, N-acetylglucosaminidase, chitosanase, chitin deacetylase (CDA) are gaining importance for their biotechnological applications. Presently, chitin degrading enzymes constitute high-cost low-volume products in human health care and associated research. Indeed chitinases and CDA-chitosanase complex possesss tremendous potential in agriculture to control plant pathogenic fungi and insects. The success in exploring chitinases especially for agriculture, i.e. as a high-volume low-cost product, depends on the availability of highly active preparations with a reasonable cost. Therefore, a reconsideration in terms of understanding the roles of chitinolytic enzymes in applications, e.g. host-pathogen interaction for biocontrol, different mechanisms of chitin degradation, and identification of new enzymes with varying specificities, may make them more useful in a variety of commercial processes in the near future. The possible issues and challenges encountered in the translation of proof of concept into a commercial product will be appraised in this review.

Flocculation of dimorphic yeast Benjaminiella poitrasii is altered by modulation of NAD-glutamate dehydrogenase.

A strategy to control flocculation is investigated using dimorphic yeast, Benjaminiella poitrasii as a model. Parent form of this yeast (Y) exhibited faster flocculation (11.1 min) than the monomorphic yeast form mutant Y-5 (12.6 min). Atomic force microscopy revealed higher surface roughness of Y (439.34 rms) than Y-5 (52 rms). Also, the former had a zeta potential of -65.97+/-3.45 as against -50.21+/-2.49 for the latter. Flocculation of both Y and Y-5 could be altered by supplementing either substrates or inhibitor of NAD-glutamate dehydrogenase (NAD-GDH) in the growth media. The rate of flocculation was promoted by alpha-ketoglutarate or isophthalic acid and decelerated by glutamate with a statistically significant inverse correlation to corresponding NAD-GDH levels. These interesting findings open up new possibilities of using NAD-GDH modulating agents to control flocculation in fermentations for easier downstream processing.

A rapid and sensitive method for screening of chitinase inhibitors using Ostazin Brilliant Red labelled chitin as a substrate for chitinase assay.

AIMS: The development of a simple, rapid and sensitive method for the screening of chitinase inhibitors using Ostazin Brilliant Red labelled chitin as a substrate for chitinase assay. METHODS AND RESULTS: The colloidal substrate prepared from the Ostazin Brilliant Red labelled chitin was used in the estimation of chitinase activity with and without the addition of potential chitinase inhibitor. The dye labelled N-acetylglucosamine (GlcNAc) released because of hydrolysis of the substrate was measured spectrophotometrically at 530 nm. The release of dye labelled GlcNAc from the colloidal Red chitin was proportional to the chitinase activity measured using other methods. The activities measured using acid swollen chitin and -Red chitin were comparable in the range 0.1-0.5 U ml(-1). The addition of chitinase inhibitor(s) (1000 ppm) proportionally reduced the release of dye labelled GlcNAc from the substrate in the reaction mixture. CONCLUSIONS: The assay for chitinase activity measuring the release of dye-labelled GlcNAc was simple, rapid and sensitive. There was no need to measure GlcNAc using a separate colorimetric method. SIGNIFICANCE AND IMPACT OF THE STUDY: The method provides a simple, one-step procedure for quantitative estimation of total chitinolytic activity and for screening of endochitinase and/or N-acetylglucosaminidase inhibitors acting singly or in combination. The method has a great potential in developing medium throughput procedure for the screening of chitinase inhibitors using 96-well plate reader.

Proteinases in fungal morphogenesis.

Fungal morphogenesis is a regulated series of events, leading to changes from one state to another, in which proteolysis could be regarded as one of the controlling functions. Proteinases are essential for the supply of amino acids, selective inactivation of specific growth phase proteins not required during development and for the activation and modification of the enzymes involved in cell wall synthesis. A critical evaluation of the role of proteinases as a biochemical correlate in fungal morphogenesis is discussed.

Mycopesticide production by fermentation: potential and challenges.

The agriculture industry is in need of novel biopesticides and development of large-scale production of mycopesticide, either fungal cells themselves or cell-free fungal components. The identification of a fungal strain with pesticide activity, and its improvement, is the primary step in developing infective propagules such as conidia, blastospores, chlamydospores, oospores, and zygospores as well as in preparing hydrolytic enzyme mixtures. This review discusses various parameters for submerged and solid state fermentation to produce fungal structures, particularly of mycoparasitic and entomopathogenic species that are prospective candidates for use as mycopesticides. The understanding of the molecular aspects of fungus-fungus and fungus-insect interactions, the role of hydrolytic enzymes especially chitinases in the killing process, and the possible use of chitin synthesis inhibitors are the prime areas of research aimed at making fungi more effective either singly or in combination as mycopesticides.

Dimorphism in Benjaminiella poitrasii: cell wall chemistry of parent and two stable yeast mutants.

In the dimorphic zygomycetous fungus Benjaminiella poitrasii, the cell wall compositions of mycelial phase (M), yeast phase (Y) and its yeast form mutants (Y-2 and Y-5) were studied. Chitosan was abundant in M-phase (26.6%) whereas lesser amounts were present in Y-phase (17.3%) and in mutants Y-2 (19.6%) or Y-5 (17.3%). Although chitin was present as a smaller fraction of the total glucosaminoglycan in each of different cell wall preparations, it was almost 3 times more prevalent in M-phase than the Y-phase cells. Cross-linking studies among the various cell wall components in B. poitrasii, suggest linkages among mannans and proteins and glucans and glucosaminoglycans.

Chitinolytic enzymes: their contribution to basic and applied research.

After cellulose, chitin is the second most abundant renewable resource available in nature. Marine invertebrates and fungal biomass are the two main sources of chitinous waste, which is commercially exploited. The enzymes involved in chitin degradation have been particularly well studied. Such enzymes have applications in ultrastructural studies, in the preparation of chitooligosaccharides which show anti-tumour activity, as biocontrol agents and in single-cell protein production. Here, the contribution chitin enzymology can make to basic and applied research is discussed.

Possible involvement of cyclic adenosine 3',5'-monophosphate in the regulation of NADP-/NAD-glutamate dehydrogenase ratio and in yeast-mycelium transition of Benjaminiella poitrasii.

The effect of different adenine-containing compounds on the NADP-/NAD-glutamate dehydrogenase (GDH) ratio was studied as a function of yeast-mycelium transition in Benjaminiella poitrasii. Under in vivo conditions, at a 5.0 mM concentration, cyclic AMP (cAMP) and dibutyryl cAMP maintained the cells in the yeast form for up to 7 and 5 h, respectively, and this was reflected in the patterns of GDH ratios observed. In vitro studies of phosphorylation and dephosphorylation have also been carried out, and the results suggest a possible correlation between cAMP, the GDH ratio, and cell form in B. poitrasii.