Psychrotrophic Fungi Research Articles

Comparative omics directed gene discovery and rewiring for normal temperature-adaptive red pigment synthesis by polar psychrotrophic fungus Geomyces sp. WNF-15A

The Antarctic fungus Geomyces sp. WNF-15A can produce high-quality red pigments (AGRP) with good prospects for the use in food and cosmetic area. However, efficient AGRP synthesis relies on low-temperature and thus limits its industrial development. Here genome sequencing and comparative analysis were performed on the wild-type versus to four mutants derived from natural mutagenesis and transposon insertion mutation. Eleven mutated genes were identified from 2309 SNPs and 256 Indels. A CRISPR-Cas9 gene-editing system was established for functional analysis of these genes. Deficiency of scaffold1.t692 and scaffold2.t704 with unknown functions highly improved AGRP synthesis at all tested temperatures. Of note, the two mutants produced comparable levels of AGRP at 20 °C to the wild-type at 14 °C. They also broke the normal-temperature limitation and effectively synthesized AGRP at 25 °C. Comparative metabolomic analysis revealed that deficiency of scaffold1.t692 improved AGRP synthesis by regulation of global metabolic pathways especially downregulation of the competitive pathways. Knockout of key genes responsible for the differential metabolites confirmed the metabolomic results. This study shows new clues for cold-adaptive regulatory mechanism of polar fungi. It also provides references for exploitation and utilization of psychrotrophic fungal resources.

Minos and Restless transposon insertion mutagenesis of psychrotrophic fungus for red pigment synthesis adaptive to normal temperature

The polar psychrotrophic fungus Geomyces sp. WNF-15A can produce high-quality natural red pigment for the potential use as edible pigment. However, it shows low-temperature-dependent synthesis of red pigment, which limits its large-scale industrial applications due to the difficult and high-cost bioprocess control. This study aims to develop transposon-mediated mutagenesis methods to generate mutants that are able to synthesize red pigment at normal temperature. Four transposable systems, including single and dual transposable systems, were established in this fungus based on the Minos from Drosophila hydei and the Restless from Tolypocladium inflatum. A total of 23 production-dominant mutants and 12 growth-dominant mutants were thus obtained by constructed transposable systems. At 14 °C and 20 °C, the MPS1 mutant strain achieved the highest level of red pigment (OD520 of 43.3 and 29.7, respectively), which was increased by 78.4% and 128.7% compared to the wild-type, respectively. Of note, 4 mutants (MPS1, MPS3, MPS4 and MPD1) successfully synthesized red pigment (OD520 of 5.0, 5.3, 4.7 and 4.9, respectively) at 25 °C, which broke the limit of the wild-type production under normal temperature. Generally, the dual transposable systems of Minos and Restless were more efficient than their single transposable systems for mutagenesis in this fungus. However, the positive mutation ratios were similar between the dual and single transposable systems for either Minos or Restless. This study provides alternative tools for genetic mutagenesis breeding of fungi from extreme environments.Graphical

Transposon insertion mutation of Antarctic psychrotrophic fungus for red pigment production adaptive to normal temperature.

Polar regions are rich in microbial and product resources. Geomyces sp. WNF-15A is an Antarctic psy chrotrophic filamentous fungus producing high quality red pigment with potential for industrial use. However, efficient biosynthesis of red pigment can only realize at low temperature, which brings difficult control and high cost for the large-scale fermentation. This study aims to develop transposon insertion mutation method to improve cell growth and red pigment production adaptive to normal temperature. Genetic manipulation system of this fungus was firstly developed by antibiotic marker screening, protoplast preparation and transformation optimization, by which transformation efficiency of ∼50% was finally achieved. Then transposable insertion systems were established using Helitron, Fot1, and Impala transposons. The transposition efficiency reached 11.9%, 9.4%, and 4.6%, respectively. Mutant MP1 achieved the highest red pigment production (OD520 of 39) at 14°C, which was 40% higher than the wild-type strain. Mutant MP14 reached a maximum red pigment production (OD520 of 14.8) at 20°C, which was about twofold of the wild-type strain. Mutants MP2 and MP10 broke the repression mechanism of red pigment biosynthesis in the wild-type and allowed production at 25°C. For cell growth, eight mutants grew remarkably better (12%∼30% biomass higher) than the wild-type at 25°C. This study established an efficient genetic manipulation and transposon insertion mutation platform for polar filamentous fungus. It provides reference for genetic breeding of psychrotrophic fungi from polar and other regions.

Isolation of a novel psychrotrophic fungus for efficient low-temperature composting

This study aimed to isolate psychrotrophic cellulose-degrading fungi and to investigate their application potential for composting in cold climate regions in China. One out of five psychrotrophic cellulose-degrading fungal isolates was identified as a novel fungal species, Aureobasidium paleasum sp. nov., with a strong straw degradation potential. Enzyme activity assays and FITR spectroscopy revealed high cellulolytic activities of this psychrotrophic fungus at lower temperatures, with high thermal adaptability from 5 °C to 50 °C (optimum at 10 °C). A. paleasum efficiently decomposed rice straws and cellulose at 10 °C compared to the common cellulose-degrading fungus Penicillium oxalicum. In comparison to P. oxalicum, A. paleasum shortened the thermophilic stage, enhanced compost maturity and improved compost quality. Our work suggests that the psychrotrophic fungus A. paleasum is efficient for rice straw degradation and composting at low temperatures, highlighting its application potential for composting in colder regions.

Total Synthesis and Conformational Analysis of Naturally Occurring Lipovelutibols along with Lead Optimization of Lipovelutibol D.

Four lipopeptaibols, namely, lipovelutibols A–D, were recently isolated from psychrotrophic fungus Trichoderma velutinum and reported to have significant cytotoxic activity against HL-60, MDA-MD-231, A549, and LS180 cancer cell lines. In the present study, these peptides were synthesized in a solution using a segment condensation approach. The conformational analysis of these peptides carried out using CD spectrophotometry revealed the formation of 310-helix, and the NMR-VT experiments showed intramolecular hydrogen bonding for NH-5, NH-6, and NH-7. Lipovelutibol D showed potent cytotoxic activity and was chosen for lead optimization. It involved N- and C-terminal truncation, N- and C-terminal modification, random deletion, l/d configuration replacement, and other synthetic analogues. These were tested against various breast cancer cell lines. The C-terminal aldehyde analogue resulting from lead optimization of lipovelutibol D was found to have almost twofold enhanced cytotoxicity against MDA-MB-231 breast cancer cell lines.

Mutagenesis and Adaptation of the Psychrotrophic Fungus Chrysosporium pannorum A-1 as a Method for Improving β-pinene Bioconversion.

Mutagenesis and adaptation of the psychrotrophic fungus Chrysosporium pannorum A-1 to the toxic substrate β-pinene were used to obtain a biocatalyst with increased resistance to this terpene and improved bioconversion properties. Mutants of the parental strain were induced with UV light and N-methyl-N′-nitro-N-nitrosoguanidine. Mutants resistant to β-pinene were isolated using agar plates with a linear gradient of substrate concentrations. Active mutants were selected based on their general metabolic activity (GMA) expressed as oxygen consumption rate. Compared to the parental strain, the most active mutant showed an enhanced biotransformation ability to convert β-pinene to trans-pinocarveol (315 mg per g of dry mycelium), a 4.3-fold greater biocatalytic activity, and a higher resistance to H2O2-induced oxidative stress. Biotransformation using adapted mutants yielded twice as much trans-pinocarveol as the reaction catalyzed by non-adapted mutants. The results indicate that mutagenesis and adaptation of C. pannorum A-1 is an effective method of enhancing β-bioconversion of terpenes.

Biodiversity and enzymes bioprospection of Antarctic filamentous fungi

AbstractAntarctica is one of the most suitable locations for the bioprospecting of psychrotrophic fungi, which play a key role in the nutrient cycle and organic material mineralization in cold environments. These actions mainly take place via the production of several cold-active extracellular enzymes. The aim of this study was to investigate the diversity of filamentous fungi from King George Island (25 De Mayo Island), Antarctica and their ability to produce extracellular hydrolytic enzymes at low temperatures. A total of 51 fungal isolates were obtained from 31 samples. Twelve genera were identified, with seven among the Ascomycota (Cadophora, Helotiales, Monographella, Oidodendron, Penicillium, Phialocephala, Phialophora, Phoma and Pseudogymnoascus), one Basidiomycota (Irpex) and two Mucoromycota (Mortierella and Mucor). Monographella lycopodina and Mucor zonatus, not previously reported in Antarctica, were identified. Nine isolates could not be identified to genus level and may represent novel species. Most of the studied fungi were psychrotrophic (76.5%). Nevertheless, only five isolates were able to grow at 35°C, 15°C being the optimal growth temperature for 65% of the fungal isolates. Results from enzyme production at low temperature revealed that the Antarctic environment contains metabolically diverse fungi, which represent potential tools for biotechnological applications in cold regions.

Study on the potential of cold-active lipases from psychrotrophic fungi for detergent formulation.

Lipases from psychrotrophic fungal isolates BPF4 and BPF6 identified as Penicilium canesense and Pseudogymnoascus roseus respectively were characterized for their compatibility towards laundry detergent. BPF4 and BPF6 lipases showed maximum activity at pH 11 and 9 respectively and at 40 °C. The residual activities at 20 °C and 4 °C of BPF4 lipase were 35% and 20% and of BPF6 lipase were 70% and 20 °C respectively. Both the enzymes were stable at 4 °C, 20 °C and 40 °C for 2 h losing at the most 20% of activities. Both the enzymes were metalloenzymes with activity enhancement by nearly threefold by Ca2+. Contrary to BPF6 lipase, BPF4 enzyme was not stimulated by EDTA nor inhibited, rather stimulated by SDS and Triton X-100 by 125% and 330% respectively. Both the lipases showed minor to moderate inhibition by NaClO3 and H2O2, and exhibited nearly 90% residual activity after 1 h of incubation in selected detergent brands thus indicating potential for their inclusion in detergent formulation thereby facilitating cold-washing as a step towards mitigation of climate change.

Lipovelutibols A-D: Cytotoxic Lipopeptaibols from the Himalayan Cold Habitat Fungus Trichoderma velutinum.

Four novel lipovelutibols A (1), B (2), C (3), and D (4) containing six amino acid residues with leucinol at the C-terminus and a fatty acyl moiety (n-octanoyl) at its N-terminus were isolated from the psychrotrophic fungus Trichoderma velutinum collected from the Himalayan cold habitat. The structures (1-4) were determined by NMR and MS/MS, and the stereochemistry of amino acids by Marfey's method. Lipopeptaibols 2 and 4 were found to contain d-isovaline, a nonproteinogenic amino acid, but lacked α-aminoisobutyric acid, characteristic of peptaibols. Cytotoxic activity of 2 and 4 was observed against HL-60, LS180, MDA-MB-231, and A549 cancer cell lines.

Antagonistic potential of a psychrotrophic fungus: Trichoderma velutinum ACR-P1

Trichoderma is a well explored genus for plethora of bioactivities due to their cosmopolitan existence, diverse metabolomics, reproductive and competitive efficacies. Mycoparasitism and antibiotics production seems to be the probable mechanisms underlying their use as biocontrol agents. The aim of this study is to investigate the antagonistic potential of a new psychrotrophic fungus Trichoderma velutinum ACR-P1 as a promising biocontrol agent against destructive phytopathogens with probable underlying mechanisms. Antagonistic potential of T. velutinum ACR-P1 against the important phytopathogens, that is, Fusarium oxysporum, Verticillium dahliae, Alternaria alternata and Colletotrichum capsici was demonstrated by the in vitro dual culturing experiments. Also, probable mechanism underlying antagonism was studied by investigating enzymatic cell wall hydrolytic potential of the strain and potential for production of secondary metabolites i.e. non-ribosomal peptides (NRPs). The putative strain ACR-P1 showed immense potential of inhibiting the growth of all the four test phytopathogens and its capability of producing cell wall degrading enzymes (CWDE) i.e. chitinase, cellulase and protease utilizing colloidal chitin, carboxymethyl cellulose and milk protein respectively as sole source of carbon and energy as illustrated by growth and agar well diffusion assays. Also, metabolic profiling firstly revealed the production of two groups of the NRPs by T. velutinum ACR-P1 as reported in our earlier study on the strain (Sharma et al., 2016). Mycoparasitism supported by NRPs and CWDEs substantiate their use as biocontrol agents. The psychrotrophic strain is endowed with immense potential to be included as a new candidate in the list of biocontrol agents (BCA). The potential antagonism against the important destructive phytopathogens signifies T. velutinum ACR-P1 as a BCA and can be exploited in fields to get rid of devastating impacts of these phytopathogens on the crops.

Thermoduric psychrotrophic proteolytic microbiota from refrigerated raw milk

Thermoduric microorganisms may withstand high temperatures during the pasteurization of milk. Therefore, the microbiota in pasteurized milk consist of thermoduric microbes and directly influence the shelf-life of the milk. The aim of this study was to identify thermoduric psychrotrophic proteolytic microbiota in refrigerated raw milk. Twenty samples, previously heat-treated, were streaked and incubated at 7°C for 10 days. The strains isolated were streaked on milk agar to assess proteolytic activity and were initially analyzed morphologically by light microscopy and then by molecular techniques to identify the species. In 40% of the samples analyzed was observed only one bacterial growth and others 10 thermoduric psychrotrophic fungi. All isolates were proteolytic. The sequencing of 16S rRNA gene identified the bacterial strain as Bacillus pumilus and analysis of the ITS1-5.8S-ITS2 region of fungal isolates revealed the Cladosporium cladosporioides (60%), Curvularia geniculatus (10%), and the 3 remaining strains were identified as Geotrichum candidum (30%). This is a fist description of B. pumilus in Brazilian raw milk. Considering the spoilage potential of all isolates and of the fungi present in raw milk and their survival in pasteurized milk, it is extremely important to carry out further studies to your resistant to heat, their impact on the shelf-life of pasteurized milk, ultra-high temperature (UHT) milk and dairy products.

Psychrophilic and psychrotrophic fungi: a comprehensive review

This article reviews the comparative diversity of psychrophilic and psychrotrophic fungi, their adaptability mechanisms for survival and potential applications in biotechnology and pharmaceuticals. Fungi are able to grow and survive at low temperature and exist widely in polar and non-polar habitats. These cold regions are known for very low temperature, high ultra violet-B radiation, frequent freeze and thaw cycles and low water and nutrient availability. Most of the fungi adapt to such harsh conditions by evolving various strategies in their metabolism and physiology. Psychrophilic and psychrotrophic fungi are of importance in biotechnological and pharmaceutical fields due to their diverse characteristics developed or evolved due to their adaptation and survival in extreme environments, like; production of cold-active enzymes, pharmaceutical or bioactive metabolites and exo-polysaccharides, have potential for bioremediation and can also be used as biofertilizer.

Bioconversion of α-pinene by a novel cold-adapted fungus Chrysosporium pannorum.

The psychrotrophic fungus Chrysosporium pannorum A-1 is reported for the first time as a novel biocatalyst for O2-promoted oxidation of α-pinene. GC–MS analysis indicated that the main products of the reaction were compounds of a high commercial value, verbenol (1) and verbenone (2). Exponentially growing cells (days 2–3) were about twice as active as cells in the late stationary phase in terms of the total concentration of products. The highest yields of 1 and 2 were obtained using three-day and two-day-old mycelia and a medium containing 1.5 and 1 % (v/v) of the substrate, respectively. The optimal time for the bioconversion of α-pinene varied from 1 to 3 days, and depended on the kind of product desired. Most of 1 was produced at a relatively high concentration of 360 mg/L after the first six hours of α-pinene bioconversion [with an average yield of 69 mg/(g dry cell L aqueous phase)]. The oxidative activity of C. pannorum was identified across a wide temperature range of 5–25 °C, 10 °C being the optimum for the production of 1 and 20 °C for the production of 2. Sequential addition of the substrate during 3 days of the biotransformation resulted in a significant increase in 1 and 2 up to 722 and 176 mg/L, respectively, and a 2-fold enhancement of product yield as compared to bioconversion with a single supply of α-pinene. The concentration of total conversion products in the culture medium reached 1.33 g/L [which corresponded product yield of 225 mg/(g dry cell L)]. This represents probably the most promising result reported to date for oxidative biotransformation of α-pinene by a wild-type microorganism.Electronic supplementary materialThe online version of this article (doi:10.1007/s10295-014-1550-0) contains supplementary material, which is available to authorized users.

Purification and Properties of a Psychrotrophic Trichoderma sp. Xylanase and its Gene Sequence

A psychrotrophic fungus identified as Trichoderma sp. SC9 produced 36.7 U/ml of xylanase when grown on a medium containing corncob xylan at 20 °C for 6 days. The xylanase was purified 37-fold with a recovery yield of 8.2%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 20.5 kDa. The enzyme had an optimal pH of 6.0, and was stable over pH 3.5-9.0. The optimal temperature of the xylanase was 42.5 °C and it was stable up to 35 °C at pH 6.0 for 30 min. The xylanase was thermolabile with a half-life of 23.9 min at 45 °C. The apparent K(m) values of the xylanase for birchwood, beechwood, and oat-spelt xylans were found to be 3, 2.1, and 16 mg/ml respectively. The xylanase hydrolyzed beechwood xylan and birchwood xylan to yield mainly xylobiose as end products. The enzyme-hydrolysed xylotriose, xylotetraose, and xylopentose to produce xylobiose, but it hardly hydrolysed xylobiose. A xylanase gene (xynA) with an open reading frame of 669 nucleotide base pairs (bp), encoding 222 amino acids, from the strain was cloned and sequenced. The deduced amino acid sequence of XynA showed 85% homology with Xyn2 from a mesophilic strain of Trichoderma viride.

Culture Conditions of Psychrotrophic Fungus, Penicillium chrysogenum and Its Lipase Characteristics

Among 97 fungal strains from the soil collected from the high mountain areas in the Jeju Island, Korea, Penicillium chrysogenum 9 was found to be the best lipase producer. Its lipase productivity reached 42 U/ml in the culture medium. Factors affecting lipase production by Penicillium chrysogenum 9 were studied using fermentation media of different chemical compositions. Under optimal conditions we noted a 1.6-fold increase of lipase activity. The maximum lipase activity was 68 U/ml of culture medium on the 5th day of incubation at pH 6.0 and 20 ˚C; therefore, Penicillium chrysogenum 9 was classified as a psy- chrotrophic microorganism. The enzyme from Penicillium chrysogenum 9 showed a maximum activity at 30 ˚C and pH 5.0 for synthetic substrates (triacylglycerols) or at pH 7.0 for natural substrates (oils).

A novel psychrotrophic fungus, Mortierella minutissima, for D-limonene biotransformation

Of 98 strains of moulds, isolated from arctic soils, Mortierella minutissima 01, grew the best on agar plates with limonene vapor. Perillyl alcohol and perillic acid were the main products of limonene biotransformation. Maximal yield of perillyl alcohol (125 mg l(-1)) occurred in medium containing 0.8% substrate, at 15 degrees C, pH 6 and after 4-5 d.

Effect of temperature on the production of sclerotia by the psychrotrophic fungus Typhula incarnata in Poland.

Effect of temperature on the production of sclerotia by the psychrotrophic fungus <i>Typhula incarnata</i> in Poland.

Temporal Distribution of Geoaquatic Fungi at a Nearshore Station in Lake Ontario

In order to better understand the role of fungi in large lakes, the temporal distribution of fungi at a nearshore Lake Ontario station was examined. Water samples were taken at three depths during May to December, 1977. Mean numbers of psychrotrophic fungi in the water samples were significantly less than the numbers of mesophilic fungi to which they were correlated. The mesophilic fungal distribution pattern was correlated (P<0.05) with rainfall in the neighbouring shore area. Apart from coincident autumn peaks, little relationship was observed between the fungi and water temperature. No significant correlation was detected between geoaquatic fungi and several other parameters: heterotrophic bacteria, chlorophyll a, total bacteria, paniculate organic carbon, and total paniculate nitrogen.

Environmental reservoirs of psychrotrophic meat spoilage fungi

Summary The numbers of psychrotrophic fungi in various environments have been investigated as these fungi are potential meat spoilage organisms, carried into the freezing works on stock awaiting slaughter. Of a number of soils examined, those from urban and high-altitude areascontained the fewest psychrotrophic fungi. In rural soil samples the total psychrotrophic fungal population varied with the stocking intensity in the paddocks from which samples were obtained. Highest fungal counts were obtained for soils from stockyards. Cowhair, wool,and sheepskin all contained a variety of psychrotrophic species in moderate numbers. A total of 17 mould and 6 yeast species were cultured, Cladosporium cladosporioides being the most frequent mould and Torulopsis Candida the most frequent yeast. Several of the species demonstrated in soils and on stock were also present in the air.