Comparison of the synergistic action of two thermostable xylanases from GH families 10 and 11 with thermostable cellulases in lignocellulose hydrolysis

Recombinant xylanase preparations from Nonomuraea flexuosa (Nf Xyn, GH11) and Thermoascus aurantiacus (Ta Xyn, GH10) were evaluated for their abilities to hydrolyze hydrothermally pretreated wheat straw. The GH family 10 enzyme Ta Xyn was clearly more efficient in solubilizing xylan from pretreated wheat straw. Improvement of the hydrolysis of hydrothermally pretreated wheat straw by addition of the thermostable xylanase preparations to thermostable cellulases was evaluated. Clear synergistic enhancement of hydrolysis of cellulose was observed when cellulases were supplemented even with a low amount of pure xylanases. Xylobiose was the main hydrolysis product from xylan. It was found that the hydrolysis of cellulose increased nearly linearly with xylan removal during the enzymatic hydrolysis. The results also showed that the xylanase preparation from T. aurantiacus, belonging to GH family 10 always showed better hydrolytic capacity of solubilizing xylan and acting synergistically with thermostable cellulases in the hydrolysis of hydrothermally pretreated wheat straw.     

Cloning, expression, and characterization of a thermostable GH7 endoglucanase from Myceliophthora thermophila capable of high-consistency enzymatic liquefaction

An endoglucanase gene from the thermophilic fungus Myceliophthora thermophila, belonging to the glycoside hydrolase family 7, was functionally expressed in methylotrophic yeast Pichia pastoris. The putative endoglucanase from the genomic DNA was successfully cloned in P. pastoris X-33 and the recombinant enzyme was purified to its homogeneity (65 kDa) and subsequently characterized. Substrate specificity analysis revealed that the enzyme exhibits high activity on substrates containing β-1,4-glycosidic bonds such as carboxymethyl cellulose, barley β-glucan, and cello-oligosaccharides, as well as activity on xylan-containing substrates, including arabinoxylan and oat spelt xylan. MtEG7a was proved to liquefy rapidly and efficiently pretreated wheat straw, indicating its key role to the initial step of hydrolysis of high-solids lignocellulose substrates. High thermostability of the endoglucanase reflects potential commercial significance of the enzyme.     

Screening of ecologically diverse fungi for their potential to pretreat lignocellulosic bioenergy feedstock

A widespread and hitherto by far underexploited potential among ecologically diverse fungi to pretreat wheat straw and digestate from maize silage in the future perspective of using such lignocellulosic feedstock for fermentative bioenergy production was inferred from a screening of nine freshwater ascomycetes, 76 isolates from constructed wetlands, nine peatland isolates and ten basidiomycetes. Wheat straw pretreatment was most efficient with three ascomycetes belonging to the genera Acephala (peatland isolate) and Stachybotrys (constructed wetland isolates) and two white-rot fungi (Hypholoma fasciculare and Stropharia rugosoannulata) as it increased the amounts of water-extractable total sugars by more than 50 % and sometimes up to 150 % above the untreated control. The ascomycetes delignified wheat straw at rates (lignin losses between about 31 and 40 % of the initial content) coming close to those observed with white-rot fungi (about 40 to 57 % lignin removal). Overall, fungal delignification was indicated as a major process facilitating the digestibility of wheat straw. Digestate was generally more resistant to fungal decomposition than wheat straw. Nevertheless, certain ascomycetes delignified this substrate to extents sometimes even exceeding delignification by basidiomycetes. Total sugar amounts of about 20 to 60 % above the control value were obtained with the most efficient fungi (one ascomycete of the genus Phoma, the unspecific wood-rot basidiomycete Agrocybe aegerita and one unidentified constructed wetland isolate). This was accompanied by lignin losses of about 47 to 56 % of the initial content. Overall, digestate delignification was implied to be less decisive for high yields of fermentable sugars than wheat straw delignification.     

Production,partial characterization,and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus <Emphasis Type="Italic">Myceliophthora</Emphasis> sp.

Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50°C, but the optimum pH shifted from 7 (SmF) to 9(SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.     

A new thermophilic species of Myceliophthora from China

Myceliophthora guttulata sp. nov. is described and illustrated based on strains isolated from soil in China. This species is thermophilic with optimal growth temperature of 40–45 °C, and minimum growth temperature of 25 °C. Morphologically, this species is characterized by smooth, guttulate, pyriform to obovoid blastoconidia born directly on the side of hyphae, on long or short pedicels or in groups of 1–4 on ampulliform swellings. Phylogenetic analysis based on multi-locus alignment of internal transcribed spacer (ITS), elongation factor 1-alpha (EF1-α) and RNA polymerase II subunit (RPB2) regions showed that M. guttulata clustered within the genus Myceliophthora, and is closely related to four thermophilic species, i.e. M. fergusii, M. thermophila, M. heterothallic, and M. hinnulea.     

Genome-wide identification,annotation and characterization of novel thermostable cytochrome P450 monooxygenases from the thermophilic biomass-degrading fungi Thielavia terrestris and Myceliophthora thermophila

Cytochrome P450 monooxygenases (P450s) are ubiquitous heme-thiolate proteins that have potential biotechnological application. Thermostable-P450s that can withstand hostile industrial conditions, such as high temperatures, extremes of pH and organic solvents, are needed for biotechnological usage. Here, for the first time, we report a large number of thermostable-P450s from two thermophilic biomass-degrading fungi, Myceliophthora thermophila and Thielavia terrestris. Genome-wide P450 analysis revealed the presence of 79 and 70 P450s (P450ome) in T. terrestris and M. thermophila. Authentic P450s containing both the P450 signature domains (EXXR and CXG) were classified as follows: T. terrestris (50 families and 56 subfamilies) and M. thermophila (49 families and 53 subfamilies). Bioinformatics analysis of P450omes suggested the presence of a large number of thermostable-P450s. Based on aliphatic index cut-off (>90), 14 and 11 P450s were determined to be thermostable in T. terrestris and M. thermophila. Among the thermostable P450s, six P450s from T. terrestris and three from M. thermophila had a melting temperature (Tm) of >65 °C, suggesting their hyperthermal tolerance. Analysis of the instability index of two ascomycete P450omes revealed the presence of 12 and 19 in vitro stable P450s in T. terrestris and M. thermophila. Overall, six P450s from T. terrestris and four from M. thermophila showed both thermal tolerance and in vitro stability. Thermophilic ascomycetes P450s are of potential interest from a structural, mechanistic and biotechnological point of view, as five P450s showed higher thermal tolerance and five showed higher in vitro stability compared to the well-characterized thermostable-P450s CYP175A1 (bacteria) and CYP119 (archaea).     

A molecular phylogeny of thermophilic fungi

Sequences from 86 fungal genomes and from the two outgroup genomes Arabidopsis thaliana and Drosophila melanogaster were analyzed to construct a robust molecular phylogeny of thermophilic fungi, which are potentially rich sources of industrial enzymes. To provide experimental reference points, growth characteristics of 22 reported thermophilic or thermotolerant fungi, together with eight mesophilic species, were examined at four temperatures: 22 °C, 34 °C, 45 °C, and 55 °C. Based on the relative growth performances, species with a faster growth rate at 45 °C than at 34 °C were classified as thermophilic, and species with better or equally good growth at 34 °C compared to 45 °C as thermotolerant. We examined the phylogenetic relationships of a diverse range of fungi, including thermophilic and thermotolerant species, using concatenated amino acid sequences of marker genes mcm7, rpb1, and rpb2 obtained from genome sequencing projects. To further elucidate the phylogenetic relationships in the thermophile-rich orders Sordariales and Eurotiales, we used nucleotide sequences from the nuclear ribosomal small subunit (SSU), the 5.8S gene with internal transcribed spacers 1 and 2 (ITS 1 and 2), and the ribosomal large subunit (LSU) to include additional species for analysis. These phylogenetic analyses clarified the position of several thermophilic taxa. Thus, Myriococcum thermophilum and Scytalidium thermophilum fall into the Sordariales as members of the Chaetomiaceae, Thermomyces lanuginosus belongs to the Eurotiales, Malbranchea cinnamomea is a member of the Onygenales, and Calcarisporiella thermophila is assigned to the basal fungi close to the Mucorales. The mesophilic alkalophile Acremonium alcalophilum clusters with Verticillium albo-atrum and Verticillium dahliae, placing them in the recently established order Glomerellales. Taken together, these data indicate that the known thermophilic fungi are limited to the Sordariales, Eurotiales, and Onygenales in the Ascomycota and the Mucorales with possibly an additional order harbouring C. thermophila in the basal fungi. No supporting evidence was found for thermophilic species belonging to the Basidiomycota.     

Xylanolytic activity and xylose utilization by thermophilic molds

Among thirteen thermophilic fungal strains,viz. Malbranchea pulchella var.sulfurea, Sporotrichum thermophile, Thielavia terrestris, Humicola insolens andAcremonium alabamensis produced high levels of xylanolytic enzymes. The secretion of xylanolytic enzymes was higher in wheat straw medium than in wheat straw hemicellulose. All fungi utilized xylose as the carbon source. However,Mucor pusillus, Torula thermophila andSporotrichum thermophile consumed 90–93% of xylose provided in the medium while others utilized 51–83%. The consumption of glucose by the fungi was high in comparison with that of xylose. Of all the treatments tried, xylose isomerase yield was highest when the mycelium ofHumicola insolens was homogenized with sand. The synthesis of xylose isomerase was very high in wheat straw hemicellulose as compared with that in xylose and glucose.     

Diversity of thermophilic fungi in Tengchong Rehai national park revealed by ITS nucleotide sequence analyses

The geothermal sites near neutral and alkalescent thermal springs in Tengchong Rehai National Park were examined through cultivation-dependent approach to determine the diversity of thermophilic fungi in these environments. Here, we collected soils samples in this area, plated on agar media conducive for fungal growth, obtained pure cultures, and then employed the method of internal transcribed spacer (ITS) sequencing combined with morphological analysis for identification of thermophilic fungi to the species level. In total, 102 strains were isolated and identified as Rhizomucor miehei, Chaetomium sp., Talaromyces thermophilus, Talaromyces byssochlamydoides, Thermoascus aurantiacus Miehe var. levisporus, Thermomyces lanuginosus, Scytalidium thermophilum, Malbranchea flava, Myceliophthora sp. 1, Myceliophthora sp. 2, Myceliophthora sp. 3, and Coprinopsis sp. Two species, T. lanuginosus and S. thermophilum were the dominant species, representing 34.78% and 28.26% of the sample, respectively. Our results indicated a greater diversity of thermophilic fungi in neutral and alkaline geothermal sites than acidic sites around hot springs reported in previous studies. Most of our strains thrived at alkaline growth conditions.     

Characterization of hemicellulases from thermophilic fungi

The thermophilic fungi Thermomyces lanuginosus, Malbranchea cinnamomea, Myceliophthora fergusii and the thermotolerant Aspergillus terreus were cultivated on various carbon sources, and hemicellulolytic and cellulolytic enzyme profiles were evaluated. All fungi could grow on locust bean galactomannan (LBG), Solka floc, wheat bran and pectin, except T. lanuginosus, which failed to utilize LBG for growth. Different levels of cellulase and hemicellulase activities were produced by these fungal strains. Depending on the carbon source, variable ratios of thermostable hydrolytic enzymes were obtained, which may be useful in various applications. All strains were found to secrete xylanolytic and mannanolytic enzymes. Generally, LBG was the most efficient carbon source to induce mannanase activities, although T. lanuginosus was able to produce mannanase only on wheat bran as a carbon source. Xylanolytic activities were usually highest on wheat bran medium, but in contrast to other investigated fungi, xylanase production by M. fergusii was enhanced on pectin medium. Preliminary thermostability screening indicated that among the investigated species, thermotolerant glycosidases can be found. Some of the accessory activities, including the α-arabinosidase activity, were surprisingly high. The capability of the produced enzymes to improve the hydrolysis of lignocellulosic pretreated substrate was evaluated and revealed potential for these enzymes.     

High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

Background

In the hydrolysis of lignocellulosic materials, thermostable enzymes decrease the amount of enzyme needed due to higher specific activity and elongate the hydrolysis time due to improved stability. For cost-efficient use of enzymes in large-scale industrial applications, high-level expression of enzymes in recombinant hosts is usually a prerequisite. The main aim of the present study was to compare the biochemical and hydrolytic properties of two thermostable recombinant glycosyl hydrolase families 10 and 11 (GH10 and GH11, respectively) xylanases with respect to their potential application in the hydrolysis of lignocellulosic substrates.

Results

The xylanases from Nonomuraea flexuosa (Nf Xyn11A) and from Thermoascus aurantiacus (Ta Xyn10A) were purified by heat treatment and gel permeation chromatography. Ta Xyn10A exhibited higher hydrolytic efficiency than Nf Xyn11A toward birchwood glucuronoxylan, insoluble oat spelt arabinoxylan and hydrothermally pretreated wheat straw, and it produced more reducing sugars. Oligosaccharides from xylobiose to xylopentaose as well as higher degree of polymerization (DP) xylooligosaccharides (XOSs), but not xylose, were released during the initial hydrolysis of xylans by Nf Xyn11A, indicating its potential for the production of XOS. The mode of action of Nf Xyn11A and Ta Xyn10A on glucuronoxylan and arabinoxylan showed typical production patterns of endoxylanases belonging to GH11 and GH10, respectively.

Conclusions

Because of its high catalytic activity and good thermostability, T. aurantiacus xylanase shows great potential for applications aimed at total hydrolysis of lignocellulosic materials for platform sugars, whereas N. flexuosa xylanase shows more significant potential for the production of XOSs.     

降解棉秸秆耐热菌株的鉴定及降解条件优化

【目的】鉴定从新疆棉花秸秆高温堆肥中分离出的两株耐热真菌Z1、Z2的属种,并通过优化影响菌株产生纤维素酶的因素来提高菌株对秸秆的降解率。【方法】经形态学和菌株的ITS区克隆与序列分析确定属种,以液体摇瓶发酵产滤纸酶活性(FPA)变化为衡量指标,对Z1、Z2以及二者混合菌(MS)的纤维素酶产生条件进行优化。【结果】菌株Z1为曲霉属烟曲霉(Aspergillus fumigatus Fresen),Z2为蚀丝霉属(Myceliophthora Cost.)。确定Z1以棉秸秆为碳源、以NaNO3为氮源、起始pH 9.5、接种量11%、50°C摇床培养10 d,对棉秸秆降解率为10.19%;Z2以麦秸秆为碳源、以NaNO3为氮源、起始pH 5.5、接种量9%、50°C摇床培养10 d,对麦秆降解率为27.50%;MS以棉花秸秆为碳源、以蛋白胨为氮源、起始pH 5.5、接种量11%、50°C摇床培养10 d,对棉秸秆的降解率为53.45%。【结论】实验表明,MS(Z1、Z2混合)对秸秆的降解效果优于单株菌,降解率达到一半以上,本研究中的两株耐热真菌在降解棉花秸秆、小麦秸秆等农作物废弃秸秆中具有较高的应用价值。     

Community dynamics of Neocallimastigomycetes in the rumen of yak feeding on wheat straw revealed by different primer sets

Anaerobic fungi (Neocallimastigomycetes) play an important role in fermenting lignin-rich plant biomass into sugars in the rumen of animals, representing a very promising enzyme resource to contribute to the conversion of plant biomass into biofuels. However, current studies about their functions mainly focus on limited species, and little is known about the coordination of different members of the anaerobic fungi in the digestion process of plant fibres. In this study, the community composition of anaerobic fungi in the rumens of yaks at five different time points (1, 3, 5, 7.5 and 24 h after feeding wheat straw) was investigated employing a cultivation-independent method using ITS clone libraries. Comparison of five pairs of primers showed that PCR primer sets could have clear amplification bias and therefore potentially affect the interpretation of the resulting fungal community structure; then two primer sets GM1/MNGM2 and ITS1/ITS4 were selected. Among the 398 sequences from 10 clone libraries, 18 operational taxonomic units (OTUs) of Neocallimastigomycetes were obtained, covering five known genera and one yet uncultured lineage. OTUs belonging to the bulbous-type morphotype (Caecomyces- or Cyllamyces-related) and the rhizoidal genus Neocallimastix were abundant and predominantly present, representing 62.7% and 19.3% OTUs respectively. In all the later samples taken from 3 h to 24 Neocallimastigomycetes h after feeding, a relatively stable community composition was revealed: members of Neocallimastix increased to represent 43.4–49.4% and the bulbous-type morphotype declined to represent 39.5–42.7%. This implies a substantial turnover and synergy between bulbous and rhizoidal morphotypes of anaerobic fungi during the process of fibre digestion. Our study provided the first insight into the in vivo temporal change in the anaerobic fungal community, and the role of Neocallimastigomycetes with a bulbous morphotype in the degradation of plant cell wall in the yak rumen.     

Microbial oil accumulation and cellulase secretion of the endophytic fungi from oleaginous plants

This research was designed to screen for strains that produce microbial oil by using straw as the substrate. One hundred and forty-one isolates of endophytic fungi were obtained from stems of seven oleaginous plant species. Sixty-nine isolates (48.9% of the total isolates) could be clearly seen having lipid bodies in their hyphae when examined with optical microscopy. Twenty-six isolates which had bigger and more oil bodies in their hyphae were selected for further research. These isolates belong to five genera includingMicrosphaeropsis, Phomopsis, Cephalosporium, Sclerocystis andNigrospora. Their oil contents ranged from 21.3 to 35.0% of dry cell weights when cultured in potato dextrose broth. When cultured on the solid-state medium composed of steam-exploded wheat straw (20% w/w), wheat bran (5%) and water (75%) they were able to produce cellulase and microbial oil with yields of 0.31≈0.69 filter paper unit and 19≈42 mg/g initial dry substrate, respectively. These results show that some endophytic fungi isolated from the oleaginous plants have the abilities of accumulating oil and producing cellulase simultaneously. They may be potential microbial oil producers by utilising straw as the substrate.     

Xylose metabolism in a thermophilic mouldMalbranchea pulchella var.sulfurea TMD-8

The thermophilic mouldMalbranchea pulchella var.sulfurea TMD-8 produced extracellular xylanases in wheat straw hemicellulose as well as wheat straw. This mould utilized xylose less efficiently than glucose. Mycelial extracts contained xylose isomerase, xylose reductase, and xylitol dehydrogenase. Xylose isomerase was less thermostable than that from other microorganisms. However, xylitol dehydrogenase and xylose reductase were relatively more thermostable in comparison with these enzymes from other microorganisms. The affinity of xylose isomerase for xylose was very high (Km 10mM), while that of xylose reductase was low (Km 23.5mM). The xylitol dehydrogenase exhibited relatively high affinity for xylitol (Km 0.02mM). The activity of this enzyme, however, declined steeply, in the alkaline range. This is the first report on the occurrence of three intracellular enzymes, xylose isomerase, xylose reductase, and xylitol dehydrogenase in a thermophilic mould, which play an important role in xylose metabolism.     

Thermostable Acid Protease Produced by Penicillium duponti K1014, a True Thermophilic Fungus Newly Isolated from Compost

A thermophilic fungus, K1014, newly derived from a compost was selected on the basis of protease productivity as the only one of 81 isolates to produce high levels of acid protease. The fungus was named Penicillium duponti K1014 based on taxonomical studies. It grew in the temperature range of 28 to 58 C, and the optimum was 45 to 50 C. These temperature characteristics showed that the fungus was the most strongly thermophilic of all the fungi next to Humicola lanuginosa. When P. duponti K1014 was grown on moistened wheat bran, maximal accumulation of acid protease occurred after 2 days at 45 to 50 C. The addition of ammonium salts, but not nitrate, was effective for the production of the acid protease. The acid protease of P. duponti K1014 was stable at 60 C for 1 hr and retained more than 65% of original activity after the treatment for 1 hr at 70 C at pH 4.7. This thermal property was different from those of the ordinary acid proteases, indicating that the enzyme is a thermostable protein.     

Polysaccharide-Degrading Activity in Marine and Terrestrial Strains of Mycelial Fungi

The activity of extracellular polysaccharide-degrading enzymes and glycosidases from mycelial fungi towards various carbohydrates and carbohydrate derivatives from plant and algal cell walls has been screened. Twenty-three strains of mycelial fungi isolated from the marine sediment and dung were grown by submerged cultivation on a plant-based substrate (a by-product of the grain processing industry) for previous screening for their biomass and protein productivity. Molecular identification allowed for the assignment of marine fungal strains to the following species: Sirastachys phyllophila, Ochroconis mirabilis, Pseudallescheria boydii, Pseudallescheria ellipsoidea, Beauveria felina, Scopulariopsis brevicaulis, Cladosporium sp., and Trichoderma sp. The terrestrial strains belonged to the species Thermomyces thermophilus, Thermomyces dupontii, Thermomyces lanuginosus, Fusarium avenaceum, Mycothermus thermophilum, and Thermothelomyces thermophila. Seven strains of thermophilic terrestrial fungal species T. thermophila, T. thermophilus, T. dupontii and M. thermophilus and two marine fungal strains of S. brevicaulis and Beauveria felina exhibited the highest protein yields and a wide range of polysaccharide-degrading activity when the cultures were cultivated at 22–25°C. The cellulolytic thermophilic strain M. thermophilus 55 isolated from dung demonstrated unusual specificity, most intensive increase of mycelial biomass, and high activity towards algal polysaccharides after seven days of cultivation. The specific activity of laminarinase was one order of magnitude higher than in the marine strains and amounted to 1180 U/mg, and the alginate lyase, carrageenase, polymannuronate lyase, agarase, and fucoidanase activity levels (from 208 to 500 U/mg) were also higher than in all marine strains. All active polysaccharide-degrading strains of thermophilic terrestrial and marine fungi identified in the present study are of considerable interest, as the potential of these fungi for polysaccharide degradation can be applied in the transformation of various agricultural and maricultural waste of plant origin and in the modification of carbohydrate-containing substances in structural research and biotechnology.     

Enhanced endoxylanase production by Myceliophthora thermophila using rice straw and its synergism with phytase in improving nutrition

The goal of the present investigation was to attain the enhanced production of endoxylanase in submerged fermentation using different approaches followed by its utility in improving nutrition of wheat and rice flours along with phytase. Myceliophthora thermophila BJTLRMDU3 produced 51.70 U/mL of xylanase using rice straw as a substrate after optimization with ‘one variable at a time’ approach. After Plackett-Burman design study, sodium nitrate, K₂HPO₄ and Tween 20 were selected as critical factors and further optimized by response surface methodology. Increased xylanase production (80.15 U/mL) was attained with 2.5 % (w/v) sodium nitrate, 1.25 % (w/v) K₂HPO₄, and 2 % (v/v) Tween 20 at 40 °C. An overall 1.5-fold increase in xylanase production was achieved after statistical optimization. Applicability of M. thermophila xylanase (200 U/g flour) alone and in combination with phytase (15 U/g flour) from Aspergillus oryzae SBS50 in wheat and rice flours showed enhancement in nutritional qualities of both flours. About 45.67 %, 29.73 %, and 107.91 % increase in reducing sugars, soluble proteins and inorganic phosphate, respectively in wheat flour, while 94.16 %, 134.52 %, and 473.33 % increase in reducing sugars, soluble proteins and inorganic phosphate, respectively in rice flour was achieved at 60 °C and pH 5.0 by synergistic action of xylanase and phytase as compared to control having only xylanase.     

Fungi of wheat and broad-bean straw composts

13 thermophilic genera and 19 species in addition to one variety of each of M. pulchella and H. grisea were collected from wheat and broad-bean straw composts at 45 °C. In wheat and broad-bean, all thermophilic fungi were completely checked between 4–9 days, and 1–8 days composting when the temperature ranged between 58° and 67 °C, and 58 ° and 70 °C respectively, and reappeared, represented by P. duponti, M. albomyces, T. lanuginosus and S. thermophile, after 9 or 10 days composting when the temperature decreased to 51.5° –54 ° C. Wheat and broad-bean straw composts were analysed biochemically to follow the changes in ethanol and diastase soluble, hemicellulose, cellulose and lignin fractions during composting.     

Metabolic engineering of the thermophilic filamentous fungus <Emphasis Type="Italic">Myceliophthora thermophila</Emphasis> to produce fumaric acid

Background

Fumaric acid is widely used in food and pharmaceutical industries and is recognized as a versatile industrial chemical feedstock. Increasing concerns about energy and environmental problems have resulted in a focus on fumaric acid production by microbial fermentation via bioconversion of renewable feedstocks. Filamentous fungi are the predominant microorganisms used to produce organic acids, including fumaric acid, and most studies to date have focused on Rhizopus species. Thermophilic filamentous fungi have many advantages for the production of compounds by industrial fermentation. However, no previous studies have focused on fumaric acid production by thermophilic fungi.

Results

We explored the feasibility of producing fumarate by metabolically engineering Myceliophthora thermophila using the CRISPR/Cas9 system. Screening of fumarases suggested that the fumarase from Candida krusei was the most suitable for efficient production of fumaric acid in M. thermophila. Introducing the C. krusei fumarase into M. thermophila increased the titer of fumaric acid by threefold. To further increase fumarate production, the intracellular fumarate digestion pathway was disrupted. After deletion of the two fumarate reductase and the mitochondrial fumarase genes of M. thermophila, the resulting strain exhibited a 2.33-fold increase in fumarate titer. Increasing the pool size of malate, the precursor of fumaric acid, significantly increased the final fumaric acid titer. Finally, disruption of the malate–aspartate shuttle increased the intracellular malate content by 2.16-fold and extracellular fumaric acid titer by 42%, compared with that of the parental strain. The strategic metabolic engineering of multiple genes resulted in a final strain that could produce up to 17 g/L fumaric acid from glucose in a fed-batch fermentation process.

Conclusions

This is the first metabolic engineering study on the production of fumaric acid by the thermophilic filamentous fungus M. thermophila. This cellulolytic fungal platform provides a promising method for the sustainable and efficient-cost production of fumaric acid from lignocellulose-derived carbon sources in the future.