Microbial α-L-arabinofuranosidases: diversity, properties, and biotechnological applications.

Arabinoxylans (AXs) are hemicellulosic polysaccharides consisting of a linear backbone of ß-1,4-linked xylose residues branched by high content of α-L-arabinofuranosyl (Araf) residues along with other side-chain substituents, and are abundantly found in various agricultural crops especially cereals. The efficient bioconversion of AXs into monosaccharides, oligosaccharides and/or other chemicals depends on the synergism of main-chain enzymes and de-branching enzymes. Exo-α-L-arabinofuranosidases (ABFs) catalyze the hydrolysis of terminal non-reducing α-1,2-, α-1,3- or α-1,5- linked α-L-Araf residues from arabinose-substituted polysaccharides or oligosaccharides. ABFs are critically de-branching enzymes in bioconversion of agricultural biomass, and have received special attention due to their application potentials in biotechnological industries. In recent years, the researches on microbial ABFs have developed quickly in the aspects of the gene mining, properties of novel members, catalytic mechanisms, methodologies, and application technologies. In this review, we systematically summarize the latest advances in microbial ABFs, and discuss the future perspectives of the enzyme research.

Functional characterization of a new 3-dehydroshikimate dehydratase from Eupenicillium parvum and its potential for protocatechuic acid production.

Protocatechuic acid (PCA) is an important phenolic compound with diverse industrial values. Conversion of 3-dehydroshikimate (DHS) to PCA by dehydroshikimate dehydratase (DSD) provides an efficient approach for the production of the molecule. Herein, a new DSD from fungus Eupenicillium parvum was functionally investigated after recombinant expression in Escherichia coli. The DSD displayed 30%-35% sequence identities with the known fungal DSDs. The recombinant protein showed catalysis activity against DHS, with the optimal temperature of 40 °C and pH of 7.5. The specific activity and Km of the protein were 910 mU per mg protein and 0.83 m m, respectively. Metal ion (Mg2+ or Mn2+) played a critical role in the enzymatic activity. Meanwhile, the thermal stability of the protein was improved by Mg2+ or Mn2+. Furthermore, the expression of the protein in E. coli resulted in de novo synthesis of 491 mg/L PCA in a modified M9 medium with glycerol as a carbon source.

Comparison of the Biochemical Properties and Roles in the Xyloglucan-Rich Biomass Degradation of a GH74 Xyloglucanase and Its CBM-Deleted Variant from Thielavia terrestris.

Xyloglucan is closely associated with cellulose and still retained with some modification in pretreated lignocellulose; however, its influence on lignocellulose biodegradation is less understood. TtGH74 from Thielavia terrestris displayed much higher catalytic activity than previously characterized fungal GH74 xyloglucanases. The carbohydrate-binding module 1 (CBM1) deleted variant (TtGH74ΔCBM) had the same optimum temperature and pH but an elevated thermostability. TtGH74 displayed a high binding affinity on xyloglucan and cellulose, while TtGH74ΔCBM completely lost the adsorption capability on cellulose. Their hydrolysis action alone or in combination with other glycoside hydrolases on the free xyloglucan, xyloglucan-coated phosphoric acid-swollen cellulose or pretreated corn bran and apple pomace was compared. CBM1 might not be essential for the hydrolysis of free xyloglucan but still effective for the associated xyloglucan to an extent. TtGH74 alone or synergistically acting with the CBH1/EG1 mixture was more effective in the hydrolysis of xyloglucan in corn bran, while TtGH74ΔCBM showed relatively higher catalytic activity on apple pomace, indicating that the role and significance of CBM1 are substrate-specific. The degrees of synergy for TtGH74 or TtGH74ΔCBM with the CBH1/EG1 mixture reached 1.22-2.02. The addition of GH10 xylanase in TtGH74 or the TtGH74ΔCBM/CBH1/EG1 mixture further improved the overall hydrolysis efficiency, and the degrees of synergy were up to 1.50-2.16.

Two C1-oxidizing AA9 lytic polysaccharide monooxygenases from Sordaria brevicollis differ in thermostability, activity, and synergy with cellulase.

Cellulolytic fungi usually have multiple genes for C1-oxidizing auxiliary activity 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) in their genomes, but their potential functional differences are less understood. In this study, two C1-oxidizing AA9 LPMOs, SbLPMO9A and SbLPMO9B, were identified from Sordaria brevicollis, and their differences, particularly in terms of thermostability, reducing agent specificity, and synergy with cellulase, were explored. The two enzymes exhibited weak binding to cellulose and intolerance to hydrogen peroxide. Their oxidative activity was influenced by cellulose crystallinity and surface morphology, and both enzymes tended to oxidize celluloses of lower crystallinity and high surface area. Comparably, SbLPMO9A had much better thermostability than SbLPMO9B, which may be attributed to the presence of a carbohydrate binding module 1 (CBM1)-like sequence at its C-terminus. In addition, the two enzymes exhibited different specificities and responsivities toward electron donors. SbLPMO9A and SbLPMO9B were able to boost the catalytic efficiency of endoglucanase I (EGI) on physically and chemically pretreated substrates but with different degrees of synergy. Substrate- and enzyme-specific synergism was observed by comparing the synergistic action of SbLPMO9A or SbLPMO9B with commercial Celluclast 1.5L on three kinds of cellulosic substrates. On regenerated amorphous cellulose and PFI (Papirindustriens Forskningsinstitut)-fibrillated bleached eucalyptus pulp, SbLPMO9B showed a higher synergistic effect than SbLPMO9A, while on delignified wheat straw, the synergistic effect of SbLPMO9A was higher than that of SbLPMO9B. On account of its excellent thermostability and boosting effect on the enzymatic hydrolysis of delignified wheat straw, SbLPMO9A may have high application potential in biorefineries for lignocellulosic biomass. KEY POINTS: • C1-oxidizing SbLPMO9A displayed higher thermostability than SbLPMO9B, probably due to the presence of a CBM1-like module. • The oxidative activity of the two SbLPMO9s on celluloses increased with decreasing cellulose crystallinity or increasing beating degree. • The two SbLPMO9s boosted the catalytic efficiency of cellulase, but the synergistic effect was substrate- and enzyme-specific.

Characterization and functional analysis of two novel thermotolerant α-L-arabinofuranosidases belonging to glycoside hydrolase family 51 from Thielavia terrestris and family 62 from Eupenicillium parvum.

Arabinofuranose substitutions on xylan are known to interfere with enzymatic hydrolysis of this primary hemicellulose. In this work, two novel α-L-arabinofuranosidases (ABFs), TtABF51A from Thielavia terrestris and EpABF62C from Eupenicillium parvum, were characterized and functionally analyzed. From sequences analyses, TtABF51A and EpABF62C belong to glycoside hydrolase (GH) families 51 and 62, respectively. Recombinant TtABF51A showed high activity on 4-nitrophenyl-α-L-arabinofuranoside (83.39 U/mg), low-viscosity wheat arabinoxylan (WAX, 39.66 U/mg), high-viscosity rye arabinoxylan (RAX, 32.24 U/mg), and sugarbeet arabinan (25.69 U/mg), while EpABF62C preferred to degrade arabinoxylan. For EpABF62C, the rate of hydrolysis of RAX (94.10 U/mg) was 2.1 times that of WAX (45.46 U/mg). The optimal pH and reaction temperature for the two enzymes was between 4.0 and 4.5 and 65 °C, respectively. Calcium played an important role in the thermal stability of EpABF62C. TtABF51A and EpABF62C showed the highest thermal stabilities at pH 4.5 or 5.0, respectively. At their optimal pHs, TtABF51A and EpABF62C retained greater than 80% of their initial activities after incubation at 55 °C for 96 h or 144 h, respectively. 1H NMR analysis indicated that the two enzymes selectively removed arabinose linked to C-3 of mono-substituted xylose residues in WAX. Compared with the singular application of the GH10 xylanase EpXYN1 from E. parvum, co-digestions of WAX including TtABF51A and/or EpABF62C released 2.49, 3.38, and 4.81 times xylose or 3.38, 1.65, and 2.57 times of xylobiose, respectively. Meanwhile, the amount of arabinose released from WAX by TtABF51A with EpXYN1 was 2.11 times the amount with TtABF51A alone. KEY POINTS: • Two novel α-l-arabinofuranosidases (ABFs) displayed high thermal stability. • The thermal stability of GH62 family EpABF62C was dependent on calcium. • Buffer pH affects the thermal stability of the two ABFs. • Both ABFs enhance the hydrolysis of WAX by a GH10 xylanase.

Expression and characterization of two glucuronoyl esterases from Thielavia terrestris and their application in enzymatic hydrolysis of corn bran.

The thermophilic fungus Thielavia terrestris when cultured on cellulose produces a cocktail of thermal hydrolases with potential application in saccharification of lignocellulosic biomass and other biotechnological areas. Glucuronoyl esterases are considered to play a unique role as accessory enzymes in lignocellulosic material biodegradation by cleaving the covalent ester linkage between 4-O-methyl-D-glucuronic acid (MeGlcA) and lignin in lignin-carbohydrate complexes (LCCs). Two glucuronoyl esterases from T. terrestris named TtGE1 and TtGE2 were expressed in Pichia pastoris. Both esterases displayed features of thermophilic enzymes, with the optimal temperature at 45 °C and 55 °C. TtGE1 and TtGE2 exhibited activity towards methyl (4-nitrophenyl ß-D-glucopyranosid) uronate (Me-GlcA-pNP) but no catalytic activity to benzyl-D-glucuronate (BnzGlcA), indicating the difference in substrate specificity from previously studied fungal GEs. A substantial increase in the release of monomeric sugars and glucuronic acid from autohydrolysis of corn bran was observed by the supplementing TtGEs into commercial xylanase; the results clearly demonstrated that the TtGEs played a significant role in this degradation process. This research on TtGEs enriches our knowledge of this novel class of fungal GEs. These newly characterized TtGEs could be used as promising accessory enzymes to improve the hydrolysis efficiency of commercial enzymes in saccharification of lignocellulosic materials due to their thermophilic characteristics.

A Myb transcription factor represses conidiation and cephalosporin C production in Acremonium chrysogenum.

Acremonium chrysogenum is the industrial producer of cephalosporin C (CPC). We isolated a mutant (AC554) from a T-DNA inserted mutant library of A. chrysogenum. AC554 exhibited a reduced conidiation and lack of CPC production. In consistent with it, the transcription of cephalosporin biosynthetic genes pcbC and cefEF was significantly decreased in AC554. Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed and sequence analysis indicated that a T-DNA was inserted upstream of an open reading frame (ORF) which was designated AcmybA. On the basis of sequence analysis, AcmybA encodes a Myb domain containing transcriptional factor. Observation of red fluorescent protein (RFP) tagged AcMybA showed that AcMybA is naturally located in the nucleus of A. chrysogenum. Transcriptional analysis demonstrated that the AcmybA transcription was increased in AC554. In contrast, the AcmybA deleted mutant (ΔAcmybA) overproduced conidia and CPC. To screen the targets of AcmybA, we sequenced and compared the transcriptome of ΔAcmybA, AC554 and the wild-type strain at different developmental stages. Twelve differentially expressed regulatory genes were identified. Taken together, our results indicate that AcMybA negatively regulates conidiation and CPC production in A. chrysogenum.

Heterologous expression of two Aspergillus niger feruloyl esterases in Trichoderma reesei for the production of ferulic acid from wheat bran.

Feruloyl esterase (FAE)-encoding genes AnfaeA and AnfaeB were isolated from Aspergillus niger 0913. For overexpression of the two genes in Trichoderma reesei, constitutive and inductive expression plasmids were constructed based on parental plasmid pAg1-H3. The constructed plasmids contained AnfaeA or AnfaeB gene under the control of glyceraldehyde-3-phosphate dehydrogenase A gene (gpdA) promoter (from A. nidulans) or cellobiohydrolases I (cbh I) gene promoter (from T. reesei), and cbh I terminator from T. reesei. The target plasmids were transferred into T. reesei D-86271 (Rut-C30) by Agrobacterium tumefaciens-mediated transformation (ATMT), respectively. A high level of feruloyl esterase was produced by the recombinant fungal strains under solid-state fermentation, and the cbh I promoter was more efficient than the gpdA promoter in the expression of AnfaeA. The optimum temperatures and pH values were 50 °C and 5.0 for AnFAEA, and 35 °C and 6.0 for AnFAEB. The maximum production levels were 20.69 U/gsd for AnFAEA and 15.08 U/gsd for AnFAEB. The recombinant fungal enzyme systems could release 62.9% (for AnFAEA) and 52.2% (for AnFAEB) of total ferulic acids from de-starched wheat bran, which was higher than the 46.3% releasing efficiency of A. niger 0913. The supplement of xylanase from T. longibrachiatum in the enzymatic hydrolysis led to a small increment of the ferulic acids release.

Evaluation of different agricultural wastes for the production of fruiting bodies and bioactive compounds by medicinal mushroom Cordyceps militaris.

BACKGROUND: In commercial production of Cordyceps militaris (a famous Chinese medicine), cereal grains are usually utilized as cultivation substrates. This study aimed to evaluate the efficiency of agricultural wastes as substitute materials in the low-cost production of C. militaris. Cottonseed shells (CS), corn cob particles (CCP), Italian poplar sawdusts (IPS) and substrates spent by Flammulina velutipes (SS) were employed to cultivate C. militaris, using rice medium as control. RESULTS: CS and CCP were suitable for fruit body formation of C. militaris, with yields of 22 and 20 g per bottle respectively. Fruit bodies grown on CCP showed the highest levels of cordycepin and adenosine, up to 9.45 and 5.86 mg g-1 respectively. The content of d-mannitol in fruit bodies obtained on CS was 120 mg g-1 (80% of the control group), followed by that on CCP, 100 mg g-1 . Fruit bodies cultivated on CCP displayed a high crude polysaccharide level of 26.9 mg g-1 , which was the closest to that of the control group (34.5 mg g-1 ). CONCLUSION: CS and CCP are effective substrates for the production of fruit bodies and bioactive compounds by C. militaris. This study provides a new approach to decreasing the cost of C. militaris cultivation and dealing with these agricultural wastes. © 2016 Society of Chemical Industry.

N- and C-terminal truncations of a GH10 xylanase significantly increase its activity and thermostability but decrease its SDS resistance.

XynII from Volvariella volvacea has high sodium dodecyl sulfate (SDS) resistance, with the potential for industrial applications under harsh conditions. It consists of a single glycoside hydrolase family 10 (GH10) catalytic domain but contains an additional unique 10 and 4 amino acid residues at the N- and C-terminus, respectively. In this study, five XynII derivatives with N- and/or C-terminus deletions were constructed to determine the effects of these regions on enzyme activity, substrate specificity, thermostability, and SDS resistance. Our results revealed that N- and/or C-terminal truncations significantly increased enzyme activity and thermostability, but reduced SDS resistance. Specifically, the XynIIΔNC4 mutant had 2.53-fold more catalytic efficiency (k cat/K m) towards beechwood xylan than wild-type and 3.0-fold more thermostability (t 1/2 [55°C]). XynIIΔNC4 displayed 3.33-, 4.38-, 1.37-, and 1.98-fold more activity against xylotriose, xylotetraose, xylopentaose, and xylohexaose, respectively, than XynII did. However, its half-life (t 1/2) in 4 % SDS was only 1.72 h, while that of XynII was 4.65 h. Circular dichroism analysis revealed that deletion of N- and C-terminal segments caused minor changes in secondary structure. Our observations suggest that the extra N- and C-terminal segments in wild-type XynII evolved to strengthen the interaction between these regions of the protein, making the local structure more rigid and reducing structural flexibility. In this way, N- and C-terminal truncations increased the thermostability and activity of XynII on different xylans and linear xylooligosaccharides, but reduced its resistance to SDS.

Biochemical characteristics of an alkaline pectate lyase PelA from Volvariella volvacea: roles of the highly conserved N-glycosylation site in its secretion and activity.

Alkaline pectate lyases have great application potential in the bioscouring of textiles. They are isolated predominantly from bacteria and a few fungi. Here, we report the biochemical characteristics of a novel alkaline pectate lyase PelA from the basidiomycete Volvariella volvacea. The full-length pelA encodes a 321-amino-acid polypeptide containing a putative 18-residue signal peptide and a pectate lyase family 1 catalytic domain. It contains one conserved and one non-conserved potential N-glycosylation site (N-X-S/T) at the residues N95 and N198, respectively. The enzyme showed optimal activity at 60 °C and pH 10, although it was stable between pH 4 and pH 11. Additional Ca(2+) was not required to measure PelA activity in vitro, but it could significantly enhance its activity and thermal stability. The V max values using polygalacturonic acid as substrate were increased from 50.71 to 89.96 IU mg(-1) by the addition of 0.1 mM Ca(2+), whereas the K m values were decreased from 0.681 to 0.514 mg ml(-1). Site-directed mutagenesis revealed PelA has only one N-glycan attached to the residue N95. This N-glycan is crucial to its efficient secretion and activity possibly due to its role in maintaining the secondary structure of PelA. Amino acid substitution at the residue N198 had no effect on PelA secretion, but resulted in a slight (5.16 %) to modest (27.37 %) decrease in specific activity and less thermal stability, indicating the amino acid itself is also important for activity due to it being highly conserved and because of its proximity to the catalytic site.

A novel cellulolytic/xylanolytic SbAA14 from Sordaria brevicollis with a branched chain preference and its synergistic effects with glycoside hydrolases on lignocellulose.

In this study, the novel auxiliary activity (AA) family 14 lytic polysaccharide monooxygenase (LPMO) SbAA14 from Sordaria brevicollis was successfully characterized. It was active against heteroxylan, xyloglucan and cellulose in ß-cellulose and released native oligosaccharides and corresponding C1- and/or C4-oxidized products. SbAA14 showed a branched chain preference, because partial removal of arabinosyl substituents from heteroxylan led to a decrease in activity. SbAA14 had synergistic effects with the debranching enzyme EpABF62C in an enzyme- and ascorbic acid-dependent manner. SbAA14 had synergistic effects with the GH10 endoxylanase EpXYN1, and the degree of synergy was greater with step-by-step addition than with simultaneous addition. SbAA14 could also synergize with Celluclast® 1.5 L on NaOH-pretreated wheat straw and on NaOH-pretreated and hydrogen peroxide-acetic acid (HPAC)-H2SO4-pretreated bamboo substrates. The greatest synergistic effect between SbAA14 and Celluclast® 1.5 L was observed for HPAC-H2SO4-200 mM pretreated bamboo, in which the degree of synergy reached approximately 1.61. The distinctive substrate preference of SbAA14 indicated that it is a novel AA14 LPMO that may act mainly on heteroxylan with numerous arabinosyl substituents between cellulose fibers rather than on recalcitrant xylan tightly associated with cellulose. These findings broaden the understanding of enigmatic AA14 LPMOs and provide new insights into the substrate specificities and biological functionalities of AA14 LPMOs in fungi.

A novel AA14 LPMO from Talaromyces rugulosus with bifunctional cellulolytic/hemicellulolytic activity boosted cellulose hydrolysis.

BACKGROUND: The recently discovered PcAA14A and B from white-rot basidiomycete Pycnoporus coccineus enriched our understanding of the oxidative degradation of xylan in fungi, however, the unusual mode of action of AA14 LPMOs has sparked controversy. The substrate specificity and functionality of AA14 LPMOs still remain enigmatic and need further investigation. RESULTS: In this study, a novel AA14 LPMO was characterized from the ascomycete Talaromyces rugulosus. TrAA14A has a broad substrate specificity with strong oxidative activity on pure amorphous cellulose and xyloglucan. It could simultaneously oxidize cellulose, xylan and xyloglucan in natural hemi/cellulosic substrate such as fibrillated eucalyptus pulp, and released native and oxidized cello-oligosaccharides, xylo-oligosaccharides and xyloglucan oligosaccharides from this substrate, but its cellulolytic/hemicellulolytic activity became weaker as the contents of xylan increase in the alkaline-extracted hemi/cellulosic substrates. The dual cellulolytic/hemicellulolytic activity enables TrAA14A to possess a profound boosting effect on cellulose hydrolysis by cellulolytic enzymes. Structure modelling of TrAA14A revealed that it exhibits a relatively flat active-site surface similar to the active-site surfaces in AA9 LPMOs but quite distinct from PcAA14B, despite TrAA14A is strongly clustered together with AA14 LPMOs. Remarkable difference in electrostatic potentials of L2 and L3 surfaces was also observed among TrAA14A, PcAA14B and NcLPMO9F. We speculated that the unique feature in substrate-binding surface might contribute to the cellulolytic/hemicellulolytic activity of TrAA14A. CONCLUSIONS: The extensive cellulolytic/hemicellulolytic activity on natural hemi/cellulosic substrate indicated that TrAA14A from ascomycete is distinctively different from previously characterized xylan-active AA9 or AA14 LPMOs. It may play as a bifunctional enzyme to decompose some specific network structures formed between cellulose and hemicellulose in the plant cell walls. Our findings shed new insights into the novel substrate specificities and biological functionalities of AA14 LPMOs, and will contribute to developing novel bifunctional LPMOs as the booster in commercial cellulase cocktails to efficiently break down the hemicellulose-cellulose matrix in lignocellulose.

A septation related gene AcsepH in Acremonium chrysogenum is involved in the cellular differentiation and cephalosporin production.

T-DNA inserted mutants of Acremonium chrysogenum were constructed by Agrobacterium tumefaciens-mediated transformation (ATMT). One mutant 1223 which grew slowly was selected. TAIL-PCR and sequence analysis indicated that a putative septation protein encoding gene AcsepH was partially deleted in this mutant. AcsepH contains nine introns, and its deduced protein AcSEPH has a conserved serine/threonine protein kinase catalytic (S_TKc) domain at its N-terminal region. AcSEPH shows high similarity with septation H proteins from other filamentous fungi based on the phylogenetic analysis of S_TKc domains. In sporulation (LPE) medium, the conidia of AcsepH mutant was only about one-seventh of the wild-type, and more than 20% of conidia produced by the mutant contain multiple nuclei which were rare in the wild-type. During fermentation, the AcsepH disruption mutant grew slowly and its cephalosporin production was only about one quarter of the wild-type, and the transcription analysis showed that pcbC expression was delayed and the expressions of cefEF, cefD1 and cefD2 were significantly decreased. The vegetative hyphae of AcsepH mutant swelled abnormally and hardly formed the typical yeast-like cells. The amount of yeast-like cells was about one-tenth of the wild-type after fermentation for 5days. Comparison of hyphal viabilities revealed that the cells of AcsepH mutant died easily than the wild-type at the late stage of fermentation. Fluorescent stains revealed that the absence of AcsepH in A. chrysogenum led to reduction of septation and formation of multinucleate cells. These data indicates that AcsepH is required for the normal cellular septation and differentiation of A. chrysogenum, and its absence may change the cellular physiological status and causes the decline in cephalosporin production.

The thioredoxin reductase-encoding gene ActrxR1 is involved in the cephalosporin C production of Acremonium chrysogenum in methionine-supplemented medium.

The thioredoxin system including thioredoxin and thioredoxin reductase (TrxR) is used for oxidative stress defenses in fungi. Based on the genomic sequence, a thioredoxin reductase-encoding gene (ActrxR1) was isolated from Acremonium chrysogenum CGMCC3.3795. Like other TrxRs, AcTrxR1 contains FAD binding domain, Redox domain, and NADPH binding domain. Disruption of ActrxR1 in A. chrysogenum led to the formation of smaller colonies and hyphal swelling in Tryptic soy agar (TSA). In chemically defined medium, the spore germination of ActrxR1 disruption mutant was strongly inhibited, which was recovered by the addition of DL-methionine. The disruption mutant grew slowly on TSA compared with the wild-type strain, but it did not show to be more sensitive to exogenous hydrogen peroxide or menadione. In defined medium of fermentation supplemented with DL-methionine, the ActrxR1 disruption mutant grew normally, and its cephalosporin C production increased by about onefold compared with the wild type (73 µg/ml for wild-type strain and 136 µg/ml for the mutant at 5 days of fermentation). Real-time polymerase chain reaction (RT-PCR) showed that the transcriptional levels of pcbC, cefEF, and cefG were obviously enhanced in the ActrxR1 mutant at the early stage of fermentation. These results indicate that ActrxR1 is required for the normal growth of A. chrysogenum and related with cephalosporin C production in methionine-supplemented medium.

Insights into the capability of the lignocellulolytic enzymes of Penicillium parvum 4-14 to saccharify corn bran after alkaline hydrogen peroxide pretreatment.

BACKGROUND: Corn bran is a major agro-industrial byproduct from corn starch processing. It contains abundant arabinoxylan that can be converted into value-added chemicals via biotechnology. Corn bran arabinoxylan (CBAX) is one of the most recalcitrant xylans for enzymatic degradation due to its particular heterogeneous nature. The present study aimed to investigate the capability of the filamentous fungus Penicillium parvum 4-14 to enzymatically saccharify CBAX and reveal the fungal carbohydrate-active enzyme (CAZyme) repertoire by genome sequencing and secretome analysis. RESULTS: CBAX1 and CBAX2 with different branching degrees, together with corn bran residue (CBR) were generated from corn bran after alkaline hydrogen peroxide (AHP) pretreatment and graded ethanol precipitation. The protein blends E_CBAX1, E_CBAX2, and E_CBR were produced by the fungus grown on CBAX1, CBAX2, or CBR, respectively. Under the optimal conditions, E_CBAX1 released more than 80% xylose and arabinose from CBAX1 and CBAX2. Almost complete saccharification of the arabinoxylans was achieved by combining E_CBAX1 and a commercial enzyme cocktail Cellic®CTec3. Approximately 89% glucose, 64% xylose, and 64% arabinose were liberated from CBR by E_CBR. The combination of E_CBR with Cellic®CTec3 enhanced the saccharification of CBR, with conversion ratios of 97% for glucose, 81% for xylose, and 76% for arabinose. A total of 376 CAZymes including plentiful lignocellulolytic enzymes were predicted in P. parvum based on the fungal genomic sequence (25.8 Mb). Proteomic analysis indicated that the expression of CAZymes in P. parvum varied between CBAX1 and CBR, and the fungus produced complete cellulases, numerous hemicellulases, as well as high levels of glycosidases under the culture conditions. CONCLUSIONS: This investigation disclosed the CAZyme repertoire of P. parvum at the genomic and proteomic levels, and elaborated on the promising potential of fungal lignocellulolytic enzymes upon saccharification of corn bran biomass after AHP pretreatment.

High-level production of cordycepin by the xylose-utilising Cordyceps militaris strain 147 in an optimised medium.

Cordycepin is an important active metabolite of Cordyceps militaris. Xylose, an attractive feedstock for producing chemicals through microbial fermentation, cannot be effectively utilised by many reported C. militaris strains. Herein, a xylose-utilising C. militaris strain 147 produced the highest level of cordycepin (3.03 g/L) in xylose culture. Xylose, alanine, and ammonium citrate were determined as the main affecting factors on the cordycepin production using a Plackett-Burman design. The combination of these factors was optimised using response surface methodology, and the maximal 6.54 g/L of cordycepin was produced by the fungus in the optimal medium. Transcriptome analysis revealed that xylose utilisation upregulated the transcriptional levels of genes participating in purine and energy metabolisms in the fungus, which may facilitate the formation of precursors for cordycepin biosynthesis. This investigation provides new insights into the efficient production of cordycepin and is conducive to the valorisation of biomass rich in xylose.

Disruption of a glutathione reductase encoding gene in Acremonium chrysogenum leads to reduction of its growth, cephalosporin production and antioxidative ability which is recovered by exogenous methionine.

Glutathione is a ubiquitous thiol in eukaryotic cells, and its high intracellular ratio of reduced form (GSH) to oxidized form (GSSG) is largely maintained by glutathione reductase (GR) using NADPH as electron donor. glrA, a glutathione reductase encoding gene, was found and cloned from Acremonium chrysogenum by searching its genomic sequence based on similarity. Its deduced protein exhibits high similarity to GRs of other eukaryotic organisms. Disruption of glrA resulted in lack of GR activity and accumulation of a high level of GSSG in A. chrysogenum. Overexpression of glrA dramatically enhanced GR activity and the ratio of GSH/GSSG in this fungus. The spore germination and hyphal growth of glrA disruption mutant was strongly reduced in chemical defined medium. Meanwhile, the mutant was more sensitive to hydrogen peroxide than the wild-type strain. We found that the glrA mutant recovered normal germination and growth by adding exogenous methionine (Met). Exogenous Met also enhanced the antioxidative ability of both the mutant and wild-type strain. GSH determination indicated that the total GSH and ratio of GSH/GSSG in the mutant or wild-type strain were significantly increased when addition of Met into the medium. The glrA mutant grew poorly and could not produce detectable cephalosporin in the fermentation medium without Met. However, its growth and cephalosporin production was restored with addition of exogenous Met. These results indicate that glrA is required for the normal growth and protection against oxidative damage in A. chrysogenum, and its absence can be complemented by exogenous Met.

Genomic Sequence and Transcriptome Analysis of the Medicinal Fungus Keithomyces neogunnii.

The filamentous fungus Keithomyces neogunnii can infect the larvae of Lepidoptera (Hepialus sp.) and form an insect-fungi complex, which is utilized as an important traditional Chinese medicine. As a valuable medicinal fungus, K. neogunnii produces diverse bioactive substances (e.g., polysaccharide, vitamins, cordycepic acid, and adenosine) under cultivation conditions. Herein, we report the first high-quality genome of the K. neogunnii single-spore isolate Cg7.2a using single-molecule real-time sequencing technology in combination with Illumina sequencing. The assembled genome was 32.6 Mb in size, containing 8,641 predicted genes and having a GC content of 52.16%. RNA sequencing analysis revealed the maximum number of differentially expressed genes in the fungus during the stroma formation stage compared with those during the mycelium stage. These data are valuable to enhance our understanding of the biology, development, evolution, and physiological metabolism of K. neogunnii.

Functional characterization of a GH62 family α-L-arabinofuranosidase from Eupenicillium parvum suitable for monosaccharification of corncob arabinoxylan in combination with key enzymes.

Corncob rich in arabinoxylan is an important raw material widely used in bio-refinery. Complete saccharification of arabinoxylan depends on the synergism of different enzymes including α-L-arabinofuranosidase (ABF). This study aimed to investigate the functional characteristics of a new ABF EpABF62A belonging to glycoside hydrolase (GH) 62 family from the fungus Eupenicillium parvum, and to explore its potential in the saccharification of corncob arabinoxylan. The recombinant EpABF62A showed high activity against wheat arabinoxylan and rye arabinoxylan, with the optimal temperature of 55 °C and pH of 4.5. The protein contains an N-terminal cellulose-binding domain family 1 (CBM_1) domain, and displayed a 59.5% absorption rate to phosphoric acid swollen cellulose. Regioselectivity analysis indicated that the enzyme selectively removed α-1,2 or α-1,3 linked arabinofuranosyl residues on mono-substituted xylose residues on arabinoxylan. Corncob arabinoxylans (CAX1 or CAX2) with different (low or high) branching degrees were extracted from the raw material by alkaline hydrogen peroxide pretreatment and graded ethanol precipitation. Single EpABF62A removed 69.5% or 67.1% arabinose from CAX1 or CAX2, respectively. EpABF62A combined with a GH10 xylanase, a GH43 ß-D-xylosidase and a GH67 α-glucuronidase released 75.0% or 64.5% xylose from CAX1 or CAX2, respectively. The addition of the four hemicellulases enhanced the saccharification the solid fraction of the pretreated corncob by the commercial cellulase Cellic® CTec2, and the conversion ratios of glucose, xylose and arabinose were up to 94.0%, 91.8% and 82.6%, respectively.