Two uridine auxotrophic mutants of Trichoderma reesei were isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. One mutant, called M23, was complemented with the Aspergillus niger pyrG gene carried by p...Two uridine auxotrophic mutants of Trichoderma reesei were isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. One mutant, called M23, was complemented with the Aspergillus niger pyrG gene carried by plasmid pAB4-1. A mutated pyrG gene of M23 was cloned and DNA sequencing analysis indicated that a cytosine was inserted into the 934―939 oligo dC position of the pyrG coding region, resulted in a frameshift mutation. Transformation efficiency was approximately 200―300 transformants per microgram of DNA with plasmid pAB4-1. Stable transformants were obtained by monosporic culture and showed to be prototroph after successive propagation. Vitreoscilla hemoglobin expression plasmid pUCVHb was cotransformed with plasmid pAB4-1 and attained a transformation efficiency of 71.8% or of 26.1% with pAN7-1. Southern blot analysis of the transformants demonstrated that plasmid pUCVHb was integrated into the chromosomal DNA. The experimental results demonstrated that the pyrG-based system was more efficient and timesaving than the conventional hygromycin B resistance-based transformation system.展开更多
β-glucanase was purified from a solid-state culture of Trichoderma reesei on wheat bran in three steps which comprised ammonium sulfate precipitation, Sephadex G-100 chromatography, and DEAE-Sepha-dex A-50 chromatogr...β-glucanase was purified from a solid-state culture of Trichoderma reesei on wheat bran in three steps which comprised ammonium sulfate precipitation, Sephadex G-100 chromatography, and DEAE-Sepha-dex A-50 chromatography, rIlae molecular mass was determined to be 35.21 kilodahons by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis. The β-glucanase at low pHs was more stable than that at high pHs, and optimum pH was 5.0. The optimum temperature was 60℃, and β-glueanase was relatively stable at below 40° for 60min. The Km of the enzyme on β-glucan was 10.86 mg/ml, and the Vmax on β-glucanwas 14286 pmol of glucose equivalents per nag of the pure enzyme per rain. The β-glucanase activity was significantly inhibited by Fe^3+ ions, and was reduced in the presence of Cu^2+ ions, Mn^2+ ions and Mg^2+ ions at 5mmol/L and 10mmol/L, respectively. The β-glucanase activity was stimulated by Co^2+ ions, Ca^2 + ions,Zn^2+ ions, and Fe^2+ ions at 1mmol/L and 5mmol/L, respectively.展开更多
The filamentous fungus Trichoderma reesei is widely used for the production of lignocellulolytic enzymes in industry.XYR1 is the major transcriptional activator of cellulases and hemicellulases in T.reesei.However,rat...The filamentous fungus Trichoderma reesei is widely used for the production of lignocellulolytic enzymes in industry.XYR1 is the major transcriptional activator of cellulases and hemicellulases in T.reesei.However,rational engineering of XYR1 for improved lignocellulolytic enzymes production has been limited by the lack of structure information.Here,alanine 873 was identified as a new potential target for the engineering of XYR1 based on its structure predicted by AlphaFold2.The mutation of this residue to tyrosine enabled significantly enhanced production of xylanolytic enzymes in the medium with cellulose as the carbon source.Moreover,xylanase and cellulase production increased by 56.7-and 3.3-fold,respectively,when glucose was used as the sole carbon source.Under both conditions,the improvements of lignocellulolytic enzyme production were higher than those in the previously reported V821F mutant.With the enriched hemicellulases and cellulases,the crude enzymes secreted by the A873Y mutant strain produced 51%more glucose and 52%more xylose from pretreated corn stover than those of the parent strain.The results provide a novel strategy for engineering the lignocellulolytic enzyme-producing capacity of T.reesei,and would be helpful for understanding the molecular mechanisms of XYR1 regulation.展开更多
Cellulose degradation results from the synergistic effect of different enzymes,but which enzyme is involved in the initial stage of cellulose degradation is still not well understood.Cellobiohydrolase 2(CBH2)attached ...Cellulose degradation results from the synergistic effect of different enzymes,but which enzyme is involved in the initial stage of cellulose degradation is still not well understood.Cellobiohydrolase 2(CBH2)attached to the conidial surface is possibly associated with the initial stage.However,its specific mechanism is still incompletely known.This study explored the potential role of CBH2 in initiating cellulose degradation using a constitutive overexpression strategy.First,the CBH2-overexpression Trichoderma reesei strains Qgc2-5 and Qrc2-40 were constructed using the constitutive promoters P gpd1 and P rpS30,respectively.It was found that cbh2 was ex-pressed at a high level under the glucose conditions and was significantly higher than that of the parental strain QM9414 at the early stage of 29 h when cellulose was used as the carbon source.Particularly,the constitutive overexpression of cbh2 caused the strong expression of major cellulase-encoding genes(cbh1,eg1,and eg2)and the rapid decomposition of cellulosic material.Meanwhile,the scanning electron microscope showed that the groove-like structure of the cellulose surface was eroded seriously owing to CBH2 overexpression,which caused the cellulose surface to be smooth.These results showed that the overexpression of CHB2 caused the major cel-lulase enzymes to be expressed and contributed to cellulose degradation,showing the potential role of CBH2 in the initial stage of the cellulose hydrolytic process.展开更多
Trichoderma reesei Rut-C-30 is a well-known robust producer of cellulolytic enzymes,which are used to degrade lignocellulosic biomass for the sustainable production of biofuels and biochemicals.However,studies of its ...Trichoderma reesei Rut-C-30 is a well-known robust producer of cellulolytic enzymes,which are used to degrade lignocellulosic biomass for the sustainable production of biofuels and biochemicals.However,studies of its sec-ondary metabolism and regulation remain scarce.Ypr1 was previously described as a regulator of the biosynthesis of the yellow pigment sorbicillin(a bioactive agent with great pharmaceutical interest)in T.reesei and several other fungi.However,the manner in which this regulator affects global gene transcription has not been explored.In this study,we report the effect of Ypr1 on the regulation of both the secondary and primary metabolism of T.reesei Rut-C30.A global gene transcription profile was obtained using a comparative transcriptomic analysis of the wild-type strain T.reesei Rut-C-30 and its ypr1 deletion mutant.The results of this analysis suggest that,in addition to its role in regulating sorbicillin and the major extracellular(hemi)cellulases,Ypr1 also affects the transcription of genes encoding several other secondary metabolites.Although the primary metabolism of T.reeseiΔypr1 became less active compared with that of T.reesei Rut-C-30,several gene clusters involved in its secondary metabolism were activated,such as the gene clusters for the biosynthesis of specific polyketides and non-ribosomal peptides,together with the“sorbicillinoid-cellulase”super cluster,indicating that specific secondary metabolites and cellulases may be co-regulated in T.reesei Rut-C-30.The results presented in this study may benefit the development of genetic engineering strategies for the production of sorbicillin by T.reesei Rut-C-30,and provide insights for enhancing sorbicillin production in other filamentous fungal producers.展开更多
Carbon catabolite repression(CCR),which is mainly mediated by Cre1 and triggered by glucose,leads to a decrease in cellulase production in Trichoderma reesei.Many studies have focused on modifying Cre1 for alleviating...Carbon catabolite repression(CCR),which is mainly mediated by Cre1 and triggered by glucose,leads to a decrease in cellulase production in Trichoderma reesei.Many studies have focused on modifying Cre1 for alleviating CCR.Based on the homologous alignment of CreA from wild-type Penicillium oxalicum 114–2(Po-0)and cellulase hyperproducer JUA10-1(Po-1),we constructed a C-terminus substitution strain—Po-2—with decreased transcriptional levels of cellulase and enhanced CCR.Results revealed that the C-terminal domain of CreAPo−1 plays an important role in alleviating CCR.Furthermore,we replaced the C-terminus of Cre1 with that of CreAPo−1 in T.reesei(Tr-0)and generated Tr-1.As a control,the C-terminus of Cre1 was truncated and Tr-2 was generated.The transcriptional profiles of these transformants revealed that the C-terminal chimera greatly improves cellulase transcription in the presence of glucose and thus upregulates cellulase in the presence of glucose and weakens CCR,consistent with truncating the C-terminus of Cre1 in Tr-0.Therefore,we propose constructing a C-terminal chimera as a new strategy to improve cellulase production and alleviate CCR in the presence of glucose.展开更多
To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinan...To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinant TrCel5A was screened out after gene optimization,synthesis,and expression.The biochemical and enzymatic properties of the new recombinant were characterized.As a result,optimization of shake-flask fermentation of the recombinant was obtained at 28℃,2%inoculum volume,an initial pH of 6.0,as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L,respectively.Under the above-optimized conditions,the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of fermentation.To further enhance enzyme production,pilot-scale cultivation was evaluated using 5-L bioreactors.Using high-cell-density fermentation,the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/L.In addition,the kinetic factors,including K_(m) and V_(max) values for TrCel5A,were detected to be 5.1 mg/mL and 265.9μmol/(min.mg),respectively.Thus,TrCel5A was effectively expressed in P.pastoris under the GAP promoter,and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.展开更多
Cold-active enzymes have received little research attention although they are very useful in industries. Since the structure bases of cold adaptation of enzymes are still unclear, it is also very difficult to obtain c...Cold-active enzymes have received little research attention although they are very useful in industries. Since the structure bases of cold adaptation of enzymes are still unclear, it is also very difficult to obtain cold-adapted enzymes for industrial applications using routine protein engineering methods. In this work, we employed directed evolution method to randomly mutate a mesophilic cellulase, endoglucanase III (EG III) from Trichoderma reesei, and obtained a cold- adapted mutant, designated as w-3. DNA sequence analysis indicates that w-3 is a truncated form of native EG III with a deletion of 25 consecutive amino acids at C-terminus. Further examination of enzymatic kinetics and thermal stability shows that mutant w-3 has a higher Kcat value and becomes more thermolabile than its parent. In addition, activation energies of w-3 and wild type EG III calculated from Arrhenius equation are 13.3 kJ@mol-1 and 26.2 kJ@mol-1, respectively. Therefore, the increased specific activity of w-3 at lower temperatures could result from increased Kcat value and decreased activation energy.展开更多
β-葡聚糖酶可水解大麦等谷物中的葡聚糖,降低葡聚糖在单胃动物消化道内产生的抗营养作用。为获得高产β-葡聚糖酶菌株,采用常压室温等离子体(atmospheric and room temperature plasma,ARTP)诱变里氏木霉(Trichoderma reesei),经初筛...β-葡聚糖酶可水解大麦等谷物中的葡聚糖,降低葡聚糖在单胃动物消化道内产生的抗营养作用。为获得高产β-葡聚糖酶菌株,采用常压室温等离子体(atmospheric and room temperature plasma,ARTP)诱变里氏木霉(Trichoderma reesei),经初筛、复筛、遗传稳定性及酶学特性表征,突变株ARTP-9较出发菌株β-葡聚糖酶活力提高54.38%,为43.75 U/mL,产酶能力稳定遗传,酶的最适反应温度及pH分别为50℃和6.0,耐受温度为40~80℃,pH 2.5~6.5时酶活力稳定,Fe^(2+)、Mg^(2+)对酶活力有促进作用,Fe^(3+)、Mn^(2+)、Zn^(2+)、Ca^(2+)、Cu^(2+)对酶活力有抑制作用。以β-葡聚糖为底物,该粗酶的K m值和V max值分别为1.59 mg/mL和6.99μmol/(mg·min)。体外模拟消化实验表明,ARTP-9固态发酵酶制剂在胃期4 h黏度最低,为2.23 mPa·s;肠期21 h还原糖为0.70 mg/mL,是空白的3.2倍,差异显著(P<0.05),其大麦粉体外消化率为48.17%,较空白提升9.02%,差异极显著(P<0.01)。研究结果表明ARTP诱变技术能够显著提高里氏木霉β-葡聚糖酶活力,为其工业化生产奠定基础。展开更多
基金the National Natural Science Foundation of China(No.30470052)the National Basic Research Program of China(Nos.2003CB716006 and 2004CB719702)the Natural Science Research Foundation for the Doctoral Program of Edu-cation Ministry of China(No.20040422042).
文摘Two uridine auxotrophic mutants of Trichoderma reesei were isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. One mutant, called M23, was complemented with the Aspergillus niger pyrG gene carried by plasmid pAB4-1. A mutated pyrG gene of M23 was cloned and DNA sequencing analysis indicated that a cytosine was inserted into the 934―939 oligo dC position of the pyrG coding region, resulted in a frameshift mutation. Transformation efficiency was approximately 200―300 transformants per microgram of DNA with plasmid pAB4-1. Stable transformants were obtained by monosporic culture and showed to be prototroph after successive propagation. Vitreoscilla hemoglobin expression plasmid pUCVHb was cotransformed with plasmid pAB4-1 and attained a transformation efficiency of 71.8% or of 26.1% with pAN7-1. Southern blot analysis of the transformants demonstrated that plasmid pUCVHb was integrated into the chromosomal DNA. The experimental results demonstrated that the pyrG-based system was more efficient and timesaving than the conventional hygromycin B resistance-based transformation system.
文摘β-glucanase was purified from a solid-state culture of Trichoderma reesei on wheat bran in three steps which comprised ammonium sulfate precipitation, Sephadex G-100 chromatography, and DEAE-Sepha-dex A-50 chromatography, rIlae molecular mass was determined to be 35.21 kilodahons by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis. The β-glucanase at low pHs was more stable than that at high pHs, and optimum pH was 5.0. The optimum temperature was 60℃, and β-glueanase was relatively stable at below 40° for 60min. The Km of the enzyme on β-glucan was 10.86 mg/ml, and the Vmax on β-glucanwas 14286 pmol of glucose equivalents per nag of the pure enzyme per rain. The β-glucanase activity was significantly inhibited by Fe^3+ ions, and was reduced in the presence of Cu^2+ ions, Mn^2+ ions and Mg^2+ ions at 5mmol/L and 10mmol/L, respectively. The β-glucanase activity was stimulated by Co^2+ ions, Ca^2 + ions,Zn^2+ ions, and Fe^2+ ions at 1mmol/L and 5mmol/L, respectively.
基金supported by the National Key R&D Program of China(2018YFA0900500)National Natural Science Foundation of China(No.32170037)the Key research program of China National Tobacco Corporation(No.110202102018)。
文摘The filamentous fungus Trichoderma reesei is widely used for the production of lignocellulolytic enzymes in industry.XYR1 is the major transcriptional activator of cellulases and hemicellulases in T.reesei.However,rational engineering of XYR1 for improved lignocellulolytic enzymes production has been limited by the lack of structure information.Here,alanine 873 was identified as a new potential target for the engineering of XYR1 based on its structure predicted by AlphaFold2.The mutation of this residue to tyrosine enabled significantly enhanced production of xylanolytic enzymes in the medium with cellulose as the carbon source.Moreover,xylanase and cellulase production increased by 56.7-and 3.3-fold,respectively,when glucose was used as the sole carbon source.Under both conditions,the improvements of lignocellulolytic enzyme production were higher than those in the previously reported V821F mutant.With the enriched hemicellulases and cellulases,the crude enzymes secreted by the A873Y mutant strain produced 51%more glucose and 52%more xylose from pretreated corn stover than those of the parent strain.The results provide a novel strategy for engineering the lignocellulolytic enzyme-producing capacity of T.reesei,and would be helpful for understanding the molecular mechanisms of XYR1 regulation.
基金supported by grants from the National Key R&D Program of China(2021YFC2101300,2018YFA0900503)the National Natural Science Foundation of China(31970070)the Youth Interdisciplinary Science and Innovative Research Groups of Shandong University(2020QNQT006).
文摘Cellulose degradation results from the synergistic effect of different enzymes,but which enzyme is involved in the initial stage of cellulose degradation is still not well understood.Cellobiohydrolase 2(CBH2)attached to the conidial surface is possibly associated with the initial stage.However,its specific mechanism is still incompletely known.This study explored the potential role of CBH2 in initiating cellulose degradation using a constitutive overexpression strategy.First,the CBH2-overexpression Trichoderma reesei strains Qgc2-5 and Qrc2-40 were constructed using the constitutive promoters P gpd1 and P rpS30,respectively.It was found that cbh2 was ex-pressed at a high level under the glucose conditions and was significantly higher than that of the parental strain QM9414 at the early stage of 29 h when cellulose was used as the carbon source.Particularly,the constitutive overexpression of cbh2 caused the strong expression of major cellulase-encoding genes(cbh1,eg1,and eg2)and the rapid decomposition of cellulosic material.Meanwhile,the scanning electron microscope showed that the groove-like structure of the cellulose surface was eroded seriously owing to CBH2 overexpression,which caused the cellulose surface to be smooth.These results showed that the overexpression of CHB2 caused the major cel-lulase enzymes to be expressed and contributed to cellulose degradation,showing the potential role of CBH2 in the initial stage of the cellulose hydrolytic process.
基金This work is supported by the State Key Research and Development Program(2022YFE0108500).
文摘Trichoderma reesei Rut-C-30 is a well-known robust producer of cellulolytic enzymes,which are used to degrade lignocellulosic biomass for the sustainable production of biofuels and biochemicals.However,studies of its sec-ondary metabolism and regulation remain scarce.Ypr1 was previously described as a regulator of the biosynthesis of the yellow pigment sorbicillin(a bioactive agent with great pharmaceutical interest)in T.reesei and several other fungi.However,the manner in which this regulator affects global gene transcription has not been explored.In this study,we report the effect of Ypr1 on the regulation of both the secondary and primary metabolism of T.reesei Rut-C30.A global gene transcription profile was obtained using a comparative transcriptomic analysis of the wild-type strain T.reesei Rut-C-30 and its ypr1 deletion mutant.The results of this analysis suggest that,in addition to its role in regulating sorbicillin and the major extracellular(hemi)cellulases,Ypr1 also affects the transcription of genes encoding several other secondary metabolites.Although the primary metabolism of T.reeseiΔypr1 became less active compared with that of T.reesei Rut-C-30,several gene clusters involved in its secondary metabolism were activated,such as the gene clusters for the biosynthesis of specific polyketides and non-ribosomal peptides,together with the“sorbicillinoid-cellulase”super cluster,indicating that specific secondary metabolites and cellulases may be co-regulated in T.reesei Rut-C-30.The results presented in this study may benefit the development of genetic engineering strategies for the production of sorbicillin by T.reesei Rut-C-30,and provide insights for enhancing sorbicillin production in other filamentous fungal producers.
基金This work was supported by National Key R&D Program of China(No.2018YFA0901700)National Natural Science Foundation of China(No.31870785 and 31570040)+1 种基金the 111 Project(No.B16030)the State Key Laboratory of Microbial Technology Open Projects Fund.
文摘Carbon catabolite repression(CCR),which is mainly mediated by Cre1 and triggered by glucose,leads to a decrease in cellulase production in Trichoderma reesei.Many studies have focused on modifying Cre1 for alleviating CCR.Based on the homologous alignment of CreA from wild-type Penicillium oxalicum 114–2(Po-0)and cellulase hyperproducer JUA10-1(Po-1),we constructed a C-terminus substitution strain—Po-2—with decreased transcriptional levels of cellulase and enhanced CCR.Results revealed that the C-terminal domain of CreAPo−1 plays an important role in alleviating CCR.Furthermore,we replaced the C-terminus of Cre1 with that of CreAPo−1 in T.reesei(Tr-0)and generated Tr-1.As a control,the C-terminus of Cre1 was truncated and Tr-2 was generated.The transcriptional profiles of these transformants revealed that the C-terminal chimera greatly improves cellulase transcription in the presence of glucose and thus upregulates cellulase in the presence of glucose and weakens CCR,consistent with truncating the C-terminus of Cre1 in Tr-0.Therefore,we propose constructing a C-terminal chimera as a new strategy to improve cellulase production and alleviate CCR in the presence of glucose.
基金supported by the National Key Research and Development Program of China(2019YFE0114600)National Natural Science Foundation of China(21776114).
文摘To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinant TrCel5A was screened out after gene optimization,synthesis,and expression.The biochemical and enzymatic properties of the new recombinant were characterized.As a result,optimization of shake-flask fermentation of the recombinant was obtained at 28℃,2%inoculum volume,an initial pH of 6.0,as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L,respectively.Under the above-optimized conditions,the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of fermentation.To further enhance enzyme production,pilot-scale cultivation was evaluated using 5-L bioreactors.Using high-cell-density fermentation,the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/L.In addition,the kinetic factors,including K_(m) and V_(max) values for TrCel5A,were detected to be 5.1 mg/mL and 265.9μmol/(min.mg),respectively.Thus,TrCel5A was effectively expressed in P.pastoris under the GAP promoter,and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.
基金This work was supported by the National Natural Science Foundation of China (Grant No. 39970392).
文摘Cold-active enzymes have received little research attention although they are very useful in industries. Since the structure bases of cold adaptation of enzymes are still unclear, it is also very difficult to obtain cold-adapted enzymes for industrial applications using routine protein engineering methods. In this work, we employed directed evolution method to randomly mutate a mesophilic cellulase, endoglucanase III (EG III) from Trichoderma reesei, and obtained a cold- adapted mutant, designated as w-3. DNA sequence analysis indicates that w-3 is a truncated form of native EG III with a deletion of 25 consecutive amino acids at C-terminus. Further examination of enzymatic kinetics and thermal stability shows that mutant w-3 has a higher Kcat value and becomes more thermolabile than its parent. In addition, activation energies of w-3 and wild type EG III calculated from Arrhenius equation are 13.3 kJ@mol-1 and 26.2 kJ@mol-1, respectively. Therefore, the increased specific activity of w-3 at lower temperatures could result from increased Kcat value and decreased activation energy.
