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
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
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
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
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
[ Objective ] This study aimed to identify a cellulase-producing strain selected from tropical rain forestry soils. [ Method ] Morphologic observation and sequence analysis of 18S rDNA were conducted. [Result] A cellu...[ Objective ] This study aimed to identify a cellulase-producing strain selected from tropical rain forestry soils. [ Method ] Morphologic observation and sequence analysis of 18S rDNA were conducted. [Result] A cellulase-preducing strain with high activity was obtained, and morphology of the strain was highly similar to that of Trichoderma reesei. Results of sequence analysis show that the 18S rDNA sequence shares 99% homology with Hypocreajecorina. [ Conclusion] The isolated cellulase-producing strain belongs to Trichoderma reesei.展开更多
[Objective] The study was to construct a exogenous expression vector for Trichoderma reesei.[Method] Using CBHI promoter and terminator of T.reesei strain 40359,we constructed an expression vector of T.reesei strain 4...[Objective] The study was to construct a exogenous expression vector for Trichoderma reesei.[Method] Using CBHI promoter and terminator of T.reesei strain 40359,we constructed an expression vector of T.reesei strain 40359 for expressing Hpt gene and got six strains capable of growing on basic medium containing 175 mg/L of hygromycin B,further conducted hygromycin resistance test.[Results] In comparison with the original strain（wild type）,hygromycin resistance the six engineered strains was increased by 75%;the hygromycin resistance could inherit stably.[Conclusion] Our results laid basis for biological study on T.reesei at molecular and genetically engineering levels.展开更多
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\|Sephadex A\|50 chroma...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\|Sephadex A\|50 chromatography. The molecular mass was determined to be 35.21 kilodaltons 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\%\|glucanase was relatively stable at below 40 ℃ for 60 min. The \%K\%\-m of the enzyme on \%β\%\|glucan was 10.86 mg/ml, and the \%V\%\-\{max\} on \%β\%\|glucan was 14286 μmol of glucose equivalents per mg of the pure enzyme per min. 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 5 mmol/L and 10 mmol/L, respectively. The \%β\%\|glucanase activity was stimulated by Co\+\{2+\} ions, Ca\+\{2+\} ions, Zn\+\{2+\} ions, and Fe\+\{2+\} ions at 1 mmol/L and 5 mmol/L, respectively.展开更多
[Objective] This study aimed to investigate the sporulation conditions of Tri- choderma reesei by solid fermentation. [Method] With sporulation yield as the response value, single-factor test, Plackett-Burmam design, ...[Objective] This study aimed to investigate the sporulation conditions of Tri- choderma reesei by solid fermentation. [Method] With sporulation yield as the response value, single-factor test, Plackett-Burmam design, steepest ascent test, BoxBehnken design and response surface analysis were employed to optimize the con- ditions for sporulation of Trichoderma reesei by solid fermentation. [Result] Based on single-factor test, the most appropriate carbon source for Trichoderma reesei was straw stalk powder and wheat bran with the ratio of 3：2 and optimal amount of 15 g/L; the most appropriate inorganic nitrogen was （NH4）2O4 with the optimal amount of 3 g/L. According to Plackett-Burmam design, moisture content, initial pH and incubation temperature were identified as significant factors affecting the sporulation yield of Trichoderma reeseL The maximum sporulation yield area was approached by steepest ascent test. Based on Box-Behnken design and response surface analysis, the optimal fermentation conditions for the maximum sporulation yield were determined as： straw stalk powder of 6 g/L, wheat bran of 9 g/L, （NH4）2SO4 of 3 g/L, moisture content of 65%, incubation temperature of 29 ℃, fermentation period of 72 h and initial pH of 5.5, under these conditions, the sporulation yield reached 2×10^10 spores/g, which was improved by 1.4 times compared with that before optimization. [Conclusion] In this study, the conditions for sporulation of Trichoderma reesei by solid fermentation were optimized with low-cost straw stalk powder and wheat bran as carbon sources, which was conducive to reducing the production cost of Trichoderma reesei and increasing the sporulation yield, showing certain social and economic significance.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.
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.
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Supported by 973 Program ( 2010CB126600 )863 Key Project ( 2007AA021307)+3 种基金National Natural Science Foundation ( 31000029 )Special Fund for Basic Research Operating Expenses of Central Nonprofit Research Institutions of Chinese Academy of Tropical Agriculture (ITBBKF1010,ITBBZD0951,ITTBB110103)Hainan Provincial Introduction Integrated Special Fund ( YJJC2011004 )Hainan Provincial Graduate Innovation Fund( Hyb2011-4)
文摘[ Objective ] This study aimed to identify a cellulase-producing strain selected from tropical rain forestry soils. [ Method ] Morphologic observation and sequence analysis of 18S rDNA were conducted. [Result] A cellulase-preducing strain with high activity was obtained, and morphology of the strain was highly similar to that of Trichoderma reesei. Results of sequence analysis show that the 18S rDNA sequence shares 99% homology with Hypocreajecorina. [ Conclusion] The isolated cellulase-producing strain belongs to Trichoderma reesei.
文摘[Objective] The study was to construct a exogenous expression vector for Trichoderma reesei.[Method] Using CBHI promoter and terminator of T.reesei strain 40359,we constructed an expression vector of T.reesei strain 40359 for expressing Hpt gene and got six strains capable of growing on basic medium containing 175 mg/L of hygromycin B,further conducted hygromycin resistance test.[Results] In comparison with the original strain（wild type）,hygromycin resistance the six engineered strains was increased by 75%;the hygromycin resistance could inherit stably.[Conclusion] Our results laid basis for biological study on T.reesei at molecular and genetically engineering levels.
文摘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\|Sephadex A\|50 chromatography. The molecular mass was determined to be 35.21 kilodaltons 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\%\|glucanase was relatively stable at below 40 ℃ for 60 min. The \%K\%\-m of the enzyme on \%β\%\|glucan was 10.86 mg/ml, and the \%V\%\-\{max\} on \%β\%\|glucan was 14286 μmol of glucose equivalents per mg of the pure enzyme per min. 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 5 mmol/L and 10 mmol/L, respectively. The \%β\%\|glucanase activity was stimulated by Co\+\{2+\} ions, Ca\+\{2+\} ions, Zn\+\{2+\} ions, and Fe\+\{2+\} ions at 1 mmol/L and 5 mmol/L, respectively.
基金Supported by General Project of Science and Technology in Hunan Province(No.2012NK3080)~~
文摘[Objective] This study aimed to investigate the sporulation conditions of Tri- choderma reesei by solid fermentation. [Method] With sporulation yield as the response value, single-factor test, Plackett-Burmam design, steepest ascent test, BoxBehnken design and response surface analysis were employed to optimize the con- ditions for sporulation of Trichoderma reesei by solid fermentation. [Result] Based on single-factor test, the most appropriate carbon source for Trichoderma reesei was straw stalk powder and wheat bran with the ratio of 3：2 and optimal amount of 15 g/L; the most appropriate inorganic nitrogen was （NH4）2O4 with the optimal amount of 3 g/L. According to Plackett-Burmam design, moisture content, initial pH and incubation temperature were identified as significant factors affecting the sporulation yield of Trichoderma reeseL The maximum sporulation yield area was approached by steepest ascent test. Based on Box-Behnken design and response surface analysis, the optimal fermentation conditions for the maximum sporulation yield were determined as： straw stalk powder of 6 g/L, wheat bran of 9 g/L, （NH4）2SO4 of 3 g/L, moisture content of 65%, incubation temperature of 29 ℃, fermentation period of 72 h and initial pH of 5.5, under these conditions, the sporulation yield reached 2×10^10 spores/g, which was improved by 1.4 times compared with that before optimization. [Conclusion] In this study, the conditions for sporulation of Trichoderma reesei by solid fermentation were optimized with low-cost straw stalk powder and wheat bran as carbon sources, which was conducive to reducing the production cost of Trichoderma reesei and increasing the sporulation yield, showing certain social and economic significance.
