APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
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2024
·Vol. 108
·期 1
·1-13
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IF 3.9
Liu Shuai,Quan Lin,Yang Mei,Wang Dan,Wang Yong-Zhong
In this study, the effect of polyethylene glycol 8000 (PEG8000) stress on cellulase biosynthesis in Trichoderma reesei CICC2626 via calcium signaling was investigated, and a plausible mechanism by which intracellular Ca 2+ regulates the transcription of cellulase genes was proposed. The results indicated that the total cellulase (filter paper-hydrolyzing activity [FPase]), endoglucanase (carboxymethyl cellulase activity [CMCase]), and β-glucosidase activities of the strain were 1.3-, 1.2-, and 1.3-fold higher than those of the control (no PEG8000 addition) at a final concentration of 1.5% (w/v) PEG8000. Moreover, the transcriptional levels of cellulase genes, protein concentrations, and biomass increased. With the synergistic use of commercial cellulase and T. reesei CICC2626 cellulase to hydrolyze alkali-pretreated rice straw, the released reducing sugar concentration reached 372.7 mg/g, and the cellulose content (22.7%, 0.32 g) was significantly lower than the initial content (62.5%, 1.88 g). Transcriptome data showed that 12 lignocellulose degradation–related genes were significantly upregulated in the presence of 1.5% PEG8000. Furthermore, the addition of Ca 2+ inhibitors and deletion of crz1 (calcineurin-responsive zinc finger 1-encoding gene, which is related to the calcium signaling pathway) demonstrated that calcium signaling plays a dominant role in PEG8000-induced cellulase genes overexpression. These results revealed a link between PEG8000 induction and calcium signaling transduction in T. reesei CICC2626. Moreover, this study also provides a novel inducer for enhanced cellulase production. Key points • Cellulase biosynthesis in Trichoderma reesei could be enhanced by PEG8000 • PEG8000 could induce a cytosolic Ca 2+ burst in Trichoderma reesei • The activated calcium signaling was involved in cellulase biosynthesis Graphical abstract
研究领域:
微生物学
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生物化学
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DOI
高影响因子
Microbial Cell Factories
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2024
·Vol. 23
·期 1
·1-12
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IF 4.3
Wang Jiajia,Chen Yumeng,Cong Jiawei,Wang Wei
Background Trichoderma reesei is renowned for its cellulase-producing ability and is used for biofuel production from lignocellulose. In plants and fungi, cellulase production is induced by cellulose and suppressed by glucose; however, whether metformin can enhance cellulase production and mitochondrial function in T. reesei remains unclear. Metformin reduces blood glucose levels by inhibiting hepatic gluconeogenesis; therefore, it is worth investigating whether metformin transmission modulates cellulase biosynthesis in T. reesei . Results Metformin increased cellulase production and the transcription of cellulase-related genes. It also enhanced the concentrations of Ca 2+ in the cytosol and mitochondria and regulated the transcription levels of cellulase-related genes by modulating calcium homeostasis in T. reesei QM6a. In addition, metformin was identified as an antioxidant that can enhance cellulase activity by reducing reactive oxygen species (ROS). Our results demonstrated that metformin influences the state of mitochondria by enhancing mitochondrial activity and membrane potential to promote cellulase production. Conclusion Collectively, these results indicate that metformin enhances cellulase production, scavenges ROS, and protects mitochondrial activity in T. reesei . Graphical Abstract
研究领域:
微生物学
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生物化学
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生物能源
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PubMed
高影响因子
Biotechnology for Biofuels
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2019
·Vol. 12
·期 1
·1-17
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IF 5.452
Chen Yumeng,Wu Chuan,Shen Yaling,Ma Yushu,Wei Dongzhi,Wang Wei
Background The filamentous fungus Trichoderma reesei produces cellulase enzymes that are widely studied for lignocellulose bioconversion to biofuel. N,N-dimethylformamide (DMF) is a versatile organic solvent used in large quantities in industries.Results In this study, we serendipitously found that biologically relevant concentrations of extracellular DMF-induced cellulase production in the T. reesei hyper-cellulolytic mutant Rut-C30 and wild-type strain QM6a. Next, by transcriptome analysis, we determined that plc-e encoding phospholipase C was activated by DMF and revealed that cytosolic Ca2+ plays a vital role in the response of T. reesei to DMF. Using EGTA (a putative extracellular Ca2+ chelator) and LaCl3 (a plasma membrane Ca2+ channel blocker), we demonstrated that DMF induced a cytosolic Ca2+ burst via extracellular Ca2+ and Ca2+ channels in T. reesei, and that the cytosolic Ca2+ burst induced by DMF-mediated overexpression of cellulase through calcium signaling. Deletion of crz1 confirmed that calcium signaling plays a dominant role in DMF-induced cellulase production. Additionally, 0.5–2% DMF increases the permeability of T. reesei mycelia for cellulase release. Simultaneous supplementation with 1% DMF and 10 mM Mn2+ to T. reesei Rut-C30 increased cellulase activity approximately fourfold compared to that without treatment and was also more than that observed in response to either treatment alone.Conclusion sOur results reveal that DMF-induced cellulase production via calcium signaling and permeabilization. Our results also provide insight into the role of calcium signaling in enzyme production for enhanced cellulase production and the development of novel inducers of cellulase.
研究领域:
生物化学
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微生物学
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工业酶学
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PubMed
高影响因子
Biotechnology for Biofuels
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2018
·Vol. 11
·期 1
·1-19
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IF 5.497
Chen Yumeng,Shen Yaling,Wang Wei,Wei Dongzhi
Background The filamentous fungus Trichoderma reesei Rut-C30 is one of the most vital fungi for the production of cellulases, which can be used for biofuel production from lignocellulose. Nevertheless, the mechanism of transmission of external stimuli and signals in modulating cellulase production in T. reesei Rut-C30 remains unclear. Calcium is a known second messenger regulating cellulase gene expression in T. reesei.Results In this study, we found that a biologically relevant extracellular Mn2+ concentration markedly stimulates cellulase production, total protein secretion, and the intracellular Mn2+ concentration of Rut-C30, a cellulase hyper-producing strain of T. reesei. Furthermore, we identified two Mn2+ transport proteins, designated as TPHO84-1 and TPHO84-2, indicating that they are upstream in the signaling pathway that leads to cellulase upregulation. We also found that Mn2+ induced a significant increase in cytosolic Ca2+ concentration, and that this increased cytosolic Ca2+ might be a key step in the Mn2+-mediated regulation of cellulase gene transcription and production. The utilization of LaCl3 to block plasma membrane Ca2+ channels, and deletion of crz1 (calcineurin-responsive zinc finger transcription factor 1) to interrupt calcium signaling, showed that Mn2+ exerts the induction of cellulase genes via calcium channels and calcium signaling. To substantiate this, we identified a Ca2+/Mn2+ P-type ATPase, TPMR1, which could play a pivotal role in Ca2+/Mn2+ homeostasis and Mn2+ induction of cellulase genes in T. reesei Rut-C30.Conclusion sTaken together, our results revealed for the first time that Mn2+ stimulates cellulase production, and demonstrates that Mn2+ upregulates cellulase genes via calcium channels and calcium signaling. Our research also provides a direction to facilitate enhanced cellulase production by T. reesei.
研究领域:
微生物学
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分子生物学
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生物化学
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PubMed
高影响因子
Biotechnology for Biofuels and Bioproducts
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2024
·Vol. 17
·期 1
·1-17
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IF 3.3
Liu Lin,Wang Zhixing,Fang Yu,Yang Renfei,Pu Yi,Meng Xiangfeng,Liu Weifeng
Background The saprophytic filamentous fungus Trichoderma reesei represents one of the most prolific cellulase producers. The bulk production of lignocellulolytic enzymes by T. reesei not only relies on the efficient transcription of cellulase genes but also their efficient secretion after being translated. However, little attention has been paid to the functional roles of the involved secretory pathway in the high-level production of cellulases in T. reesei. Rab GTPases are key regulators in coordinating various vesicle trafficking associated with the eukaryotic secretory pathway. Specifically, Rab7 is a representative GTPase regulating the transition of the early endosome to the late endosome followed by its fusion to the vacuole as well as homotypic vacuole fusion. Although crosstalk between the endosomal/vacuolar pathway and the secretion pathway has been reported, the functional role of Rab7 in cellulase production in T. reesei remains unknown.Results A TrRab7 was identified and characterized in T. reesei. TrRab7 was shown to play important roles in T. reesei vegetative growth and vacuole morphology. Whereas knock-down of Trrab7 significantly compromised the induced production of T. reesei cellulases, overexpression of the key transcriptional activator, Xyr1, restored the production of cellulases in the Trrab7 knock-down strain (Ptcu-rab7KD) on glucose, indicating that the observed defective cellulase biosynthesis results from the compromised cellulase gene transcription. Down-regulation of Trrab7 was also found to make T. reesei more sensitive to various stresses including carbon starvation. Interestingly, overexpression of Snf1, a serine/threonine protein kinase known as an energetic sensor, partially restored the cellulase production of Ptcu-rab7KD on Avicel, implicating that TrRab7 is involved in an energetic adaptation to carbon starvation which contributes to the successful cellulase gene expression when T. reesei is transferred from glucose to cellulose.Conclusion sTrRab7 was shown to play important roles in T. reesei development and a stress response to carbon starvation resulting from nutrient shift. This adaptation may allow T. reesei to successfully initiate the inducing process leading to efficient cellulase production. The present study provides useful insights into the functional involvement of the endosomal/vacuolar pathway in T. reesei development and hydrolytic enzyme production.
研究领域:
微生物学
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分子生物学
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酶工程
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高影响因子
INDUSTRIAL CROPS AND PRODUCTS
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2017
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IF 3.181
Jun Xia,Aiyong He,Rongqing Li,Yang Zhang,Jiaxing Xu,Xiaoyan Liu,Jiming Xu
Oxygen mass transfer is one of the most essential factors affecting microbial cell growth and cellulase fermentation. In this study, an effective approach capable of improving oxygen mass transfer was developed by adding oxygen vectors into the fermentation broth by Trichoderma reesei . With the addition of 1% (v/v) n -dodecane, the volumetric mass transfer coefficient ( k L a ) was increased from 142 h −1 to 185 h −1 at the beginning of the fermentation. In response to the improved oxygen transfer, 44.6% higher FPase activity, 29.2% higher xylanase activity and 42.5% higher saccharification yield of rice straw were obtained. The scanning electron microscopy showed that the addition of n -dodecane did not cause obvious variations on the cell morphology, whereas the comparative proteomics analysis identified 30 protein spots with different expression levels. Among them, exoglucanases (CBH I, CBH II), endoglucanase (EG VI), xylanases (Xyn I, Xyn II, Xyn III), acetyl xylan esterase, swollenin and cellulose induced protein (Cip1) were highly expressed after the addition of n -dodecane, whereas more EG III and β -xylosidase were produced without the addition of n -dodecane. The present study indicated that the addition of n -dodecane was advantageous for cellulase fermentation by T. reesei .
研究领域:
生物工程
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微生物学
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高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2023
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IF 6.1
Jing Li,Yumeng Chen,Ao Gao,Liujing Wei,Dongzhi Wei,Wei Wang
β-Carotene is an indispensable additive in beverage, cosmetic, feed, and pharmaceutical production. The fermentation industry annually generates abundant waste mycelia from Trichoderma reesei (T. reesei), a pivotal industrial strain for cellulase and heterologous protein production. In this study, we constructed a T. reesei cell factory for β-carotene production for the first time. Four key enzymes, CarRP, CarB, GGS1/CrtE, and HMG1, were overexpressed in T. reesei. The concentrations of medium components, including tryptone and glucose, were optimized. The modified strain accumulated β-carotene at a titer of 218.8 mg/L in flask culture. We achieved cellulase production (FPase, 22.33 IU/mL) with the concomitant production of β-carotene (286.63 mg/L) from T. reesei in a jar. Overall, this study offers a novel and unique approach to address the costly waste mycelium management process using T. reesei industrial strains that simultaneously produce proteins and carotenoids.
研究领域:
生物技术
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微生物学
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发酵工程
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代谢工程
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PubMed
高影响因子
Frontiers in Bioengineering and Biotechnology
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2020
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IF 3.644
Han Lijuan,Tan Yinshuang,Ma Wei,Niu Kangle,Hou Shaoli,Guo Wei,Liu Yucui,Fang Xu
In Trichoderma reesei, carbon catabolite repression (CCR) significantly downregulates the transcription of cellulolytic enzymes, which is usually mediated by the zinc finger protein Cre1. It was found that there is a conserved region at the C-terminus of Cre1/CreA in several cellulase-producing fungi that contains up to three continuous S/T phosphorylation sites. Here, S387, S388, T389, and T390 at the C-terminus of Cre1 in T. reesei were mutated to valine for mimicking an unphosphorylated state, thereby generating the transformants Tr_Cre1S387V, Tr_Cre1S388V, Tr_Cre1T389V, and Tr_Cre1T390V, respectively. Transcription of cel7a in Tr_ Cre1S388V was markedly higher than that of the parent strain when grown in glucose-containing media. Under these conditions, both filter paperase (FPase) and p-nitrophenyl-β-D-cellobioside (pNPCase) activities, as well as soluble proteins from Tr_Cre1S388V were significantly increased by up to 2- to 3-fold compared with that of other transformants and the parent strain. The results suggested that S388 is critical site of phosphorylation for triggering CCR at the terminus of Cre1. To our knowledge, this is the first report demonstrating an improvement of cellulase production in T. reesei under CCR by mimicking dephosphorylation at the C-terminus of Cre1. Taken together, we developed a precision engineering strategy based on the modification of phosphorylation sites of Cre1 transcription factor to enhance the production of cellulase in T. reesei under CCR.
研究领域:
分子生物学
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生物技术
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微生物学
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PubMed
高影响因子
BIORESOURCE TECHNOLOGY
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2018
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IF 5.807
Jian Du,Xiu Zhang,Xuezhi Li,Jian Zhao,Guodong Liu,Baoyu Gao,Yinbo Qu
Commercial cellulase preparations for lignocellulose bioconversion are mainly produced by the fungus Trichoderma reesei . The maximum cellulose conversion of T. reesei cellulase mixture was 15%–20% higher than that of Penicillium oxalicum in the hydrolysis of corncob residue and Avicel. Nevertheless, both preparations hydrolyzed more than 92% of cellulose in NaOH-mercerized Avicel. When added to Avicel hydrolysis residue that was less reactive to P. oxalicum cellulases, cellobiohydrolase I (CBH I) from T. reesei resulted in a higher cellulose conversion than its homologous proteins from P. oxalicum and Aspergillus niger at the same protein loadings. Further domain exchange experiment attributed the high hydrolytic efficiency of T. reesei CBH I to its inter-domain linker and cellulose-binding domain. The results in part explained the superior performance of T. reesei cellulases on the degradation of native crystalline cellulose, and highlighted the important role of cellulose-binding region in determining the degree of hydrolysis by cellulases.
研究领域:
生物化学
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微生物学
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PubMed
高影响因子
Microbial Cell Factories
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2021
·Vol. 20
·期 1
·1-12
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IF 5.328
Jia Hexue,Sun Wan,Li Xuezhi,Zhao Jian
Background Trichoderma reesei is currently the main strain for the commercial production of cellulase. Cellulose induced protein 1 (Cip1) is one of the most abundant proteins in extracellular proteins of T. reesei. Reported literatures about Cip1 mainly focused on the regulation of Cip1 and its possible enzyme activities, but the effect of Cip1 on the enzymatic hydrolysis of lignocellulose and possible mechanism have not still been reported.Results In this study, Cip1 from T. reesei was cloned, expressed and purified, and its effects on enzymatic hydrolysis of several different pretreated lignocellulose were investigated. It was found that Cip1 could promote the enzymatic hydrolysis of pretreated lignocellulose, and the promoting effect was significantly better than that of bovine serum albumin (BSA). And especially for the lignocellulosic substrate with high lignin content such as liquid hot water pretreated corn stover and corncob residue, the promoting effect of Cip1 was even better than that of the commercial cellulase when adding equal amount protein. It was also showed that the metal ions Zn2+ and Cu2+ influenced the promoting effect on enzymatic hydrolysis. The Cip1 protein had no lyase activity, but it could destroy the crystal structure of cellulose and reduce the non-productive adsorption of cellulase on lignin, which partly interpreted the promoting effect of Cip1 on enzymatic hydrolysis of lignocellulose.Conclusion The Cip1 from T. reesei could significantly promote the enzymatic hydrolysis of pretreated lignocellulose, and the promotion of Cip1 was even higher than that of commercial cellulase in the enzymatic hydrolysis of the substrates with high lignin content. This study will help us to better optimize cellulase to improve its ability to degrade lignocellulose, thereby reducing the cost of enzymes required for enzymatic hydrolysis.
研究领域:
生物化学
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生物技术
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微生物学
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酶学
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DOI
PubMed
高影响因子
LANGMUIR
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2020
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IF 3.557
Zihuayuan Yang,Ying Qiao,Junying Li,Fu-Gen Wu,Fengming Lin
Antimicrobial photodynamic therapy (APDT) is a promising alternative to traditional antibiotics for the treatment of bacterial infections, which inactivates a broad spectrum of bacteria. However, many traditional photosensitizers (PSs) are hydrophobic with poor water solubility and easy aggregation. On the other hand, some light sources such as ultraviolet (UV) have poor penetration and high cytotoxicity. Both issues lead to undesired photodynamic therapy efficacy. To overcome these issues, we develop a novel water-soluble natural PS (sorbicillinoids) obtained by microbial fermentation using recombinant filamentous fungus Trichoderma reesei. Sorbicillinoids could effectively generate singlet oxygen (1O2) under UV light irradiation and ultimately display photoinactivation activity on Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteus but not Gram-negative ones such as Escherichia coli and Proteus vulgaris. Sorbicillinoids were found to enter S. aureus but not E. coli. S. aureus treated with sorbicillinoids and UV light displayed high levels of intracellular reactive oxygen species (ROS), notable DNA photocleavage, and compromised cell semipermeability without overt cell membrane disruption, none of which was found in the treated E. coli. All these contribute to the sorbicillinoid-based photoinactivation of Gram-positive bacteria. Moreover, the dark toxicity and phototoxicity on mammalian cells or hemolysis activity of sorbicillinoids is negligible, showing its excellent biocompatibility. This study expands the utilization of UV light for surface sterilization to disinfection in solution. Therefore, sorbicillinoids, a type of secondary metabolite from fungus, have a promising future as a new PS for APDT using a nontoxic dose of UV irradiation.
研究领域:
微生物学
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生物技术
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药理学
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光动力治疗
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PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
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IF 6.2
Xiaozan Dai,Guanchen Liu,Quan Zhang,Chao Zhu,Huipeng Gao,Youduo Wu,Chi Cheng,Hailong Lin,Chuang Xue
The fungus Trichoderma reesei is widely employed for industrial enzyme production. However, the mechanisms coordinating enzyme production with amino acid biosynthesis and metabolism remain incompletely understood. Here, we characterized a novel C2H2 zinc finger transcription factor, TrTRC-1, in T. reesei. The deletion of TrTRC-1 enhanced cellulase, xylanase, and extracellular protein production by 9.8–32.4% compared to that in T. reesei Rut C30, while simultaneously stimulating mycelial growth and conidiation. Transcriptomic analysis revealed that the deletion of TrTRC-1 significantly altered the expression of genes associated with primary metabolic pathways, amino acid biosynthesis/metabolism, and carbohydrate-active enzymes. In a 5 L bioreactor, T. reesei ΔTrTRC-1 achieved an FPase activity of 17.35 IU/mL and exhibited robust saccharification efficiency on lignocellulosic biomass. These findings demonstrate that TrTRC-1 serves as a multifunctional regulator in T. reesei, advancing our understanding of the regulatory network that balances enzyme production with cellular metabolism and providing a strategic basis for constructing microbial chassis strains with industrial application prospects.
研究领域:
微生物学
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分子生物学
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工业生物技术
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真菌遗传学
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代谢工程
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PubMed
高影响因子
危险品化学品经营许可证（带存储）