共查询到20条相似文献，搜索用时 31 毫秒
1.
Objectives
To develop a versatile Trichoderma reesei (teleomorph Hypocrea jecorina) expression system for the high-purity production of heterologous proteins.Results
The versatile T. reesei expression system is based on xyn1 and xyn2 promoters, A824V transition in XYRI, and a bicomponent carbon source strategy. Red fluorescent protein gene rfp and alkaline endoglucanase EGV gene egv3 from Humicola insolens were used as reporter genes to test our versatile expression systemConclusions
The versatile T. reesei expression system can be applied to produce heterologous proteins with high purity and high yield.2.
Lili Wei Leixi Cao Yanyan Miao Shuju Wu Shumei Xu Ruisheng Wang Jun Du Aihua Liang Yuejun Fu 《Biotechnology letters》2017,39(8):1129-1139
3.
4.
Jinxiang Zhu Qiaoyun Zhu Ruiqing Gong Qin Xu Menghao Cai Tianyi Jiang Xiangshan Zhou Mian Zhou Yuanxing Zhang 《Biotechnology letters》2018,40(9-10):1365-1376
Objective
Around one-fourth of the Komagataella phaffii genes encode hypothetical proteins with unknown functions. However, lack of powerful tools for genetic screening in K. phaffii significantly limits the functional analysis of these unknown genes. Transposon mutagenesis has been utilized as an insertional mutagenesis tool in many other organisms and would be extremely valuable if it could be applied in K. phaffii.Results
In this study, we investigated in K. phaffii the transposition activity and efficiency of piggyBac (PB) transposon, a DNA transposon from the cabbage looper moth Trichoplusia ni through the integrated-plasmid system. We also designed a binary-plasmid system which could generate stable mutants. Finally we evaluated the quality of this mutagenesis system by a simple screening for functional genes involved in K. phaffii carbon catabolite repression.Conclusions
Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.5.
Takuya Katayama Yuki Tanaka Tomoya Okabe Hidetoshi Nakamura Wataru Fujii Katsuhiko Kitamoto Jun-ichi Maruyama 《Biotechnology letters》2016,38(4):637-642
Objectives
To develop a genome editing method using the CRISPR/Cas9 system in Aspergillus oryzae, the industrial filamentous fungus used in Japanese traditional fermentation and for the production of enzymes and heterologous proteins.Results
To develop the CRISPR/Cas9 system as a genome editing technique for A. oryzae, we constructed plasmids expressing the gene encoding Cas9 nuclease and single guide RNAs for the mutagenesis of target genes. We introduced these into an A. oryzae strain and obtained transformants containing mutations within each target gene that exhibited expected phenotypes. The mutational rates ranged from 10 to 20 %, and 1 bp deletions or insertions were the most commonly induced mutations.Conclusions
We developed a functional and versatile genome editing method using the CRISPR/Cas9 system in A. oryzae. This technique will contribute to the use of efficient targeted mutagenesis in many A. oryzae industrial strains.6.
Ningning Sun Yuanchao Qian Weiwei Wang Yaohua Zhong Meixue Dai 《Biotechnology letters》2018,40(7):1119-1126
Objective
Improve the hydrolysis efficiency of the Trichoderma reesei cellulase system by heterologously expressing cellobiohydrolase Cel7A (Te-Cel7A) from the thermophilic fungus Talaromyces emersonii.Results
Te-Cel7A was expressed in T. reesei under control of the cdna1 promoter and the generated transformant QTC14 could successfully secrete Te-Cel7A into the supernatant using glucose as carbon source. The recombinant Te-Cel7A had a temperature optimum at 65 °C and an optimal pH of 5, which were similar to those from the native host. The culture supernatant of QTC14 exhibited a 28.8% enhancement in cellobiohydrolase activity and a 65.2% increase in filter paper activity relative to that of the parental strain QP4. Moreover, the QTC14 cellulase system showed higher thermal stability than that of the parental strain QP4. In the saccharification of delignified corncob residue, the cellulose conversion of QTC14 showed 13.9% higher than that of QP4 at the end of reaction.Conclusions
The thermophilic fungus-derived cellulases could be efficiently expressed by T. reesei and the recombinant cellulases had potential applications for biomass conversion.7.
Sandra Castillo Dorothee Barth Mikko Arvas Tiina M. Pakula Esa Pitkänen Peter Blomberg Tuulikki Seppanen-Laakso Heli Nygren Dhinakaran Sivasiddarthan Merja Penttilä Merja Oja 《Biotechnology for biofuels》2016,9(1):252
Background
Trichoderma reesei is one of the main sources of biomass-hydrolyzing enzymes for the biotechnology industry. There is a need for improving its enzyme production efficiency. The use of metabolic modeling for the simulation and prediction of this organism’s metabolism is potentially a valuable tool for improving its capabilities. An accurate metabolic model is needed to perform metabolic modeling analysis.Results
A whole-genome metabolic model of T. reesei has been reconstructed together with metabolic models of 55 related species using the metabolic model reconstruction algorithm CoReCo. The previously published CoReCo method has been improved to obtain better quality models. The main improvements are the creation of a unified database of reactions and compounds and the use of reaction directions as constraints in the gap-filling step of the algorithm. In addition, the biomass composition of T. reesei has been measured experimentally to build and include a specific biomass equation in the model.Conclusions
The improvements presented in this work on the CoReCo pipeline for metabolic model reconstruction resulted in higher-quality metabolic models compared with previous versions. A metabolic model of T. reesei has been created and is publicly available in the BIOMODELS database. The model contains a biomass equation, reaction boundaries and uptake/export reactions which make it ready for simulation. To validate the model, we dem1onstrate that the model is able to predict biomass production accurately and no stoichiometrically infeasible yields are detected. The new T. reesei model is ready to be used for simulations of protein production processes.8.
Alexey А. Moskalev Anna V. Kudryavtseva Alexander S. Graphodatsky Violetta R. Beklemisheva Natalya A. Serdyukova Konstantin V. Krutovsky Ivan V. Kulakovskiy Andrey S. Lando Artem S. Kasianov Dmitry A. Kuzmin Yuliya A. Putintseva Sergey I. Feranchuk Mikhail V. Shaposhnikov Vadim E. Fraifeld Dmitri Toren Anastasia V. Snezhkina Vasily V. Sitnik 《BMC evolutionary biology》2017,17(2):258
9.
Seyed Omid Ranaei Siadat Hamidreza Mollasalehi Narjes Heydarzadeh 《Biotechnology letters》2016,38(3):483-488
Objectives
To modify two main N-glycosylation residues of cellobiohydrolase I from Trichoderma reesei by site-directed mutagenesis for decreasing the extent of glycosylation and exploring possible effects on its properties.Results
Asparagine 45 and 64 residues were mutated to alanine to make single/double mutants and expressed in P. pastoris. Decreasing N-glycosylation of the recombinant CBH I resulted in an increased affinity of the enzyme for carboxymethylcellulose and also improved the Kcat/Km while the specific activity was decreased. Also, the enzymes were stable up to 80 °C. There was no significant change of the optimum pH and temperature by decrease of glycosylation in the mutated enzymes in comparison to the wild-type at constant incubation time of assay.Conclusion
Post-translational glycan-modification of CBH I in P. pastoris has different impacts on the properties of the secreted enzymes. Substrate affinity and catalytic efficiency were improved significantly while the activity and high temperature stability were negatively affected.10.
Yingtong Zhang Haiqin Chen Eusebio Navarro Sergio López-García Yong Q. Chen Hao Zhang Wei Chen Victoriano Garre 《Biotechnology letters》2017,39(3):439-446
Objectives
To generate lycopene-overproducing strains of the fungus Mucor circinelloides with interest for industrial production and to gain insight into the catalytic mechanism of lycopene cyclase and regulatory process during lycopene overaccumulation.Results
Three lycopene-overproducing mutants were generated by classic mutagenesis techniques from a β-carotene-overproducing strain. They carried distinct mutations in the carRP gene encoding lycopene cyclase that produced loss of enzymatic activity to different extents. In one mutant (MU616), the lycopene cyclase was completely destroyed, and a 43.8% (1.1 mg/g dry mass) increase in lycopene production was observed in comparison to that by the previously existing lycopene overproducer. In addition, feedback regulation of the end product was suggested in lycopene-overproducing strains.Conclusions
A lycopene-overaccumulating strain of the fungus M. circinelloides was generated that could be an alternative for the industrial production of lycopene. Vital catalytic residues for lycopene cyclase activity and the potential mechanism of lycopene formation and accumulation were identified.11.
Objectives
To improve production of lipids and carotenoids by the oleaginous yeast Rhodosporidium toruloides by screening mutant strains.Results
Upon physical mutagenesis of the haploid strain R. toruloides np11 with an atmospheric and room temperature plasma method followed by chemical mutagenesis with nitrosoguanidine, a mutant strain, R. toruloides XR-2, formed dark-red colonies on a screening plate. When cultivated in nitrogen-limited media, XR-2 cells grew slower but accumulated 0.23 g lipids/g cell dry wt and 0.75 mg carotenoids/g CDW. To improve its production capacity, different amino acids and vitamins were supplemented. p-Aminobenzoic acid and tryptophan had beneficial effects on cell growth. When cultivated in nitrogen-limited media in the presence of selected vitamins, XR-2 accumulated 0.41 g lipids/g CDW and 0.69 mg carotenoids/g CDW.Conclusions
A mutant R. toruloides strain with improved production profiles for lipids and carotenoids was obtained, indicating its potential to use combined mutagenesis for a more productive phenotype.12.
13.
14.
15.
Objectives
To improve the stability and sweetness of the sweet-tasting protein, monellin, by using site-directed mutagenesis and a Pichia pastoris expression system with a GAPDH constitutive promoter.Results
Both wild-type and E2 N mutant of single-chain monellin gene were cloned into the PGAPZαA vector and expressed in Pichia pastoris. The majority of the secreted recombinant protein, at 0.15 g/l supernatant, was monellin. This was purified by Sephadex G50 chromatography. The sweetness threshold of wild-type and E2 N were 30 μg/ml and 20 μg/ml, respectively. Compared with the proteins expressed in Escherichia coli, the thermostability of both proteins was improved. The N-terminal sequence is determinative for the sweetness of the proteins expressed in yeast strains.Conclusions
Site-directed mutagenesis, modification of the N-terminus of monellin, and without the need of methanol induction in P. pastoris expression system, indicate the possibility for large-scale production of this sweet-tasting protein.16.
Edwin Barrios-Villa Gerardo Cortés-Cortés Patricia Lozano-Zaraín Margarita María de la Paz Arenas-Hernández Claudia Fabiola Martínez de la Peña Ygnacio Martínez-Laguna Carmen Torres Rosa del Carmen Rocha-Gracia 《Annals of clinical microbiology and antimicrobials》2018,17(1):42
Background
The widespread Escherichia coli clone ST131 implicated in multidrug-resistant infections has been recently reported, the majority belonging to O25:H4 serotype and classified into five main virotypes in accordance with the virulence genes carried.Methods
Pathogenicity Islands I and II (PAI-I and PAI-II) were determined using conventional PCR protocols from a set of four E. coli CTXR ST131 O25:H4/H30-Rx strains collected from healthy donors’ stool. The virulence genes patterns were also analyzed and compared them with the virotypes reported previously; then adherence, invasion, macrophage survival and biofilm formation assays were evaluated and AIEC pathotype genetic determinants were investigated.Findings
Non-reported virulence patterns were found in our isolates, two of them carried satA, papA, papGII genes and the two-remaining isolates carried cnfI, iroN, satA, papA, papGII genes, and none of them belonged to classical ST131 virotypes, suggesting an endemic distribution of virulence genes and two new virotypes. The presence of PAI-I and PAI-II of Uropathogenic E. coli was determined in three of the four strains, furthermore adherence and invasion assays demonstrated higher degrees of attachment/invasion compared with the control strains. We also amplified intI1, insA and insB genes in all four samples.Interpretation
The results indicate that these strains own non-reported virotypes suggesting endemic distribution of virulence genes, our four strains also belong to an AIEC pathotype, being this the first report of AIEC in México and the association of AIEC with healthy donors.17.
18.
Objectives
To investigate single nucleotide polymorphism (SNP) in the transformation process of phytosterol to valuable steroid intermediates in three steroid-producing Mycobacterium neoaurum strains using deep sequencing and bioinformation analysis.Results
The assembled contig sequences from RNA sequencing of strains producing 9α-hydroxy-4-androstene-3,17-dione (9OHAD), 1,4-androstadiene-3,17-dione (ADD), and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (1,4-BNA) were analyzed for the presence of putative SNPs for steroid catabolism. 413, 375, and 491 SNPs were detected in the coding domain sequences and non-coding domain sequences of RNA sequencing reads of M. neoaurum strains producing 9OHAD, ADD, and BNA, respectively. Special attention was focused on SNPs associated with genes showing differential expression at proteome level, including the genes for sterol catabolism, glycerol catabolic process, signal transduction systems, transport system and energy metabolism.Conclusions
The work facilitates the understanding of underlying genetic changes that may be responsible for steroid accumulation in M. neoaurum and is useful for its targeted genetic engineering.19.