Biotechnology Letters最新文献_第10页

Metabolic engineering of Saccharomyces cerevisiae for high-level production of (+)-ambrein from glucose. 利用代谢工程改造酿酒酵母，从葡萄糖中高水平生产 (+)-ambrein 蛋白。

IF 2 4区生物学

Biotechnology Letters Pub Date : 2024-08-01 Epub Date: 2024-06-17 DOI: 10.1007/s10529-024-03502-2

Chumin Lin, Xiaopeng Zhang, Zhongju Ji, Baolian Fan, Yaman Chen, Yuhong Wu, Yuhong Gan, Zhengping Li, Yi Shang, Lixin Duan, Feng Wang

{"title":"Metabolic engineering of Saccharomyces cerevisiae for high-level production of (+)-ambrein from glucose.","authors":"Chumin Lin, Xiaopeng Zhang, Zhongju Ji, Baolian Fan, Yaman Chen, Yuhong Wu, Yuhong Gan, Zhengping Li, Yi Shang, Lixin Duan, Feng Wang","doi":"10.1007/s10529-024-03502-2","DOIUrl":"10.1007/s10529-024-03502-2","url":null,"abstract":"(+)-Ambrein is the primary component of ambergris, a rare product found in sperm whales (Physeter microcephalus). Microbial production using sustainable resources is a promising way to replace animal extraction and chemical synthesis. We constructed an engineered yeast strain to produce (+)-ambrein de novo. Squalene is a substrate for the biosynthesis of (+)-ambrein. Firstly, strain LQ2, with a squalene yield of 384.4 mg/L was obtained by optimizing the mevalonate pathway. Then we engineered a method for the de novo production of (+)-ambrein using glucose as a carbon source by overexpressing codon-optimized tetraprenyl-β-curcumene cyclase (BmeTC) and its double mutant enzyme (BmeTCY167A/D373C), evaluating different promoters, knocking out GAL80, and fusing the protein with BmeTC and squalene synthase (AtSQS2). Nevertheless, the synthesis of (+)-ambrein is still limited, causing low catalytic activity in BmeTC. We carried out a protein surface amino acid modification of BmeTC. The dominant mutant BmeTCK6A/Q9E/N454A for the first step was obtained to improve its catalytic activity. The yield of (+)-ambrein increased from 35.2 to 59.0 mg/L in the shake flask and finally reached 457.4 mg/L in the 2 L fermenter, the highest titer currently available for yeast. Efficiently engineered strains and inexpensive fermentation conditions for the industrial production of (+)-ambrein. The metabolic engineering tools provide directions for optimizing the biosynthesis of other high-value triterpenes.","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":" ","pages":"615-626"},"PeriodicalIF":2.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141330356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Plant production of recombinant antigens containing the receptor binding domain (RBD) of two SARS-CoV-2 variants 用植物生产含有两种 SARS-CoV-2 变体受体结合域 (RBD) 的重组抗原

IF 2.7 4区生物学

Biotechnology Letters Pub Date : 2024-07-27 DOI: 10.1007/s10529-024-03517-9

Flavia Fagiani, Rachele Frigerio, Anna Maria Salzano, Andrea Scaloni, Carla Marusic, Marcello Donini

{"title":"Plant production of recombinant antigens containing the receptor binding domain (RBD) of two SARS-CoV-2 variants","authors":"Flavia Fagiani, Rachele Frigerio, Anna Maria Salzano, Andrea Scaloni, Carla Marusic, Marcello Donini","doi":"10.1007/s10529-024-03517-9","DOIUrl":"https://doi.org/10.1007/s10529-024-03517-9","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Objectives</h3>The aim of this work was to rapidly produce in plats two recombinant antigens (RBDw-Fc and RBDo-Fc) containing the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 variants Wuhan and Omicron as fusion proteins to the Fc portion of a murine IgG2a antibody constant region (Fc).<h3 data-test=\"abstract-sub-heading\">Results</h3>The two recombinant antigens were expressed in Nicotiana benthamiana plants, engineered to avoid the addition of N-linked plant-typical sugars, through vacuum agroinfiltration and showed comparable purification yields (about 35 mg/kg leaf fresh weight).<h3 data-test=\"abstract-sub-heading\">Conclusions</h3>Their Western blotting and Coomassie staining evidenced the occurrence of major in planta proteolysis in the region between the RBD and Fc, which was particularly evident in RBDw-Fc, the only antigen bearing the HRV 3C cysteine protease recognition site. The two RBD N-linked glycosylation sites showed very homogeneous profiles free from plant-typical sugars, with the most abundant glycoform represented by the complex sugar GlcNAc4Man3. Both antigens were specifically recognised in Western Blot analysis by the anti-SARS-CoV-2 human neutralizing monoclonal antibody J08-MUT and RBDw-Fc was successfully used in competitive ELISA experiments for binding to the angiotensin-converting enzyme 2 receptor to verify the neutralizing capacity of the serum from vaccinated patients. Both SARS-Cov-2 antigens fused to a murine Fc region were rapidly and functionally produced in plants with potential applications in diagnostics.","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"39 1","pages":""},"PeriodicalIF":2.7,"publicationDate":"2024-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141783584","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Enhanced hypoxanthine utilization for cAMP salvage synthesis efficiently by Arthrobacter sp. CCTCC 2013431 via xanthine oxidase inhibition 节杆菌 CCTCC 2013431 通过抑制黄嘌呤氧化酶提高利用次黄嘌呤合成 cAMP 的效率

IF 2.7 4区生物学

Biotechnology Letters Pub Date : 2024-07-27 DOI: 10.1007/s10529-024-03513-z

Baofeng Chen, Hai Tan, Chang Li, Linbo Li, Zhonghua Zhang, Zhigang Li

{"title":"Enhanced hypoxanthine utilization for cAMP salvage synthesis efficiently by Arthrobacter sp. CCTCC 2013431 via xanthine oxidase inhibition","authors":"Baofeng Chen, Hai Tan, Chang Li, Linbo Li, Zhonghua Zhang, Zhigang Li","doi":"10.1007/s10529-024-03513-z","DOIUrl":"https://doi.org/10.1007/s10529-024-03513-z","url":null,"abstract":"When hypoxanthine was utilized as the activator for the salvage pathway in cAMP synthesis, xanthine oxidase would generate in quantity leading to low hypoxanthine conversion ratios and cell viability. To enhance cAMP salvage synthesis, fermentations with citrate/luteolin and hypoxanthine coupling added were conducted in a 7 L bioreactor and then multiple physiological indicators of fermentation with luteolin addition were assayed. Due to hypoxanthine feeding, cAMP productivity reached 0.066 g/(L·h) with 43.5% higher than control, however, cAMP synthesis, cell growth and glucose uptake all ceased at 50 h which was shortened by 22 h in comparison to control. The addition of citrate resulted in the cessation of fermentation at 61 h, on the contrary, owing to luteolin addition, cAMP fermentation performance was enhanced significantly during the whole fermentation period (72 h) with higher hypoxanthine conversion ratios and cAMP contents when compared with citrate and only hypoxanthine added batches. Multiple physiological indicators revealed that luteolin inhibited xanthine oxidase activity reducing hypoxanthine decomposition and ROS generation. ATP/AMP, NADH/NAD+ and NADPH/NADP+ were significantly increased especially at the late phase. Moreover, HPRT, PUP expression contents and corresponding gene transcription levels were also elevated. Luteolin could inhibit xanthine oxidase activity and further decrease hypoxanthine decomposition and ROS generation leading to higher hypoxanthine conversion and less cell damage for cAMP salvage synthesis efficiently.","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"26 1","pages":""},"PeriodicalIF":2.7,"publicationDate":"2024-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141783585","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Establishment of a semi-continuous scale-down clone screening model for intensified perfusion culture 为强化灌注培养建立半连续缩减克隆筛选模型

IF 2.7 4区生物学

Biotechnology Letters Pub Date : 2024-07-27 DOI: 10.1007/s10529-024-03512-0

Tao Sun, Yu Zhang, Hengrui Liang, Wenjing Fang, Zichen Qian, Kee Wee Tan, Junjie Li, Xiang Zheng, Mingyue Fang, Hang Zhou, Weichang Zhou, Sam Zhang

{"title":"Establishment of a semi-continuous scale-down clone screening model for intensified perfusion culture","authors":"Tao Sun, Yu Zhang, Hengrui Liang, Wenjing Fang, Zichen Qian, Kee Wee Tan, Junjie Li, Xiang Zheng, Mingyue Fang, Hang Zhou, Weichang Zhou, Sam Zhang","doi":"10.1007/s10529-024-03512-0","DOIUrl":"https://doi.org/10.1007/s10529-024-03512-0","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Purpose</h3>Perfusion cultures have been extensively used in the biotechnology industry to achieve high yields of recombinant products, especially those with stability issue. The WuXiUP™ platform represents a novel intensified perfusion that can achieve ultra‐high productivity. This study describes a representative scale-down 24-deep well plate (24-DWP) cell culture model for intensified perfusion clone screening.<h3 data-test=\"abstract-sub-heading\">Methods</h3>Clonal cell lines were expanded and evaluated in 24-DWP semi-continuous culture. Cell were sampled and counted daily with the aid of an automated liquid handler and high-throughput cell counter. To mimic perfusion culture, 24-DWP plates were spun down and resuspended with fresh medium daily. Top clones were ranked based on growth profiles and productivities. The best performing clones were evaluated on bioreactors.<h3 data-test=\"abstract-sub-heading\">Results</h3>The selected clones achieved volumetric productivity (Pv) up to 5 g/L/day when expressing a monoclonal antibody, with the accumulative harvest Pv exceeding 60 g/L in a 21-day cell culture. Product quality attributes of clones cultured in 24-DWP were comparable with those from bioreactors. A high seeding strategy further shortened the clone screening timeline.<h3 data-test=\"abstract-sub-heading\">Conclusion</h3>In this study, a 24-DWP semi-continuous scale-down model was successfully developed to screen for cell lines suitable for intensified perfusion culture.","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"70 1","pages":""},"PeriodicalIF":2.7,"publicationDate":"2024-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141783586","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Native CRISPR-Cas-based programmable multiplex gene repression in Klebsiella variicola 变异克雷伯氏菌中基于 CRISPR-Cas 的原生可编程多重基因抑制

IF 2.7 4区生物学

Biotechnology Letters Pub Date : 2024-07-27 DOI: 10.1007/s10529-024-03516-w

Zhifeng Mo, Siying Lin, Ting Li, Guohui Yu, Yunhao Sun, Jianuan Zhou, Zeling Xu

{"title":"Native CRISPR-Cas-based programmable multiplex gene repression in Klebsiella variicola","authors":"Zhifeng Mo, Siying Lin, Ting Li, Guohui Yu, Yunhao Sun, Jianuan Zhou, Zeling Xu","doi":"10.1007/s10529-024-03516-w","DOIUrl":"https://doi.org/10.1007/s10529-024-03516-w","url":null,"abstract":"Klebsiella variicola is a Gram-negative bacterium that is frequently isolated from a wide variety of natural niches. It is a ubiquitous opportunistic pathogen that can cause diverse infections in plants, animals, and humans. It also has significant biotechnological potential. However, due to the lack of efficient genetic tools, the molecular basis contributing to the pathogenesis and beneficial activities of K. variicola remains poorly understood. In this study, we found and characterized a native type I-E CRISPR-Cas system in a recently isolated K. variicola strain KV-1. The system cannot cleave target DNA sequences due to the inactivation of the Cas3 nuclease by a transposable element but retains the activity of the crRNA-guided Cascade binding to the target DNA sequence. A targeting plasmid carrying a mini-CRISPR to encode a crRNA was designed and introduced into the KV-1 strain, which successfully repurposed the native type I-E CRISPR-Cas system to inhibit the expression of the target gene efficiently and specifically. Moreover, by creating a mini-CRISPR to encode multiple crRNAs, multiplex gene repression was achieved by providing a single targeting plasmid. This work provides the first native CRISPR-Cas-based tool for programmable multiplex gene repression in K. variicola, which will facilitate studying the pathogenic mechanism of K. variicola and enable metabolic engineering to produce valuable bioproducts.","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 1","pages":""},"PeriodicalIF":2.7,"publicationDate":"2024-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141786056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Redifferentiation of genetically modified dedifferentiated chondrocytes in a microcavitary hydrogel. 转基因再分化软骨细胞在微腔水凝胶中的再分化。

IF 2.7 4区生物学

Biotechnology Letters Pub Date : 2024-06-01 Epub Date: 2024-03-25 DOI: 10.1007/s10529-024-03475-2

Yongchang Yao, Ke Chen, Qian Pan, Hui Gao, Weixian Su, Shicong Zheng, Weiqiang Dong, Dongyang Qian

{"title":"Redifferentiation of genetically modified dedifferentiated chondrocytes in a microcavitary hydrogel.","authors":"Yongchang Yao, Ke Chen, Qian Pan, Hui Gao, Weixian Su, Shicong Zheng, Weiqiang Dong, Dongyang Qian","doi":"10.1007/s10529-024-03475-2","DOIUrl":"10.1007/s10529-024-03475-2","url":null,"abstract":"Objectives: We genetically modified dedifferentiated chondrocytes (DCs) using lentiviral vectors and adenoviral vectors encoding TGF-β3 (referred to as transgenic groups below) and encapsulated these DCs in the microcavitary hydrogel and investigated the combinational effect on redifferentiation of the genetically manipulated DCs.Results: The Cell Counting Kit-8 data indicated that both transgenic groups exhibited significantly higher cell viability in the first week but inferior cell viability in the subsequent timepoints compared with those of the control group. Real-time polymerase chain reaction and western blot analysis results demonstrated that both transgenic groups had a better effect on redifferentiation to some extent, as evidenced by higher expression levels of chondrogenic genes, suggesting the validity of combination with transgenic DCs and the microcavitary hydrogel on redifferentiation. Although transgenic DCs with adenoviral vectors presented a superior extent of redifferentiation, they also expressed greater levels of the hypertrophic gene type X collagen. It is still worth further exploring how to deliver TGF-β3 more efficiently and optimizing the appropriate parameters, including concentration and duration.Conclusions: The results demonstrated the better redifferentiation effect of DCs with the combinational use of transgenic TGF-β3 and a microcavitary alginate hydrogel and implied that DCs would be alternative seed cells for cartilage tissue engineering due to their easily achieved sufficient cell amounts through multiple passages and great potential to redifferentiate to produce cartilaginous extracellular matrix.","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":" ","pages":"483-495"},"PeriodicalIF":2.7,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140206322","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Accumulation of docosapentaenoic acid (n-3 DPA) in a novel isolate of the marine ichthyosporean Sphaeroforma arctica. 在海洋鱼类 Sphaeroforma arctica 的新分离物中积累二十二碳五烯酸（n-3 DPA）。

IF 2.7 4区生物学

Biotechnology Letters Pub Date : 2024-06-01 Epub Date: 2024-03-16 DOI: 10.1007/s10529-024-03472-5

Qiang Wilson Yan, Ying-Chun Liu, Christa Barrett, Kelly Haake, Daniel Seeler, Oliver May, Ross Zirkle

{"title":"Accumulation of docosapentaenoic acid (n-3 DPA) in a novel isolate of the marine ichthyosporean Sphaeroforma arctica.","authors":"Qiang Wilson Yan, Ying-Chun Liu, Christa Barrett, Kelly Haake, Daniel Seeler, Oliver May, Ross Zirkle","doi":"10.1007/s10529-024-03472-5","DOIUrl":"10.1007/s10529-024-03472-5","url":null,"abstract":"Objective: Currently, there is lack of a consistent and highly enriched source for docosapentaenoic acid (n-3 DPA, C22:5), and this work report the isolation of microorganism that naturally produces n-3 DPA.Results: In this work, we screened microorganisms in our culture collections with the goal to isolate a strain with high levels of n-3 DPA. We isolated a strain of Sphaeroforma arctica that produces up to 11% n-3 DPA in total fatty acid and has a high n-3 DPA to DHA/EPA ratio. The cell growth of the isolated strain was characterized using microscopy imaging and flow cytometer technologies to confirm the coenocytic pattern of cell divisions previously described in S. arctica. Our novel isolate of S. arctica grew more robustly and produced significantly more n-3 DPA compared to previously isolated and described strains indicating the uniqueness of the discovered strain.Conclusion: Overall, this work reports a first isolate n-3 DPA producing microorganism and establishes the foundation for future strain improvement and elucidation of the physiological function of this LC-PUFA for human nutrition and health.","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":" ","pages":"373-383"},"PeriodicalIF":2.7,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140139816","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A recombinant fungal photolyase autonomously enters human cell nuclei to fix UV-induced DNA lesions. 重组真菌光解酶可自主进入人类细胞核，修复紫外线诱导的 DNA 损伤。

IF 2.7 4区生物学

Biotechnology Letters Pub Date : 2024-06-01 Epub Date: 2024-03-25 DOI: 10.1007/s10529-024-03474-3

Yuting Bao, Weiguo Fang

{"title":"A recombinant fungal photolyase autonomously enters human cell nuclei to fix UV-induced DNA lesions.","authors":"Yuting Bao, Weiguo Fang","doi":"10.1007/s10529-024-03474-3","DOIUrl":"10.1007/s10529-024-03474-3","url":null,"abstract":"Solar ultraviolet radiations induced DNA damages in human skin cells with cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PPs) as the most frequent lesions. CPDs are repaired much slower than 6-4PPs by the nucleotide excision repair pathway, which are thus the major lesions that interfere with key cellular processes and give rise to gene mutations, possibly resulting in skin cancer. In prokaryotes and multicellular eukaryotes other than placental mammals, CPDs can be rapidly repaired by CPD photolyases in one simple enzymatic reaction using the energy of blue light. In this study, we aim to construct recombinant CPD photolyases that can autonomously enter human cell nuclei to fix UV-induced CPDs. A fly cell penetration peptide and a viral nucleus localization signal peptide were recombined with a fungal CPD photolyase to construct a recombinant protein. This engineered CPD photolyase autonomously crosses cytoplasm and nuclear membrane of human cell nuclei, which then efficiently photo-repairs UV-induced CPD lesions in the genomic DNA. This further protects the cells by increasing SOD activity, and decreasing cellular ROSs, malondialdehyde and apoptosis.","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":" ","pages":"459-467"},"PeriodicalIF":2.7,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140206320","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

ANCUT1, a novel thermoalkaline cutinase from Aspergillus nidulans and its application on hydroxycinnamic acids lipophilization. 来自裸曲霉的新型热碱性角叉菜酶 ANCUT1 及其在羟基肉桂酸脂化中的应用

IF 2.7 4区生物学

Biotechnology Letters Pub Date : 2024-06-01 Epub Date: 2024-02-28 DOI: 10.1007/s10529-024-03467-2

Carolina Peña-Montes, Eva Bermúdez-García, Denise Castro-Ochoa, Fernanda Vega-Pérez, Katia Esqueda-Domínguez, José Augusto Castro-Rodríguez, Augusto González-Canto, Laura Segoviano-Reyes, Arturo Navarro-Ocaña, Amelia Farrés

{"title":"ANCUT1, a novel thermoalkaline cutinase from Aspergillus nidulans and its application on hydroxycinnamic acids lipophilization.","authors":"Carolina Peña-Montes, Eva Bermúdez-García, Denise Castro-Ochoa, Fernanda Vega-Pérez, Katia Esqueda-Domínguez, José Augusto Castro-Rodríguez, Augusto González-Canto, Laura Segoviano-Reyes, Arturo Navarro-Ocaña, Amelia Farrés","doi":"10.1007/s10529-024-03467-2","DOIUrl":"10.1007/s10529-024-03467-2","url":null,"abstract":"One of the four cutinases encoded in the Aspergillus nidulans genome, ANCUT1, is described here. Culture conditions were evaluated, and it was found that this enzyme is produced only when cutin is present in the culture medium, unlike the previously described ANCUT2, with which it shares 62% amino acid identity. The differences between them include the fact that ANCUT1 is a smaller enzyme, with experimental molecular weight and pI values of 22 kDa and 6, respectively. It shows maximum activity at pH 9 and 60 °C under assayed conditions and retains more than 60% of activity after incubation for 1 h at 60 °C in a wide range of pH values (6-10) after incubations of 1 or 3 h. It has a higher activity towards medium-chain esters and can modify long-chain length hydroxylated fatty acids constituting cutin. Its substrate specificity properties allow the lipophilization of alkyl coumarates, valuable antioxidants and its thermoalkaline behavior, which competes favorably with other fungal cutinases, suggests it may be useful in many more applications.","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":" ","pages":"409-430"},"PeriodicalIF":2.7,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11055803/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139982262","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Recycling selectable markers via Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins. 通过 Cre/loxP 系统回收可选择标记，构建共同表达多种蛋白质的 Komagataella phaffii 菌株。

IF 2.7 4区生物学

Biotechnology Letters Pub Date : 2024-06-01 Epub Date: 2024-02-28 DOI: 10.1007/s10529-024-03466-3

Weixian Wang, Minghai Han, Guofei Zhu, Xiaohui Liu, Tianming Zhao, Xiaoyan Ma, Xun Gong, Cunbin Xu

{"title":"Recycling selectable markers via Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins.","authors":"Weixian Wang, Minghai Han, Guofei Zhu, Xiaohui Liu, Tianming Zhao, Xiaoyan Ma, Xun Gong, Cunbin Xu","doi":"10.1007/s10529-024-03466-3","DOIUrl":"10.1007/s10529-024-03466-3","url":null,"abstract":"Objective: A convenient strategy was developed to recycle selectable markers using Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins.Results: A plasmid in this strategy was generated from pPICZαA with integration of lox71-Sh ble-lox66. Firstly, the plasmid was inserted with one target protein gene and then transformed into K. phaffii KM71. Secondly, the auxiliary plasmid pPICZαA/cre/his4 containing CRE recombinase gene was further chromosomally inserted to Sh ble gene therein. Finally, methanol induction was conducted to produce CRE for Cre/loxP-mediated recombination, and consequently, the sequence between lox71 and lox66 was deleted, leading to recycling of ZeoR and His- markers. Then the resulted strain expressing the one target protein was used as the host to which another target protein gene could be inserted by the same procedures.Conclusions: With easy manipulation, the method was effective in recycling of the selectable markers, and consequently two protein genes were sequential integrated chromosomally and successfully co-expressed in the yeast.","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":" ","pages":"399-407"},"PeriodicalIF":2.7,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139982263","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0