{"title":"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。","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":null,"url":null,"abstract":"<p><strong>Objective: </strong>A convenient strategy was developed to recycle selectable markers using Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins.</p><p><strong>Results: </strong>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 Zeo<sup>R</sup> and His<sup>-</sup> 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.</p><p><strong>Conclusions: </strong>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.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":null,"pages":null},"PeriodicalIF":2.0000,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"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\":null,\"url\":null,\"abstract\":\"<p><strong>Objective: </strong>A convenient strategy was developed to recycle selectable markers using Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins.</p><p><strong>Results: </strong>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 Zeo<sup>R</sup> and His<sup>-</sup> 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.</p><p><strong>Conclusions: </strong>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.</p>\",\"PeriodicalId\":8929,\"journal\":{\"name\":\"Biotechnology Letters\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.0000,\"publicationDate\":\"2024-06-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biotechnology Letters\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1007/s10529-024-03466-3\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/2/28 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q3\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnology Letters","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1007/s10529-024-03466-3","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/2/28 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
目的利用Cre/loxP系统构建可循环选择性标记，构建共表达多种蛋白的Komagataella phaffii菌株：结果：该策略中的质粒由整合了lox71-Sh ble-lox66的pPICZαA产生。首先，质粒插入一个目的蛋白基因，然后转化到 K. phaffii KM71 中。其次，将含有 CRE 重组酶基因的辅助质粒 pPICZαA/cre/his4 进一步染色体插入其中的 Sh ble 基因。最后，通过甲醇诱导产生用于 Cre/loxP 介导重组的 CRE，从而删除 lox71 和 lox66 之间的序列，实现 ZeoR 和 His- 标记的循环。然后，以表达一种目的蛋白的菌株为宿主，用同样的方法插入另一种目的蛋白基因：结论：该方法操作简便，能有效回收可选择标记，从而使两个蛋白质基因在染色体上依次整合，并成功地在酵母中共同表达。
Recycling selectable markers via Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins.
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.
期刊介绍：
Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them.
All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included.
Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields.
The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories.
Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.
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