Design and construction of light-regulated gene transcription and protein translation systems in yeast P. Pastoris
Introduction: P. pastoris is a common host for effective biosynthesis of heterologous proteins as well as small molecules. Accurate regulation of gene transcription and protein synthesis is necessary to coordinate synthetic gene circuits and optimize cellular energy distribution. Traditional methano...
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Elsevier
2025-07-01
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| Series: | Journal of Advanced Research |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2090123224003308 |
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| author | Siyu Zhang Jiazhen Zhang Ru Lin Chaoyu Lu Bohao Fang Jiacheng Shi Tianyi Jiang Mian Zhou |
| author_facet | Siyu Zhang Jiazhen Zhang Ru Lin Chaoyu Lu Bohao Fang Jiacheng Shi Tianyi Jiang Mian Zhou |
| author_sort | Siyu Zhang |
| collection | DOAJ |
| description | Introduction: P. pastoris is a common host for effective biosynthesis of heterologous proteins as well as small molecules. Accurate regulation of gene transcription and protein synthesis is necessary to coordinate synthetic gene circuits and optimize cellular energy distribution. Traditional methanol or other inducible promoters, natural or engineered, have defects in either fermentation safety or expression capacity. The utilization of chemical inducers typically adds complexity to the product purification process, but there is no other well-controlled protein synthesis system than promoters yet. Objective: The study aimed to address the aforementioned challenges by constructing light-regulated gene transcription and protein translation systems with excellent expression capacity and light sensitivity. Methods: Trans-acting factors were designed by linking the N. crassa blue-light sensor WC-1 with the activation domain of endogenous transcription factors. Light inducible or repressive promoters were then constructed through chimeric design of cis-elements (light-responsive elements, LREs) and endogenous promoters. Various configurations of trans-acting factor/LRE pairs, along with different LRE positions and copy numbers were tested for optimal promoter performance. In addition to transcription, a light-repressive translation system was constructed through the “rare codon brake” design. Rare codons were deliberately utilized to serve as brakes during protein synthesis, which were switched on and off through the light-regulated changes in the expression of the corresponding pLRE-tRNA. Results: As demonstrated with GFP, the light-inducible promoter 4pLRE-cPAOX1 was 70 % stronger than the constitutive promoter PGAP, with L/D ratio = 77. The light-repressive promoter PGAP-pLRE was strictly suppressed by light, with expression capacity comparable with PGAP in darkness. As for the light-repressive translation system, the “triple brake” design successfully eliminated leakage and achieved light repression on protein synthesis without any impact on mRNA expression. Conclusion: The newly designed light-regulated transcription and translation systems offer innovative tools that optimize the application of P. pastoris in biotechnology and synthetic biology. |
| format | Article |
| id | doaj-art-68ae9a110f5f4439ab8071fee3fcaed8 |
| institution | OA Journals |
| issn | 2090-1232 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Elsevier |
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| series | Journal of Advanced Research |
| spelling | doaj-art-68ae9a110f5f4439ab8071fee3fcaed82025-08-20T02:36:46ZengElsevierJournal of Advanced Research2090-12322025-07-017321923010.1016/j.jare.2024.08.008Design and construction of light-regulated gene transcription and protein translation systems in yeast P. PastorisSiyu Zhang0Jiazhen Zhang1Ru Lin2Chaoyu Lu3Bohao Fang4Jiacheng Shi5Tianyi Jiang6Mian Zhou7State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, ChinaState Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, ChinaState Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, ChinaState Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, ChinaState Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, ChinaState Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, ChinaChina Innovation Center of Roche, Shanghai 201203, ChinaState Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China; Corresponding author.Introduction: P. pastoris is a common host for effective biosynthesis of heterologous proteins as well as small molecules. Accurate regulation of gene transcription and protein synthesis is necessary to coordinate synthetic gene circuits and optimize cellular energy distribution. Traditional methanol or other inducible promoters, natural or engineered, have defects in either fermentation safety or expression capacity. The utilization of chemical inducers typically adds complexity to the product purification process, but there is no other well-controlled protein synthesis system than promoters yet. Objective: The study aimed to address the aforementioned challenges by constructing light-regulated gene transcription and protein translation systems with excellent expression capacity and light sensitivity. Methods: Trans-acting factors were designed by linking the N. crassa blue-light sensor WC-1 with the activation domain of endogenous transcription factors. Light inducible or repressive promoters were then constructed through chimeric design of cis-elements (light-responsive elements, LREs) and endogenous promoters. Various configurations of trans-acting factor/LRE pairs, along with different LRE positions and copy numbers were tested for optimal promoter performance. In addition to transcription, a light-repressive translation system was constructed through the “rare codon brake” design. Rare codons were deliberately utilized to serve as brakes during protein synthesis, which were switched on and off through the light-regulated changes in the expression of the corresponding pLRE-tRNA. Results: As demonstrated with GFP, the light-inducible promoter 4pLRE-cPAOX1 was 70 % stronger than the constitutive promoter PGAP, with L/D ratio = 77. The light-repressive promoter PGAP-pLRE was strictly suppressed by light, with expression capacity comparable with PGAP in darkness. As for the light-repressive translation system, the “triple brake” design successfully eliminated leakage and achieved light repression on protein synthesis without any impact on mRNA expression. Conclusion: The newly designed light-regulated transcription and translation systems offer innovative tools that optimize the application of P. pastoris in biotechnology and synthetic biology.http://www.sciencedirect.com/science/article/pii/S2090123224003308Light regulationP. pastorisPromoterCodon brake |
| spellingShingle | Siyu Zhang Jiazhen Zhang Ru Lin Chaoyu Lu Bohao Fang Jiacheng Shi Tianyi Jiang Mian Zhou Design and construction of light-regulated gene transcription and protein translation systems in yeast P. Pastoris Journal of Advanced Research Light regulation P. pastoris Promoter Codon brake |
| title | Design and construction of light-regulated gene transcription and protein translation systems in yeast P. Pastoris |
| title_full | Design and construction of light-regulated gene transcription and protein translation systems in yeast P. Pastoris |
| title_fullStr | Design and construction of light-regulated gene transcription and protein translation systems in yeast P. Pastoris |
| title_full_unstemmed | Design and construction of light-regulated gene transcription and protein translation systems in yeast P. Pastoris |
| title_short | Design and construction of light-regulated gene transcription and protein translation systems in yeast P. Pastoris |
| title_sort | design and construction of light regulated gene transcription and protein translation systems in yeast p pastoris |
| topic | Light regulation P. pastoris Promoter Codon brake |
| url | http://www.sciencedirect.com/science/article/pii/S2090123224003308 |
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