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dc.contributor.authorYang, Yankun
dc.contributor.authorZheng, Yating
dc.contributor.authorWang, Pengcheng
dc.contributor.authorLi, Xiang
dc.contributor.authorZhan, Chunjun
dc.contributor.authorLinhardt, Robert J.
dc.contributor.authorZhang, Fuming
dc.contributor.authorLiu, Xiuxia
dc.contributor.authorZhan, Jinling
dc.contributor.authorBai, Zhonghu
dc.date2020
dc.date.accessioned2022-06-27T15:40:49Z
dc.date.available2022-06-27T15:40:49Z
dc.date.issued2020-09-01
dc.identifier.citationCharacterization and application of a putative transcription factor SUT2 in Pichia pastoris, Y. Yang, Y. Zheng, P. Wang, X. Li, C. Zhan, X. Liu, C. Liu, Y. Li, Z. Bai, F. Zhang, R. J. Linhardt, Molecular Genetics and Genomics, 295, 1295–1304, 2020.
dc.identifier.issn16174623
dc.identifier.issn16174615
dc.identifier.urihttps://doi.org/10.1007/s00438-020-01697-3
dc.identifier.urihttps://hdl.handle.net/20.500.13015/5491
dc.descriptionMolecular Genetics and Genomics, 295, 1295–1304
dc.descriptionNote : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
dc.description.abstractPichia pastoris is able to metabolize methanol via a specific MUT (methanol utilization) pathway. Based on the powerful AOX1 (Alcohol Oxidase 1) promoter, the P. pastoris expression system has become one of the most widely used eukaryotic expression systems. The molecular mechanisms of methanol metabolic regulation remain unclearly understood, so it is important to identify and develop new transcriptional regulators. Our previous studies suggested that the expression of SUT2 could be induced by methanol but is repressed by glycerol, which indicates that SUT2 may be involved in methanol metabolism through an unknown mechanism. SUT2 encodes a putative transcription factor-like protein harboring a Gal4-like Zn2Cys6 DNA-binding domain in Pichia pastoris, and its homolog in Saccharomyces cerevisiae regulates sterol uptake and synthesis. This study shows that the overexpression of SUT2 promoted the expression of AOX1 and increases ergosterol content in cells. Furthermore, via truncation of the putative SUT2 promoter at diverse loci, the − 973 base pair (bp) to − 547 bp region to the ATG was shown to be the core element of the inducible promoter PSUT2, which strongly responds to the methanol signal. The transcriptional start site of SUT2, “A” at the 22nd bp upstream of ATG, was determined with 5′-rapid amplification of cDNA ends. A forward-loop cassette was constructed with MXR1 (Methanol Expression Regulator 1, a positive transcription factor of PAOX1) promoted by PSUT2, enabling moderate elevation in the expression level of Mxr1 and high activity of PAOX1 without damaging cellular robustness further boosting the production of heterologous proteins. The PAOX1-driven expression of enhanced green fluorescent protein in this novel system was improved by 18%, representing a promising method for extrinsic protein production. SUT2 may play roles in methanol metabolism by participating in sterol biosynthesis. PSUT2 was characterized as a novel inducible promoter in P. pastoris and a PSUT2-driven MXR1 forward-loop cassette was constructed to enhance the PAOX1 activity, laying a foundation for further development and application of P. pastoris expression system.
dc.description.sponsorshipNational Natural Science Foundation of China
dc.description.urihttps://login.libproxy.rpi.edu/login?url=https://doi.org/10.1007/s00438-020-01697-3
dc.languageen_US
dc.language.isoENG
dc.relation.ispartofThe Linhardt Research Labs Online Collection
dc.relation.ispartofRensselaer Polytechnic Institute, Troy, NY
dc.relation.ispartofMolecular Genetics and Genomics
dc.relation.urihttps://harc.rpi.edu/
dc.subjectBiology
dc.subjectChemistry and chemical biology
dc.subjectChemical and biological engineering
dc.subjectBiomedical engineering
dc.titleCharacterization and application of a putative transcription factor SUT2 in Pichia pastoris
dc.typeArticle
dcterms.accessRightshttps://login.libproxy.rpi.edu/login?url=https://doi.org/10.1007/s00438-020-01697-3
dcterms.isPartOfJournal
dcterms.isVersionOfhttps://doi.org/10.1007/s00438-020-01697-3
dc.rights.holderIn Copyright : this Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). https://rightsstatements.org/page/InC/1.0/
dc.creator.identifierhttps://orcid.org/0000-0003-2219-5833
dc.relation.departmentThe Linhardt Research Labs.
dc.relation.departmentThe Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
rpi.description.pages1295-1304
rpi.description.volume295


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