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Deletion analysis of Pichia pastoris alcohol dehydrogenase 2 (ADH2) promoter and development of synthetic promoters.
Erden-Karaoglan, Fidan; Karaoglan, Mert; Yilmaz, Gürkan; Yilmaz, Semiramis; Inan, Mehmet.
Afiliación
  • Erden-Karaoglan F; Department of Food Engineering, Erzincan Binali Yildirim University, Erzincan, Turkey.
  • Karaoglan M; Department of Food Engineering, Akdeniz University, Antalya, Turkey.
  • Yilmaz G; Department of Food Engineering, Erzincan Binali Yildirim University, Erzincan, Turkey.
  • Yilmaz S; Department of Food Engineering, Akdeniz University, Antalya, Turkey.
  • Inan M; Department of Food Engineering, Akdeniz University, Antalya, Turkey.
Biotechnol J ; 17(2): e2100332, 2022 Feb.
Article en En | MEDLINE | ID: mdl-34870891
Pichia pastoris (Komagataella phaffii) is a non-conventional Crabtree-negative yeast with the capability of reaching very high cell densities in a fed-batch fermentation process. The alcohol dehydrogenase (ADH) genes of P. pastoris involved in ethanol metabolism were identified and were previously characterized. This work aimed to extend current knowledge of the regulation of the ADH2 promoter. To this end, we first determined the upstream activator (UAS) and repressor (URS) sequences of the promoter by deletion assays. Two upstream activator sites have been identified, positioned between -900 and -801 bp, and -284 and -108 bp upstream of the ADH2 transcription start site. The sequences positioned between -361 and -262 bp had a negative effect on the promoter activity and designated a repressor sequence (URS). We then demonstrated that Mxr1 (methanol expression regulator 1) transcription factor activates the ADH2 promoter through the direct interaction with UAS regions in response to ethanol. Furthermore, five different synthetic promoters were constructed by adding or deleting the regulatory sites. These synthetic promoters were tested for extracellular xylanase production at shake flask level by inducing with ethanol. These promoter variants improved the xylanase production ranging between 165% and 200% of the native promoter. The synthetic promoter 5 (SNT5) that displayed the highest activity was further evaluated at the fermenter scale. The modification in the promoter features might have several implications for industrial processes where decoupling the cell growth and product formation is advantageous.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Pichia / Alcohol Deshidrogenasa / Proteínas Fúngicas / Regiones Promotoras Genéticas Tipo de estudio: Prognostic_studies Idioma: En Revista: Biotechnol J Asunto de la revista: BIOTECNOLOGIA Año: 2022 Tipo del documento: Article País de afiliación: Turquía Pais de publicación: Alemania
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Pichia / Alcohol Deshidrogenasa / Proteínas Fúngicas / Regiones Promotoras Genéticas Tipo de estudio: Prognostic_studies Idioma: En Revista: Biotechnol J Asunto de la revista: BIOTECNOLOGIA Año: 2022 Tipo del documento: Article País de afiliación: Turquía Pais de publicación: Alemania