Deletion analysis of Pichia pastoris alcohol dehydrogenase 2 (ADH2) promoter and development of synthetic promoters

Abstract

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.

Transcription regulation strategies in methylotrophs: progress and challenges

“…Five expression models driven by different synthetic promoter P ADH2 variants formed a small library in which SNT5 variant optimized by 2.2-fold compared with the original expression element has been screened out. This research indicates strong variants are powerful alternatives to the most widely used promoters in P. pastoris (Erden-Karaoglan et al 2022 ). Garg accompanied with his colleagues optimized several heterologous metabolites producing pathways by examining the strength among a series of RBS variants and diverse promoters in M. buryatense 5GB1C, up to 70 mg/L of crotonic acid and 40 mg/L butyric acids, were obtained in engineered strain from methane (Garg et al 2018 b ).…”

Advances in Metabolic Engineering of Pichia pastoris Strains as Powerful Cell Factories

“…Among these, the promoter P ADH2 is naturally present in P. pastoris to regulate the expression of the alcohol dehydrogenase gene ADH2, which is involved in the conversion of ethanol to formaldehyde. This promoter can be activated via direct interaction with the transcription factor Mxr1 [51]. By replacing the repressor region of P ADH2 with the activator region, the SNT5 promoter is obtained, which is much stronger than P ADH2 [51].…”

“…This promoter can be activated via direct interaction with the transcription factor Mxr1 [51]. By replacing the repressor region of P ADH2 with the activator region, the SNT5 promoter is obtained, which is much stronger than P ADH2 [51]. P. pastoris can use L-rhamnose as a sole carbon source, and its metabolism is heavily dependent on L-rhamnonate dehydratase and L-2-keto-3-deoxyrhamnonate aldolase encoded by LRA3 and LRA4, respectively [20].…”

Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

“…Among five promoter variants, synthetic promoter 5 (SNT5) indicated the highest promoter activitythat is, 2.2-fold higher than the wild-type, and is suggested as a powerful alternative to P. pastoris promoters. 64 Karaoglan et al 65 compared alternative promoter pADH3 to pAOX1 and pGAP promoters. Xylanase gene (XylB) from A. niger was heterologously expressed in P. pastoris under three different promoters ( pGAP, pADH3, and pAOX1), tested in a shake flask and fed-batch (5 L) fermentation process with a carbon source of glucose, ethanol, and methanol, respectively.…”