Rational design and characterization of enhanced alcohol-inducible synthetic promoters in <i>Pichia pastoris</i>

The C1 and C2 alcohols hold great promise as substrates for biomanufacturing due to their low cost and rich resources. <i>Pichia pastoris</i> is considered a preferred host for methanol and ethanol bioconversion due to its natural utilization of methanol and ethanol. However, the scarcity of strong and tightly regulated alcohol-inducible promoters limits its extended use. This study aimed to develop enhanced methanol- and ethanol-inducible promoters capable of improving gene expression in <i>P. pastoris</i>. Rational design strategies were employed to rewire the upstream regulatory sequence of the methanol-inducible P<i>_AOX1</i>, generating several high-strength methanol-inducible promoters with a stringent regulatory pattern. Eleven strong promoters were identified from 36 endogenous ethanol-inducible candidates recognized from transcriptome analysis. Core promoter regions, the crucial element influencing transcriptional strength, were also characterized. Five high-activity core promoters were then combined with four upstream regulatory sequences of high-strength promoters, resulting in four groups of synthetic promoters. Ultimately, the highly active methanol-inducible P<i>_A13</i> and ethanol-inducible P<i>₀₆₈₈</i> and P<i>_synIV-5</i> were selected for the expression of an α-amylase and yielded enzyme activity 1.6, 2.6, and 4.5 times higher as compared to that of P<i>_AOX1</i>. This work expands the genetic toolkit available for <i>P. pastoris</i>, providing more precise and efficient options for regulating gene expression. It benefits the use of <i>P. pastoris</i> as an efficient platform for the C1 and C2 alcohol-based biotransformation in industrial biotechnology.IMPORTANCE<i>P. pastoris</i> represents a preferred microbial host for the bio-utilization of C1 and C2 alcohols that are regarded as renewable carbon sources based on clean energy. However, lack of efficient and regulated expression tools highly limits the C1 and C2 alcohols based bioproduction. By exploring high-strength and strictly regulated alcohol-inducible promoters, this study expands the expression toolkit for <i>P. pastoris</i> on C1 and C2 alcohols. The newly developed methanol-inducible P<i>_A13</i> and ethanol-inducible P<i>_synIV-5</i> demonstrate significantly higher expression levels than the commercial P<i>_AOX1</i> system. The endogenous and synthetic promoter series established in this study provides new construction references and alternative tools for expression control in <i>P. pastoris</i> for C1 and C2 alcohols based biomanufacturing.