Engineered promoters are key components that allow engineered expression of genes in the cell-factory design. Promoters having exceptional strength are attractive candidates for designing metabolic engineering strategies for tailoring de novo production strategies that require directed evolution methods by engineering with de novo synthetic biology tools. Engineered promoter variants (EPVs) of naturally occurring promoters (NOPs) can be designed using metabolic engineering strategies and synthetic biology tools if the genes encoding the activating transcription factors (TFs) exist in the genome and are expressed and synthesized at non-limiting concentrations within the cell. The hybrid-architectured EPV design method targets an essential and predetermined part of the general transcription machinery. That is cis-acting DNA site(s) in coordination with the trans-acting factor(s) that must bind for the regulated transcription machinery activation. The method needs genomic and functional information that can lead to the discovery of the master TF(s) and synthetic cis-acting DNA elements, enabling the engineering of binding of master regulator TF(s). The method aims to generate EPVs that combine the advantages of being an exceptional stronger EPV(s) than the NOPs and permit \"green-and-clean production\" on a non-toxic carbon source, such as ethanol or glucose. By introducing our recent work on the engineering of ADH2 hybrid-promoter architectures to enhance recombinant protein expression on ethanol, we provide the method and protocol for the design of ADH2 hybrid-promoter architectures that can be adapted to other promoters in different substrate utilization pathways in Pichia pastoris (syn. Komagataella phaffii), as well as in other yeasts.

Hybrid-architectured promoter design to deregulate expression in yeast under modulating power of carbon sources involves replacing native cis-acting DNA sequence(s) with de novo synthetic tools in coordination with master regulator transcription factor (TF) to alter crosstalk between signaling pathways, and consequently, transcriptionally rewire the expression. Hybrid-promoter architectures can be designed to mimic native promoter architectures in yeast\'s preferred carbon source utilization pathway. The method aims to generate engineered promoter variants (EPVs) that combine the advantages of being an exceptionally stronger EPV(s) than the naturally occurring promoters and permit \"green-and-clean\" production on a non-toxic carbon source. To implement the method, a predetermined essential part of the general transcription machinery is targeted. This targeting involves cis-acting DNA sequences to be replaced with synthetic cis-acting DNA sites in coordination with the targeted TF that must bind for transcription machinery activation. The method needs genomic and functional information that can lead to the discovery of the master TF(s) and synthetic cis-acting DNA elements, which enable the engineering of binding of master regulator TF(s). By introducing our recent work on the engineering of Pichia pastoris (syn. Komagataella phaffii) alcohol oxidase 1 (AOX1) hybrid-promoter architectures, we provide the method and protocol for the hybrid-architectured EPV design to deregulate expression in yeast. The method can be adapted to other promoters in different substrate utilization pathways in P. pastoris, as well as in other yeasts.