Hybrid-architectured promoter design to engineer expression in yeast

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.