Promoter Engineering in Yeast Cell Factories: Strategies, Applications, and Perspectives

Abstract Yeasts serve as premier eukaryotic hosts for microbial cell factories, enabling the production of recombinant proteins, biofuels, and high-value natural products. Precise transcriptional control is paramount for balancing complex metabolic pathways and maximizing target product yields. Promoters, as the key regulatory elements governing transcription initiation, have thus become a focal point of metabolic and synthetic biology engineering. This review surveys promoter architecture, function, and engineering in yeast. It outlines promoter structure, from core elements to upstream regulatory sequences, and reviews current and emerging strategies for discovery, optimization, and de novo design, including random mutagenesis, combinatorial assembly, rational design based on transcription factor binding sites, intron-mediated enhancement, cross-species approaches. Importantly, this review highlights the rapidly growing role of machine learning (ML) and artificial intelligence (AI) in promoter engineering, from predictive modeling of promoter strength to in silico generation of synthetic variants. Special emphasis is placed on synthetic promoter engineering to yield tunable and inducible toolkits. The review also highlights applications in yeast cell factories, such as metabolic pathway optimization, dynamic regulation, high-throughput strain screening, and recombinant protein production. By summarizing these recent advances, it serves as a practical resource for researchers aiming to achieve precise metabolic control and improve bioproduction outcomes in yeast.