Systematic engineering of synthetic minimal promoters for tunable transgene expression in mammalian cells

Abstract

Fine-tuning transgene levels is crucial in fundamental and applied research. Native promoters are broadly utilized to regulate transgene expression. However, their large size and structural complexity hinder predictable tuning, constrain compatibility with viral vectors with limited capacity, and increase the risk of homologous recombination. Synthetic promoters are short de novo sequences, typically consisting of an enhancer region, which is positioned upstream of a minimal promoter that determines transcription initiation efficiency. Enhancer optimization typically yields coarse activation shifts, alters response curves, and requires case-by-case optimization. Synthetic minimal promoter architecture enables fine-tuning that is potentially compatible with any enhancer region. In this study, we systematically optimized minimal promoter sequences, core element composition, and regulatory motif sequences. Our findings support a modular model of minimal promoter function in which cis-regulatory elements from different minimal promoters can be mixed, altered, added, or deleted to yield predictable expression outcomes. We shortened and thereby enhanced the efficiency of known minimal promoters, engineered modular tunable minimal promoters, and generated candidate regulatory elements spanning 7–8 nucleotides that drive efficient transcription when combined with artificial ${5^{\prime}}$ untranslated regions (5'UTRs) that function as an additional regulatory layer. This framework broadens the utility of synthetic promoters for next-generation applications.