Synthetic expression system enhances recombinant protein production in Aspergillus oryzae

For centuries, the koji mold Aspergillus oryzae has played a crucial role in East Asian food fermentations, producing culinary staples such as soy sauce, miso, and sake. More recently, its proficiency in secreting enzymes has positioned it as a cornerstone in industrial biotechnology. This makes A. oryzae uniquely suitable for the heterologous production of food proteins for use in novel meat and dairy alternatives. However, there is a critical lack of well-characterized strong constitutive promoters for A. oryzae. To address this, we established a synthetic expression system for A. oryzae. This modular system involves a synthetic transcription factor in one locus and a gene of interest under control of a core promoter and upstream activating sequence (UAS) in the other. These elements interact with the synthetic transcription factor, allowing precise control over gene expression. Using the synthetic expression system, we screened a curated library of thirteen core promoters, derived from A. oryzae genes with high expression levels. This revealed a wide dynamic range, enabling the finetuning of gene expression levels. The most potent core promoter, in conjunction with six repeats of the UAS, enabled a remarkable sixfold increase in mycelial fluorescent protein levels compared to the strong native alpha-amylase promoter (PamyB) under PamyB-inducing conditions, and was equally effective on minimal glucose media. To our knowledge, this makes it the strongest promoter for A. oryzae published to date.
Furthermore, by combining UASs with two core promoters oriented in opposing directions, we engineered synthetic bidirectional promoters. These substantially reduce the time it takes to optimize production strains by enabling multiplex gene integrations in a single transformation. Collectively, these results will contribute to the establishment of A. oryzae as a reliable platform for more efficient and sustainable recombinant protein production and help pave the way for novel precision fermentation processes.