Engineering Aspergillus oryzae for the production of biosynthetic proteins

Abstract

This investigation aims to strategically engineer the biosynthesis of fungal natural products through heterologous expression. It includes modifications to the heterologous host to boost productivity; leveraging the product diversity of an individual enzyme via mutasynthesis and combining biosynthetic genes from multiple pathways in a single heterologous host to creat new compounds.

To improve tetraketide 25 titer in A. oryzae NSAR1 through squalestatin tetraketide synthase (SQTKS) expression, we co-expressed two hydrolase-encoding genes. The 7-day fermentation in DPY medium involved daily titer measurements. Although the hydrolases had a modest impact, the titer reached around 3.2 mg/L, marking a fourfold increase from the previous experiment.

We conducted protein-level engineering of the cis-ER domain in SQTKS, introducing 10 mutation groups via yeast recombination into the complete SQTKS. These mutations were designed based on isolated ER mutants of SQTKS, analyzed through a combination of computational modeling and experimental assays with various mimic substrates by previous co-worker. After expressing them in A. oryzae NSAR1, we screened for correct transformants, performed fermentation and chemical extraction, and utilized LCMS for compound detection. However, no new compounds were generated in these expression experiments.

We performed pathway-level engineering by integrating genome-mined genes from multiple biosynthetic gene clusters (BGCs) into A. oryzae NSAR1. We co-expressed genes from the four BGCs of sporogen AO1 27, hypoxylan A 73, eremoxylarin D 123 and PR-toxin 107. These four BGCs share a common core carbon skeleton, while possessing distinct tailoring enzymes. Twenty oxygenated aristolochene congeners were synthesized, and their structures were characterized, featuring notable compounds such as the natural product hypoxylan A 73 and an epimer of guignaderemophilane C 37. A novel fungal aromatase enzyme has been identified, which catalyses the production of phenols via oxidative demethylation.

We investigated fungal pathways for tetraketide multiforisins and islandic acid-related compounds. Heterologous expression experiments yielded high titers of these compounds and pathway intermediates, leading to the structure characterization of 14 isolated compounds, including multiforisin H 143h and I 143i. These results not only clarified the pathway but also laid the groundwork for the total biosynthesis of this metabolite class. Attempts to add genes from the islandic acid BGC into A. oryzae for synthesizing islandic acid 144 or Allantopyrone A 145 proved unsuccessful, as none of the transformants generated compounds beyond those already synthesized by the A. oryzae hosts.