Replicon engineering-driven modular synthetic biology toolkit for efficiently iterative genome editing in Fusarium venenatum.

Fusarium venenatum is the most widely used filamentous fungus for biomass-based microbial protein production mainly used as meat analogs. However, insufficient iterative genome editing and lack of synthetic biology tools for fine-tuning gene expression limited engineering of F. venenatum for improved traits. Here, replicon engineering-based modular synthetic biology toolkit in F. venenatum was established. Specifically, stable and curable mitochondrial DNA-derived replicons were firstly identified and characterized. The curable replicon MT07 enabled CRISPR/Cas9 genome editing system with efficient plasmid curing, shortening curing time from 22 to 8 days. Based on genome editing system, six neutral genomic integration sites were identified for gene integration expression. In contrast, the stable replicon MT071 enabled stable episomal expression system construction, facilitating the characterization of modular transcriptional and translational regulatory elements, including 21 promoters, 8 terminators, 9 Kozak sequences, and 2A peptide-mediated polycistronic expression modules, yielding 662.9-fold gene expression dynamic range. As a proof of concept, this toolkit was applied to engineering the chassis cell and construct 3-hydroxy-3-methylbutyrate (HMB) biosynthetic pathway, enabling efficient co-production of microbial protein and HMB, with titers reaching 36.2 g/L and 6.2 g/L, respectively. Overall, this work establishes a replicon engineering-driven synthetic biology platform to engineer filamentous fungi for sustainable food biomanufacturing.