Modular Yeast Engineering via Genomic Cassette Swapping for the Biomanufacturing of Plant Natural Products

Plants are a valuable source of secondary metabolites with pharmaceutical relevance, and the demand for these compounds is continuously increasing. Traditional production methods pose significant sustainability and environmental challenges. Synthetic biology offers promising alternative strategies for the sustainable production of these bioactive molecules using microbial cell factories. A key challenge in engineering these cell factories is the optimization of heterologous biosynthetic pathways through the identification and testing of the most suitable enzyme variants. Although many synthetic biology toolkits enable the genome integration of expression cassettes, they do not allow their modification or replacement after integration. This limitation is particularly relevant because enzymes from different plant species or even from distinct phylogenetic groups may catalyze the same chemical reaction, but it remains difficult to predict which variants will perform best in a heterologous host. To address this challenge, here a CRISPR-mediated system was developed to enable the rapid swapping or removal of previously integrated expression cassettes, introducing modularity at the strain level and simplifying iterative strain optimization. The system was developed as an extension of the previously established Easy-MISE toolkit and validated by exchanging fluorescent protein expression cassettes. This platform will facilitate the rapid evaluation of alternative enzyme variants, particularly for long biosynthetic pathways, accelerating enzyme selection and strain engineering.