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bioRxiv · 10.1101/2021.08.20.457072

Cre/lox-mediated chromosomal integration of biosynthetic gene clusters for heterologous expression in Aspergillus nidulans

Roux, I.·Chooi, Y.-H.
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Abstract

Building strains of filamentous fungi for stable long-term heterologous expression of large biosynthetic pathways is limited by the low transformation efficiency or genetic stability of current methods. Here, we developed a system for targeted chromosomal integration of large biosynthetic gene clusters in Aspergillus nidulans based on site-specific recombinase-mediated cassette exchange. We built A. nidulans strains harboring a chromosomal landing pad for Cre/lox-mediated recombination and demonstrated efficient targeted integration of a 21 kb DNA fragment in a single step. We further evaluated the integration at two loci by analyzing the expression of a fluorescent reporter and the production of a heterologous polyketide metabolite. We compared chromosomal expression at those landing loci to episomal AMA1-based expression, which also shed light on uncharacterized aspects of episomal expression in filamentous fungi. This is the first demonstration of site-specific recombinase-mediated integration in filamentous fungi, setting the foundations for the further development of this tool.

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Roux, I., Chooi, Y.-H.. 2021-08-20. Cre/lox-mediated chromosomal integration of biosynthetic gene clusters for heterologous expression in Aspergillus nidulans. https://doi.org/10.1101/2021.08.20.457072

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