Thread by @IndraRoux

Happy to share my third PhD paper, published at @ACSSynBio! We developed a recombinase-based system to transfer large biosynthetic gene clusters into filamentous fungi heterologous hosts for #naturalproduct discovery. @YitHengChooi @SMS_UWA #Synbio (1/8)
doi.org/10.1021/acssyn…

We often find promising cryptic biosynthetic gene clusters (BGCs) in the genomes of fungal species that are hard to cultivate or genetically intractable. Heterologous expression in hosts such as Aspergillus nidulans is increasingly being used to access the encoded compounds.(2/8)

However, targeted integration of large BGCs remained challenging. Site-specific recombinases are well suited for the integration of large constructs but remained unadopted in filamentous fungi. So, we established a Cre/lox-based system for BGC integration in A. nidulans.🛠️🧬(3/8)

We built A. nidulans strains harbouring a landing pad at different loci, and as proof-of-concept we integrated in 1-step a 21 kb BGC fragment encoding a heterologous polyketide. We found conditions for highly efficient targeted integration! 💫🧬🍄(4/8)

We further evaluated the expression of the chromosomally integrated constructs by observing a fluorescent reporter and compound production. We also benchmarked them to episomal AMA1 based expression in A. nidulans, learning some things about AMA1 vectors on the way! (5/8)

The donor, helper and landing pad vectors are now available on @Addgene addgene.org/browse/article…. The vector set is built with parts known to work across diverse filamentous fungi, so should be portable or easily adaptable to other #filamentousfungi cell factories! #synbio (6/8)

I had a lot of fun working on this story, #recombinases are so robust! And I also enjoyed setting up methods to evaluate chromosomal and episomal expression, such as the analysis of fluorescent reporters in fungal spores by flow cytometry. (7/8)

I am grateful to my reviewers at @ACSSynBio. This story was such a great opportunity to grow in my writing and story-crafting style, and I was also really lucky to have the great guidance of @YitHengChooi ! So, a very special paper personally 😊💫 ! (8/8)

Here goes the link again: pubs.acs.org/doi/10.1021/ac… PS. Anyone else also having trouble with paper links not showing the preview image? The Twitter card validator does not seem to take the links.🤔