Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeast Candida intermedia
Journal article, 2023

Genome-editing toolboxes are essential for the exploration and exploitation of nonconventional yeast species as cell factories, as they facilitate both genome studies and metabolic engineering. The nonconventional yeast Candida intermedia is a biotechnologically interesting species due to its capacity to convert a wide range of carbon sources, including xylose and lactose found in forestry and dairy industry waste and side-streams, into added-value products. However, possibilities of genetic manipulation have so far been limited due to lack of molecular tools for this species. We describe here the development of a genome editing method for C. intermedia, based on electroporation and gene deletion cassettes containing the Candida albicans NAT1 dominant selection marker flanked by 1000 base pair sequences homologous to the target loci. Linear deletion cassettes targeting the ADE2 gene originally resulted in <1% targeting efficiencies, suggesting that C. intermedia mainly uses nonhomologous end joining for integration of foreign DNA fragments. By developing a split-marker based deletion technique for C. intermedia, we successfully improved the homologous recombination rates, achieving targeting efficiencies up to 70%. For marker-less deletions, we also employed the split-marker cassette in combination with a recombinase system, which enabled the construction of double deletion mutants via marker recycling. Overall, the split-marker technique proved to be a quick and reliable method for generating gene deletions in C. intermedia, which opens the possibility to uncover and enhance its cell factory potential.

nonconventional yeast

nonhomologous end-joining

cell factory development

Author

Kameshwara Venkata Ramana Peri

Chalmers, Life Sciences, Industrial Biotechnology

Fábio Luis Da Silva Faria Oliveira

Chalmers, Biology and Biological Engineering

Adam Larsson

Chalmers, Life Sciences, Industrial Biotechnology

Alexander Plovie

Chalmers, Life Sciences, Industrial Biotechnology

Cecilia Geijer

Chalmers, Life Sciences, Industrial Biotechnology

FEMS Yeast Research

1567-1356 (ISSN) 1567-1364 (eISSN)

Vol. 23

Subject Categories

Biochemistry and Molecular Biology

DOI

10.1093/femsyr/foad016

PubMed

36893808

More information

Latest update

4/12/2023