Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeastCandida intermedia

Abstract

Genome editing toolboxes are essential for the exploration and exploitation of non-conventional yeast species as cell factories, as they facilitate both genome studies and metabolic engineering. The non-conventional 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 1,000 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 non-homologous 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.

Regulation of lactose and galactose growth: Insights from a unique metabolic gene cluster inCandida intermedia

“…To decipher the importance of the three clusters for (ga)lactose metabolism in C. intermedia , we deleted the clusters one by one using the split-marker technique previously developed for this yeast 39 . The cluster deletion mutants ( lacΔ , galΔ and gallacΔ ) grew almost as well as the wild-type strain (WT) in minimal media containing glucose (Figure 3A).…”

“…C. intermedia CBS 141442 was grown in YPD medium (1% yeast extract, 2% bactopeptone and 2% glucose) prior to yeast transformation using the split marker technique as described previously 39 . Using this technique, deletion cassettes were constructed as two partially overlapping fragments, each containing half of the selection marker fused to either upstream or downstream sequences of the target gene.…”

“…C. intermedia is one of very few yeasts in the Metschnikowia family that can grow on lactose 1 , and it has been used for cheese whey bioremediation in the past 36 . Our previous works on characterizing the in-house isolated C. intermedia strain CBS 141442 in terms of genomics, transcriptomics and physiology 33,37,38 and the development of a genome editing toolbox for this species 39 provide a stable platform for exploration of the genetic determinants of lactose metabolism in this yeast.…”

Improving homology‐directed repair by small molecule agents for genetic engineering in unconventional yeast?—Learning from the engineering of mammalian systems

“…HU has successfully been used to increase homology‐directed gene integration in yeast: The HR efficiency was increased in a species‐dependent manner by 1.2‐ to 8‐fold in Arxula adeninivorans, S. cerevisiae, Kluyveromyces lactis , and Pichia pastoris even when using short homology arms (~50 bp). In Candida intermedia integration rates were boosted from 1% to 70% using a split‐marker genome editing approach (Peri et al., 2023 ). In Y. lipolytica KI rates at 15 loci showed a large range of enhancements, from 4 to 96% for different loci (Tsakraklides et al., 2015 ).…”