Patterns of spontaneous and induced genomic alterations in Yarrowia lipolytica

Abstract

This study explored the genomic alterations in Yarrowia lipolytica, a key yeast in industrial biotechnology, under both spontaneous and mutagen-induced conditions. Our findings reveal that spontaneous mutations occur at a rate of approximately 4 × 10^-10 events per base pair per cell division, primarily manifesting as single-nucleotide variations (SNVs) and small insertions and deletions (InDels). Notably, C-to-T/G-to-A transitions and C-to-A/G-to-T transversions dominate the spontaneous SNVs, while 1 bp deletions, likely resulting from template slippage, are the most frequent InDels. Furthermore, chromosomal aneuploidy and rearrangements occur, albeit at a lower frequency. Exposure to ultraviolet (UV) light, methylmethane sulfonate (MMS), and Zeocin significantly enhances the rates of SNVs and alters their mutational spectra in distinct patterns. Notably, Zeocin-induced SNVs are predominantly T-to-A and T-to-G substitutions, often occurring within the 5'-TGT^*-3' motif (^* denotes the mutated base). Additionally, Zeocin exhibits a higher potency in stimulating InDels compared to UV and MMS. Translesion DNA synthesis is implicated as the primary mechanism behind most Zeocin-induced SNVs and some InDels, whereas non-homologous end joining serves as the main pathway for Zeocin-mediated InDels. Intriguingly, the study identifies the gene YALI1_E21053g, encoding a protein kinase, as negatively associated with Zeocin resistance. Overall, our results not only deepened our knowledge about the genome evolution in Y. lipolytica but also provided reference to develop innovative strategies to harness its genetic potential.IMPORTANCEYarrowia lipolytica exhibits high environmental stress tolerance and lipid metabolism capabilities, making it a microorganism with significant industrial application potential. In this study, we investigated the genomic variation and evolutionary patterns of this yeast under both spontaneous and induced mutation conditions. Our results reveal distinctive mutation spectra induced by different mutagenic conditions and elucidate the underlying genetic mechanisms. We further highlight the roles of non-homologous end joining and translesion synthesis pathways in Zeocin-induced mutations, demonstrating that such treatments can rapidly confer drug resistance to the cells. Overall, our research enhances the understanding of how yeast genomes evolve under various conditions and provides guidance for developing more effective mutagenesis and breeding techniques.

Keywords: Yarrowia lipolytica; Zeocin; drug resistance; genomic alterations; mutation spectrum.

Fig 1

Chromosomal rearrangements and aneuploidy event in subcultured isolates of Y. lipolytica. Red and blue lines indicate duplicated and deleted regions, respectively. The relative coverage (RC) values of 0, 1, and 2 correspond to 0, 1, and 2 copies of DNA. (A) Whole-chromosome duplication in isolate WY-1. (B) An interstitial duplication event in isolate WY-5. Terminal deletions in isolates WY-11 (C) and WY-15 (D). A terminal duplication (E) and two interstitial duplications (F) in the UV-treated isolate WU-2. A terminal duplication event in the MMS-treated isolate WM-1 (G) and an interstitial duplication in the Zeocin-treated isolate WZ-5 (H).

Fig 2

Distinct patterns of spontaneous and mutagen-induced SNVs. (A) Ratios of base substitutions at A, T, C, and G sites in Y. lipolytica isolates subcultured under spontaneous and mutagen-treated conditions. (B) Ratios of transition and transversion mutations. (C) Mutation rates of different classes of base substitutions. The data are shown as the means ± SDs. Numbers above the bars are absolute mutation rates. P values were determined using the Wilcoxon rank-sum test and are indicated with asterisks. *P < 0.05. ns, not significant.

Fig 3

DNA sequence context of signature mutations. Adjacent sequences of mutated bases under (A) spontaneous, (B) UV treatment, (C) MMS treatment, and (D) Zeocin treatment conditions. Single-nucleotide variants (SNVs) are positioned in the middle of the three-nucleotide sequence. P values were determined using the chi-squared test and are indicated with asterisks. *P < 0.01. Adjacent four-base sequences for prominent base substitutions are shown for (E) spontaneous, (F) UV treatment, (G) MMS treatment, and (H) Zeocin treatment conditions, generated using WebLogo 3.

Fig 4

The role of NHEJ and translesion synthesis (TLS) pathway mutations. (A) Zeocin-induced DNA lesions, primarily including abasic (AP) sites and DNA breaks. (B) Effects of deleting RAD30 (PPY3Δrad30), REV1 (PPY3Δrev1), REV3 (PPY3Δrev3), and KU70 (PPY3Δku70) on Zeocin resistance. (C) Comparison of SNV rates between the reference strain (PPF) and the TLS and NHEJ mutants. (D) Rates of InDels in the PPF strain compared to the TLS and NHEJ mutants.

Fig 5

Increased Zeocin resistance of subcultured isolates and underlying mechanisms in Y. lipolytica. (A) All WZ strains showed enhanced tolerance to Zeocin. Ten mutant strains (WZ1~WZ10) and the parental strain W29 were separately spotted on YPD and 800 µg/mL Zeocin-containing plates and then incubated at 30°C for 2 days. (B) Eight genes potentially associated with Zeocin tolerance. (C) Comparative growth of PPW and the gene deletion mutant on YPD and Zeocin plates. The deletion of gene YALI1_E21053g led to a higher Zeocin resistance.

Fig 6

Models of signature mutations of Y. lipolytica under various conditions. (A) C-to-T and G-to-T substitution under spontaneous conditions. (B) UV induced C-to-T was associated with cyclobutane pyrimidine dimers (CPDs) formation. (C) MMS-induced A–T was presumed to be caused by 3-methyladenine (N3-MeA). (D) Zeocin-induced T–A and T–G were dependent on AP site formation and translesion synthesis. (E) Two distinct mechanisms underlying Zeocin-induced 1 bp deletion.