Psychrophilic Yeast Strain, Plasmids and Media
The psychrophilic yeast used in this study was M. australis W7-5 isolated from the sea mud at Antarctica (Wei et al. 2021). The plasmids pGM simple-T fast and pLB-simple were used for amplification of the plasmids in E. coli DH5α. The knock-out plasmid Ma-NAT-loxp carrying nourseothricin resistance gene (NAT gene), the knock-in plasmid Ma-NATX13-loxP carrying NAT gene, and the plasmid Ma-HPTX13-CRE carrying hygromycin B resistance gene (HPT gene) and Cre recombinase gene, specific for genome editing in M. australis W7-5 were constructed in our previous studies (Wei et al. 2021). The YPD medium for cultivation of the psychrophilic yeast contained 10.0 g/l yeast extract, 20.0 g/l peptone, and 20.0 g/l glucose and the solid YPD containing different concentrations of sorbitol or Congo red were used to examine growth of the unsaturated fatty acid mutants and their wild type strain W7-5. The genes and their functions used in this study are shown in Supplementary file 1.
Isolation and Sequencing of the Genomic DNAs and Molecular Identification of the M. australis W7-5
The genomic DNAs of M. australis W7-5 were isolated and purified based on the methods described by Chi et al (2012). The purified DNAs were detected by the agarose gel electrophoresis and quantified by a Qubit® 2.0 Fluorometer (Thermo Scientific). The libraries for single-molecule real-time (SMRT) sequencing were constructed with an insert size of 20 kb using a SMRT bell TM Template kit (version 1.0). For the Illumina HiSeq sequencing, sequencing libraries were generated using a NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The whole genomic DNAs of M. australis W7-5 were sequenced using a PacBio Sequel platform and Illumina NovaSeq PE150 at the Beijing Novogene Bioinformatics Technology Co., Ltd, China. In order to ensure the accuracy of the subsequent analysis results, the low-quality reads were filtered (≤ 500 bp) to obtain clean data. The errors in the primary assembly were identified and corrected with a BLASR v5.1 (Chaisson and Tesler 2012). All the genes in the genome were predicted using an Augustus 3.2.1 and were functionally annotated using a BLAST2GO. The whole genome was online mined and analyzed using BLASTP. The whole-genome based phylogenetic tree of including M. australis W7-5 (GenBank: JAGSXI000000000) and other yeast strains was conducted through a composition vector (CV) approach on the CVTree3 website (http://tlife.fudan.edu.cn/cvtree/cvtree/).
Construction of the Disruption Vectors and Expression Vectors
The genomic DNA of M. australis strain W7-5 was prepared as described above. According to the sequenced genomic DNA (GenBank: JAGSXI000000000) of M. australis W7-5, the primers (Supplementary file 2) were designed to PCR amplify the gene FAD91 encoding Δ9 fatty acid desaturase 1, the FAD92 gene encoding Δ9 fatty acid desaturase 2, the gene FAD12 encoding Δ12 fatty acid desaturase and the gene FAD15 encoding Δ15 fatty acid desaturase using the genomic DNA of M. australis strain W7-5 as the template. Their accession numbers of these cloned genes are shown in Supplementary file 1. The 3′-arms and 5′-arms of the FAD91 gene, the FAD92 gene, the FAD12 gene, the FAD15 gene were PCR amplified using the primers shown in Supplementary file 2 and the genomic DNA of M. australis strain W7-5 as template. The cloned 3′-arms and 5′-arms were digested with the DNA restriction enzymes shown in Supplementary file 2 and the digests were ligated into plasmid Ma-NAT-loxP carrying the NAT gene digested with the same DNA restriction enzymes, forming Ma-NAT-loxP-ΔFAD91 (carrying 3′-arms and 5′-arms of the FAD91 gene) (Supplementary file 3A), Ma-NAT-loxP-ΔFAD92 (carrying 3′-arms and 5′-arms of the FAD92 gene) (Supplementary file 3B), Ma-NAT-loxP-ΔFAD12 (carrying 3′-arms and 5′-arms of the FAD12 gene) (Supplementary file 3C), Ma-NAT-loxP-FAD15 (carrying 3′-arms and 5′-arms of the FAD15 gene) (Supplementary file 3D), The FAD12 gene was PCR amplified using the primers (FAD12-F/FAD12-R) and the PCR products were digested with the corresponding enzymes in Supplementary file 2 and the digests were ligated into the knock-in plasmid MaNATX13 to yield Ma-NATX13-loxP-FAD12 (Supplementary file 3E).
Transformation of M. australis Strain W7-5 and Isolation of Various Deletants and Transformants
Preparation of the competent cells of M. australis strain W7-5 and transformation of M. australis strain W7-5 was carried out using the high efficiency transformation by electroporation as described by Wei et al (2021). The linear DNA fragments 5′-arm-PolyA-NAT-PGK-3′-arm were PCR amplified using the primers FAD91-5F/FAD91-5F, FAD92-5F/FAD92-3R, FAD12-5F/FAD12-3R, FAD15-5F/FAD15-3R (Supplementary file 2) and the plasmids Ma-NAT-loxP-ΔFAD91, Ma-NAT-loxP-ΔFAD92, Ma-NAT-loxP-ΔFAD12, Ma-NAT-loxP-ΔFAD15 constructed above as the templates. Similarly, the plasmids Ma-NATX13-loxP-FAD12 was digested with the enzyme SmaI to obtain the linear DNA fragments 18SrDNA-PGK-FAD12-polyA-NAT-PGK-26SrDNA. The linear DNA fragments 5′-arm-PolyA-NAT-PGK-3′-arm were transformed into the competent cells of M. australis strain W7-5 by using the electroporation method under the optimal conditions of voltage 2000 V, OD600 nm of the yeast culture = 1.0, and the amount of DNA = 10.0 µg as described by Wei et al. (2021) and the mutants Δfad12 in which the FAD12 gene was totally removed and Δfad15 in which the FAD15 gene was completely abolished were obtained. However, the mutants in which the FAD91 gene and the FAD92 gene were removed could not be acquired because the absence of palmitoleic (C16:1) or oleic (C18:1) acids in the medium. After the mutant Δfad12 was obtained, the linear DNA fragment PGK-HPT-polyA-CRE-PGK from the plasmid Ma-HPTX13-CRE was introduced into the disruptant cells to remove the NAT gene by the transiently expressed Cre recombinase as described by Wei et al (2021). Both the NAT gene and the HPT gene were lost after these disruptants were cultivated for 4 h and the mutant Δfad12-cre was obtained. At the same time, the linear DNA fragments 18SrDNA-PGK-FAD12-polyA-NAT-PGK-26SrDNA from the plasmid Ma-NATX13-loxP-FAD12 (Supplementary file 3E) transformed into the mutant Δfad12-cre, to acquire the complementing strain (FAD12-H). Finally, the linear DNA fragment PGKHPT-polyA-CRE-PGK from the plasmid Ma-HPTX13-CRE was again introduced into the FAD12-H to remove the NAT gene by the transiently expressed Cre recombinase and the HPT gene was lost automatically.
Determination of the Compositions of Fatty Acids in the Extracted Oil
All the disruptants, the complementing strain (FAD12-H) and their wild type strain were grown aerobically in the liquid YPD medium at 15°C for three days, The yeast cells in the culture were collected and washed by centrifugation at 5000 ⋅ g and 4°C for 10 min with sterile saline water for three times. The washed cells were dried at 80°C until the cell dried weight was constant. In order to analyze fatty acids compositions, the dried cells were added to 10.0 ml of 6.0 M hydrochloric acid solution, and the mixture was incubated in water bath at 80 ℃ for 4 h. Then, 7.0 ml of 60% methanol and 7.0 ml of chloroform were added to the mixture and the fatty acids were extracted by centrifugation at 5000 ⋅ g and 4°C for 10 min. Then, the extracted atty acids were dissolved in 2.0 ml of chloroform, 2.5 ml of 2% (v/v) sulfuric acid/methanol solution was added to the mixture. Then, the mixture was incubated in water bath at 80 ℃ for 1 h. After cooling, 2.0 ml of saturated NaCl solution was added and mixed with shaking. Fatty acid methyl esters were extracted with 1.0–2.0 ml of n-hexane, and the upper layer with the fatty acid methyl ester was filtrated with 0.22 µm filter membrane to remove the impurity. Gas chromatography analysis of the fatty acid methyl esters obtained was carried out by using 5890-II (Agilent Company, USA). The chromatography column was a fused silica AC2.0 capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness); injector temperature, 250°C; carrier gas, N2, 1.0 ml per min; temperature program, 50°C, held for 2.0 min from 150 to 200°C at 15°C per min, held for 2.0 min, then to 250°C at 2°C per min, held for 5.0 min. The fatty acid with C19:0 was used as an internal standard (Li et al. 2010).
Analysis of Expression of Various Genes
The wild-type strain M. australis strain W7-5, different disruptants, and complementing strain obtained above were aerobically grown in 50.0 ml of the YPD medium at 15°C and 180 rpm for 2 days and total RNAs of them were extracted using an E.Z.N.A. Fungal RNA Kit. The yeast RNAs were reversely transcripted into cDNA using a PrimeScriptTM RT Reagent Kit (Perfect Real Time). The transcriptional levels of various genes in the wild-type strain M. australis strain W7-5, different disruptants, and complementing strain were determined using a Real time fluorescence quantitative PCR analyzer (QIAGEN, Germany) and the primers shown in Supplementary file 4.
Cell Growth in the Liquid YPD Medium at Different Temperatures
All the disruptants, complementing strain and their wild type strain were grown aerobically in the liquid YPD medium, effects of different temperatures (5°C, 15°C and 25°C) on their cell growth (OD600nm) were examined. When the cells were grown for three days at 5°C, 15°C and 25°C, the yeast cells were harvested and washed by centrifugation at 5000 ⋅ g for 10 min with the sterile saline water. One part of the washed cells was treated at 80°C for 20 min. The washed yeast cells treated and untreated were suspended in 600.0 µl of the Annexin V-FITC binding solution and the cell suspension was thoroughly mixed with 5.0 µl of the Annexin V-FITC. Then, 10.0 µl of PI solution was added to the cell suspension and mixed well. The new cell suspension was incubated in the dark for 10–15 min. After that, the new cell suspension was centrifuged at 8000 × g for 5 min and the pellets obtained was resuspended in the Annexin V-FITC binding solution and the new cell suspension was observed under the fluorescence microscope (Olympus U-LH100HG, Japan) and images were recorded using the CellSense Standard software.
Cell Growth on the YPD Plates with Different Concentration of Congo Red and Sorbitol
At the same time, to test susceptibility to the cell wall disturbing compound (Congo red) and osmotic pressure stabilizer sorbitol, the yeast cells of M. australis strain W7-5, the disruptants and the complementing strain obtained above were cultivated on the YPD plates with Congo red (its concentrations were 200 µg/ml, 300 µg/ml, and 400 µg/ml) and sorbitol (its concentrations were 1.5 M, 2.0 M, and 2.5 M) at 5, 15 and 25°C for 4 days. After that, cell growth on the plates and cell morphology were observed and photographed.