Article Figures & Data
Figures
- FIG 1
CRISPR-Cas9 genome editing and mating-type switching in K. marxianus. (A) CRISPRNHEJ and CRISPRHDR systems. K. marxianus transformed with the pKCas plasmid generates small indels near the cut site, a common product of nonhomologous end joining (NHEJ) repair of the DNA double-strand break. When transformed with both the pKCas plasmid and a donor DNA, homologous recombination products are seen in the target site. (B) Yeast life cycle. Haploid MATa and MATα switch mating type by transposases α3 and Kat1 in K. lactis. Haploid cells conjugate to form MATa/MATα diploids. Diploids undergo meiosis to form haploid spores that germinate to complete the life cycle. (C) Mature a- and α-pheromones from K. marxianus aligned with the S. cerevisiae and K. lactis sequences. Red indicates nonconserved amino acids between K. marxianus a- and α-factors. Amino acids are marked as identical (*), with similar polarity (:), or with different polarity (.). (D) Incubation of putative heterothallic MATa and MATα strains with a cocktail of both mature α-factor pheromones (KmMfα1 and -2) results in mating projections from the MATa strain only (*).
- FIG 2
Creation of heterothallic K. marxianus strains. (A) Auxotrophic mating assay of Km1 strains. Shown are results from strains Km1 MATα α3– kat1– leu2–, Km1 MATa α3– kat1– leu2–, and homothallic Km1 leu2–, streaked through strain Km1 MATa α3– kat1– trp1– on 2% glucose plates and replica plated onto SCD − (Leu, Trp) plates after 2 days. Diploid growth is seen only upon sexual crossing between strains with opposite mating types or with homothallic haploid strains. (B) Auxotrophic mating assay of several α3– kat1– leu2– triple-inactivation strains and Km1 MATa α3– kat1– trp1– or Km1 MATα α3– kat1– trp1–. Putative heterothallic strains were spotted over the negative control (−), the Km1 MATa α3– kat1– trp1– reference (a), or the Km1 MATα α3– kat1– trp1– reference (α) on glucose plates for mating. Replica plating onto SCD − (Leu, Trp) results in diploid growth. (C) The wild homothallic isolate Km18 was made trp– by UV mutagenesis and crossed with heterothallic Km1 MATa α3– kat1– leu2–. Diploids were sporulated, 16 spores were isolated (a through p) and germinated, and the resulting haploids were screened for heterothallic strains by crossing with Km1 MATa α3– kat1– trp1– or Km1 MATα α3– kat1– trp1–. Screened haploids were auxotrophic strains unable to mate (c, d, f, i, j, and o), possible trp– revertants (h, k, n, and p), homothallic (g), or heterothallic (a, b, e, l, and m).
- FIG 3
Lipogenesis of K. marxianus strains. (A) Nile red fluorescence flow cytometry of 11 wild-type isolates after 24, 48, 72, and 96 h at 42°C in lipogenesis medium. Experiments were carried out in biological triplicate, with means and standard deviations shown. (B) DIC images superimposed with epifluorescence microscopy of Nile red-stained cells. Little or no fluorescence is seen after 24 h in 2% glucose. After 24 h in 8% glucose at 42°C (Km19 and Km6) and 48 h (Km18), fluorescence is seen encompassing the majority of the cell volume. (C) TLC analysis of Km19 total lipids after 24 h in 8% glucose at 42°C. Lane 1, ladder of standards containing steryl ester (SE), fatty acid methyl ester (FAME), triacylglycerols (TAG), and free fatty acids (FFA). Lane 2, Km19 lipids.
- FIG 4
Genetic dissection of lipogenesis of a high-producing K. marxianus strain, Km6. (A) General overview of lipid-related metabolism. Genes in red were inactivated with CRISPRNHEJ, and genes in green were overexpressed using plasmids. (B) Percentage of fatty acids in dry cell weight (DCW) after 24 h under lipogenic conditions at 42°C for several variants of Km6 (wild type and mutants). (C) Percentage of fatty acids in dry cell weight for several Km6 variants containing ACC1, DGA1, and ACL1/2 overexpression plasmids. In panels B and C, all experiments were carried out in biological triplicate, with mean values and standard deviations shown. Lipogenesis medium in panels B and C contained monosodium glutamate instead of ammonium sulfate.
- FIG 5
Selection of K. marxianus strains with combined beneficial traits. (A) Selection strategy. Km19 and Km17 were crossed, sporulated, and then germinated at 44°C to select for thermotolerant segregants. Single segregants were isolated and tested for lipid production, transformability, and high-temperature growth. (B) Fatty acid percentage in dry cell weight (DCW) for several segregants from the Km17 × Km19 cross and the parental strains. Three segregants have similar profiles to the more lipogenic parental strain (Km19). Experiments are from biological triplicates with mean and standard deviation shown. (C) Growth curves at 45°C for the segregants 4B, 5E, and 2G, as well as parental strains Km17 and Km19, in biological triplicate. Km19 is unable to grow at this temperature. Growth curves for parental strains at 30, 37, and 42°C can be found in the supplemental material (Fig. S7A), as well as for segregants at 30 and 37°C (Fig. S7B). (D) Transformation efficiency for several segregants normalized by Km17 transformation efficiency. Experiments are from 2 to 4 biological replicates with normalized mean and standard deviations shown.
Tables
- TABLE 1
List of wild-type K. marxianus strains used in this work
Straina Designation no. by: ATCC NCYC CBS NRRL Km1 Km2 10022 100 6432 Y-665 Km5 46537 851 397 Y-2415 Km6 143 608 Y-8281 Km9 26548 2597 6556 Y-7571 Km11 36907 587 Km16 10606 396 Y-1550 Km17 8635/28910 Y-1190 Km18 1089 Y-2265 Km19 26348 Km20 Km21 ↵a Km1, Km20, and Km21 were isolated from sugarcane bagasse piles. Km1 was isolated at Raceland Raw Sugar Corporation, Raceland, LA. Km20 and Km21 were isolated at the Sugarcane Growers’ Cooperative, Belle Glade, FL.
FIG S1
Cas9 editing outcomes and efficiency for several K. marxianus genes. (A) CRISPRNHEJ results for KmURA3-targeting experiments in the absence of donor DNA. Three regions were targeted, and small insertions and deletions were found upon repair of double-strand breaks by the NHEJ machinery. (B) Small indels can also be observed when targeting KmKAT1 and KmALPHA3 genes. (C) Editing efficiency across 10 different K. marxianus genes is around 75%, with some genes more editing prone than others. Download FIG S1, TIF file, 2.66 MB.
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TABLE S1
List of engineered K. marxianus strains. Download Table S1, XLSX file, 0.04 MB.
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FIG S2
K. marxianus mating. (A) K. marxianus a- and α-factor protein sequences. Mature pheromones (in red) derive from precursor proteins, mating factor a (MFA1 and MFA2) and mating factor α (MFα1). We identified two putative MFA genes as well as the MFα gene (KmMFα1 encoding 2 isotypes, KmMFα1 and KmMFα2) in the K. marxianus genome by reciprocal BLASTp using the S. cerevisiae and K. lactis protein sequences as queries. (B) Proposed architecture of K. marxianus MATa, MATα, HMRa, and HMLα loci. Black arrows indicate annealing sites for the genotyping primers (Table S2). PCR of an a-type strain yielded an ∼3,480-bp product, while the α-type yielded an ∼6,500-bp PCR product. Download FIG S2, TIF file, 0.56 MB.
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TABLE S2
DNA sequences for sgRNA spacers, primers, and genes. Download Table S2, XLSX file, 0.05 MB.
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FIG S3
Kat1 and α3 rescue of mating-type switching. Plasmid-based ectopic expression of Kat1 and α3 restores mating-type switching in MATa α3– kat1– and MATα α3– kat1–, respectively. SCD − (Leu, Trp) plates are shown where only diploid strains are able to grow. (A) Ectopic expression of Kat1 allowed a stable MATa α3– kat1– leu2– strain to switch to MATα and mate with the MATa α3– kat1– trp1– reference strain. (B) Similarly, ectopic expression of α3 allowed some MATα transformants to switch to MATa and mate with the MATα reference strain. Note that some MATα transformants did not switch to MATa, keeping the ability to mate with MATa. Download FIG S3, TIF file, 1.70 MB.
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FIG S4
Lipogenesis of K. marxianus strains. (A) Strains grown on 8% cellobiose. Shown is mean Nile red fluorescence flow cytometry of 11 wild-type isolates after 24, 48, 72, and 96 h at 42°C in lipogenesis medium containing 8% cellobiose. Maximum values do not surpass 30% of the value obtained for the top lipid-producing strain grown on glucose. Experiments were carried out in biological triplicate, with means and standard deviations shown. (B) Lipid accumulation in K. marxianus strains. Shown are Km19, Km17, and Km6 percentages of fatty acids in dry cell weight after 24 h in 8% glucose at 42°C and 250 rpm. Lipogenesis medium contained ammonium sulfate instead of monosodium glutamate for this set of measurements. Measurements were from biological triplicates, with mean and standard deviation shown. Download FIG S4, TIF file, 0.35 MB.
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FIG S5
Fatty acid composition of Km6-derived strains. Eight fatty acids were measured using GC-FID; no appreciable difference in composition was seen among the strains, except for mutant Km6 eht1– bearing the ACC1 overexpression plasmid. Download FIG S5, TIF file, 0.95 MB.
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FIG S6
Nile red staining and flow cytometry for each single segregant from the Km17 × Km19 cross. The diploids from this cross were sporulated, and spores were germinated at high temperature (44°C). Ninety-one spores were collected, grown under the lipogenesis condition, treated with Nile red, and subjected to flow cytometry. Download FIG S6, TIF file, 0.29 MB.
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FIG S7
Temperature dependence of the growth of K. marxianus strains tested for lipogenesis. (A) Growth curves of Km17 and Km19 in 50 ml YPD medium at 30, 37, 42, and 45°C and 250 rpm. Experiments are from biological triplicates. (B) Growth curves of the highly lipogenic isolates from mating Km17 and Km19. Cells were grown in 50 ml YPD medium at 250 rpm at 30 and 37°C. Experiments are from biological triplicates. Download FIG S7, TIF file, 0.80 MB.
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