Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host

Abstract

Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCE The yeast Kluyveromyces marxianus grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast Saccharomyces cerevisiae in industrial applications. Here, we describe genetic tools for genome editing and breeding K. marxianus strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using K. marxianus as a versatile synthetic biology platform organism for industrial applications.

Keywords: CRISPR-Cas9; Kluyveromyces marxianus; lipogenesis; mating; renewable chemicals; thermotolerant yeast.

FIG 1

CRISPR-Cas9 genome editing and mating-type switching in K. marxianus. (A) CRISPR_NHEJ and CRISPR_HDR 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 CRISPR_NHEJ, 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.