RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus

Abstract

The thermotolerant yeast Kluyveromyces marxianus has gained significant attention in recent years as a promising microbial candidate for industrial biomanufacturing. Despite several contributions to the expanding molecular toolbox for gene expression and metabolic engineering of K. marxianus, there remains a need for a more efficient and versatile genome editing platform. To address this, we developed a CRISPR-based editing system that enables high efficiency marker-less gene disruptions and integrations using only 40 bp homology arms in NHEJ functional and non-functional K. marxianus strains. The use of a strong RNA polymerase II promoter allows efficient expression of gRNAs flanked by the self-cleaving RNA structures, tRNA and HDV ribozyme, from a single plasmid co-expressing a codon optimized Cas9. Implementing this system resulted in nearly 100% efficiency of gene disruptions in both NHEJ-functional and NHEJ-deficient K. marxianus strains, with donor integration efficiencies reaching 50% and 100% in the two strains, respectively. The high gRNA targeting performance also proved instrumental for selection of engineered strains with lower growth rate but improved polyketide biosynthesis by avoiding an extended outgrowth period, a common method used to enrich for edited cells but that fails to recover advantageous mutants with even slightly impaired fitness. Finally, we provide the first demonstration of simultaneous, markerless integrations at multiple loci in K. marxianus using a 2.6 kb and a 7.6 kb donor, achieving a dual integration efficiency of 25.5% in a NHEJ-deficient strain. These results highlight both the ease of use and general robustness of this system for rapid and flexible metabolic engineering in this non-conventional yeast.

Keywords: CRISPR-Cas9; Kluyveromyces marxianus; Metabolic engineering; Polyketides; Thermotolerant yeast.

Fig. 1

TGR gene cassette and approximate premature transcript secondary structure. Black triangles indicate sites of tRNA and HDV ribozyme cleavage.

Fig. 2

CRISPR-mediated genome modification in K. marxianus. A, B The HIS3 and LEU2 loci were targeted using pDBtgr-Cas9 plasmids containing 20 bp targeting sequences matching locations within each ORF (HIS3: bright green band; LEU2: bright orange band). Integration of three different donors were evaluated, an 80 bp KO donor, a g2ps1 expression cassette, and an ACC1 expression cassette, each containing 40 bp homology sequences matching regions of the targeted ORF just upstream and downstream of the targeting sequence (HIS3: dark green; LEU2: dark orange). C, D Percentage of transformants with a gene knockout or donor integration in a NHEJ-functional strain, CBS 712ΔU and E,F in a NHEJ-deficient strain, CBS 712ΔUΔK. The total height of the bars represents average total gene knockout frequency ± standard deviation and the height of the blue bars represents the proportion of KOs that were created through integration of a donor ± standard deviation. Ten transformants were analyzed for each of two transformations per condition. Unpaired t-tests showed there is no significant difference in the percent integration efficiency for the CBS 712ΔU strain when HIS3 is targeted and co-transformed with the KO, 2PS, or ACC1 donor (panel C, three blue bars), nor when LEU2 is targeted and co-transformed with the KO or 2PS donor (panel D, two blue bars). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Comparison of gene integration frequency in CBS 712ΔUΔK with and without use of a two-day outgrowth prior to plating. Acquisition of four different modifications were evaluated using each post-transformation method. The missing bar for the ZWF1::KO donor and GPD1::KO donor conditions is indicative of the lack of recovery of any ZWF1 KO and GPD1 KO mutants, respectively, after use of a two-day outgrowth prior to plating. Bars represent the average frequency of integration for the respective donor ± standard deviation. Ten transformants were analyzed for each of two transformations per condition.

Fig. 4

Growth and TAL production from unengineered and engineered K. marxianus strains grown for 48 h in 3 mL SLC media at 37 °C. All strains harbor the higher-copy pKD-P2PS plasmid. A Growth (optical density at 600 nm). B TAL titer. C Specific TAL Titer. Bars represent average ± standard deviation for 2 biological replicates, *p < 0.05, **p < 0.01. TAL: triacetic acid lactone.

Fig. 5

Expansion of TGR cassette for simultaneous targeting of two loci in K. marxianus.A Double gRNA cassette structure, approximate pre-mature transcript secondary structure, and final gRNAs after self-cleavage at the black triangles. B Percentage of transformations with simultaneous, dual integrations at the HIS3 and LEU2 loci in K. marxianus using ACC1 and g2ps1 gene donors, respectively. Bar represents average ± standard deviation of two experiments comprised of two and four transformations, with up to ten transformants analyzed per transformation.