Kluyveromyces marxianus is a non-conventional yeast whose physiology and metabolism lends itself to diverse biotechnological applications. While the wild-type yeast is already in use for producing fragrances and fermented products, the lack of standardised tools for its genetic and metabolic engineering prevent it from being used as a next-generation cell factory for bio-based chemicals. In this paper, we bring together and characterise a set of native K. marxianus parts for the expression of multiple genes for metabolic engineering and synthetic biology. All parts are cloned and stored according to the MoClo/Yeast Tool Kit standard for quick sharing and rapid construction. Using available genomic and transcriptomic data, we have selected promoters and terminators to fine-tune constitutive and inducible gene expression. The collection includes a number of known centromeres and autonomously replication sequences (ARS). We also provide a number of chromosomal integration sites selected for efficiency or visible phenotypes for rapid screening. Finally, we provide a single-plasmid CRISPR/Cas9 platform for genome engineering and facilitated gene targeting, and rationally create auxotrophic strains to expand the common range of selection markers available to K. marxianus. The curated and characterised tools we have provided in this kit will serve as a base to efficiently build next-generation cell factories from this alternative yeast. Plasmids containing all parts are available at Addgene for public distribution.

Humans have used yeasts to make cheese and kefir for millennia, but the ability to ferment the milk sugar lactose is found in only a few yeast species, of which the foremost is Kluyveromyces lactis [1]. Two genes, LAC12 (lactose permease) and LAC4 (lactase), are sufficient for lactose uptake and hydrolysis to glucose and galactose [2]. Here, we show that these genes have a complex evolutionary history in the genus Kluyveromyces that is likely the result of human activity during domestication. We show that the ancestral Lac12 was bifunctional, able to import both lactose and cellobiose into the cell. These disaccharides were then hydrolyzed by Lac4 in the case of lactose or Cel2 in the case of cellobiose. A second cellobiose transporter, Cel1, was also present ancestrally. In the K. lactis lineage, the ancestral LAC12 and LAC4 were lost and a separate upheaval in the sister species K. marxianus resulted in loss of CEL1 and quadruplication of LAC12. One of these LAC12 genes became neofunctionalized to encode an efficient lactose transporter capable of supporting fermentation, specifically in dairy strains of K. marxianus, where it formed a LAC4-LAC12-CEL2 gene cluster, although another remained a cellobiose transporter. Then, the ability to ferment lactose was acquired very recently by K. lactis var. lactis by introgression of LAC12 and LAC4 on a 15-kb subtelomeric region from a dairy strain of K. marxianus. The genomic history of the LAC genes shows that strong selective pressures were imposed on yeasts by early dairy farmers.

While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade- mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies ( < 1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.