Genotypic and phenotypic diversity among Komagataella species reveals a hidden pathway for xylose utilization

Abstract

Background: The yeast genus Komagataella currently consists of seven methylotrophic species isolated from tree environments. Well-characterized strains of K. phaffii and K. pastoris are important hosts for biotechnological applications, but the potential of other species from the genus remains largely unexplored. In this study, we characterized 25 natural isolates from all seven described Komagataella species to identify interesting traits and provide a comprehensive overview of the genotypic and phenotypic diversity available within this genus.

Results: Growth tests on different carbon sources and in the presence of stressors at two different temperatures allowed us to identify strains with differences in tolerance to high pH, high temperature, and growth on xylose. As Komagataella species are generally not considered xylose-utilizing yeasts, xylose assimilation was characterized in detail. Growth assays, enzyme activity measurements and ¹³C labeling confirmed the ability of K. phaffii to utilize D-xylose via the oxidoreductase pathway. In addition, we performed long-read whole-genome sequencing to generate genome assemblies of all Komagataella species type strains and additional K. phaffii and K. pastoris isolates for comparative analysis. All sequenced genomes have a similar size and share 83-99% average sequence identity. Genome structure analysis showed that K. pastoris and K. ulmi share the same rearrangements in difference to K. phaffii, while the genome structure of K. kurtzmanii is similar to K. phaffii. The genomes of the other, more distant species showed a larger number of structural differences. Moreover, we used the newly assembled genomes to identify putative orthologs of important xylose-related genes in the different Komagataella species.

Conclusions: By characterizing the phenotypes of 25 natural Komagataella isolates, we could identify strains with improved growth on different relevant carbon sources and stress conditions. Our data on the phenotypic and genotypic diversity will provide the basis for the use of so-far neglected Komagataella strains with interesting characteristics and the elucidation of the genetic determinants of improved growth and stress tolerance for targeted strain improvement.

Keywords: Genome sequencing; Komagataella species; Pichia pastoris; Xylose assimilation; Yeast diversity.

Fig. 1

Phylogenetic tree of Komagataella species. The tree was calculated based on pairwise genomic distances between the assembled genomes as determined by Mash [22]. The genome sequence of Citeromyces matritensis NRRL Y-2407 (NCBI id ASM370516v1) was used as outgroup

Fig. 2

Regions of similarity between the reference strain K. phaffii CBS 7435 (NCBI assembly id ASM170808v1) and K. ulmi CBS 12361 (A), K. pastoris DSMZ 70877 (B), K. pastoris CBS 9178 (C), K. kurtzmanii CBS 12817 (D), K. phaffii UWOPS 03-328y3 (E), K. pseudopastoris CBS 9187 (F), K. populi CBS 12362 (G) and K. mondaviorum CBS 15017 (H) based on whole-genome comparisons. Colors refer to the four chromosomes (chr1: orange, chr2: blue, chr3: green, chr4: purple), contigs appear in their initial order and orientation of the assemblies. Only matches spanning at least 10 kbp are shown and non-matching contigs were removed

Fig. 3

Whole-genome comparison of K. pseudopastoris CBS 9187 (A) and K. mondaviorium CBS 15017 (B) against K. populi CBS 12362. The order and coloring of K. populi contigs were assigned after inspecting the comparisons with K. phaffii (remains preliminary due to heavy rearrangements). Contigs of K. pseudopastoris and K. mondaviorum were ordered and oriented manually to achieve congruence with K. populi as far as possible. ctg contig, K. pop K. populi, rev reverse

Fig. 4

A Summary of spotting assays at 30 and 37 °C. Cell growth was evaluated after 2 and 7 days of incubation according to the following criteria: 0 = no growth (white), 1–5 rating depending on the dilution down to which growth was observed, 6 = large colonies in every dilution (dark blue). All plates with inhibitors contained glucose as carbon source. B Growth on xylose as the only carbon source. Plate after incubation at 30 °C for 3 days. C Growth at pH 9. Plates after incubation at 30 °C for 7 days

Fig. 5

Growth, xylose consumption and xylitol production of K. phaffii X-33 (A), K. pastoris CBS 704 (B), and K. populi CBS 12362 (C) in YNB with 2% xylose (continuous lines with filled symbols). Dashed lines with empty symbols indicate samples from the control cultures in YNB with no addition of carbon source. Data represent the average of three biological replicates with standard deviation

Fig. 6

A Relative transcript levels of putative xylose pathway genes in K. phaffii X-33 and K. populi CBS 12362. Transcript levels are given as fold change compared to the average SOR1 transcript level in K. phaffii X-33 grown in minimal medium with glucose. Error bars represent the standard deviation of three biological replicates shown as individual data points. B Specific enzyme activities of cell-free extracts prepared from cells grown in minimal medium with xylose. Cultures of the xylose utilizing yeast S. lignohabitans grown in the same medium were used as a control. Values are given as U mg⁻¹ total protein. Errors represent the standard deviation of three biological replicates. Nd activity not detectable

Fig. 7

Xylose assimilation in K. phaffii X-33—¹³C labeling and modeling of metabolic fluxes. A Putative xylose utilization pathway. The isotopologue distribution of all colored metabolites was measured by mass spectrometry. The colors indicate the pathway to which the metabolites mainly belong (orange—glycolysis, green—oxidative pentose phosphate pathway, blue—non-oxidative pentose phosphate pathway). Absolute specific flux rates per carbon atom (mmol g⁻¹ h ⁻¹) with 95% confidence intervals were calculated by ¹³C metabolic flux analysis. Values of each reaction refer to the total number of carbon atoms involved in the corresponding reaction. For reversible fluxes, only net fluxes are shown. B Relative abundance of ¹³C labeled isotopologues in selected metabolites after cultivation on ¹²C or 1-¹³C D-xylose