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Meiotic Recombination and Genomic Diversity in the Yeast Komagataella bioRxiv preprint doi: https://doi.org/10.1101/704627; this version posted July 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 3 Tetrad analysis without tetrad dissection: Meiotic recombination and genomic 4 diversity in the yeast Komagataella phaffii (Pichia pastoris) 5 6 1 1,2 1 1 7 Stephanie Braun-Galleani , Julie A. Dias , Aisling Y. Coughlan , Adam P. Ryan , 1 1 8 Kevin P. Byrne , Kenneth H. Wolfe * 9 10 1 11 UCD Conway Institute, School of Medicine, University College Dublin, Dublin, Ireland, 2 12 Department of Mathematics and Statistics, McGill University, Montreal, Quebec, Canada. 13 14 * [email protected] (KHW) 15 16 Short title: Tetrad analysis in Komagataella phaffii 17 1 bioRxiv preprint doi: https://doi.org/10.1101/704627; this version posted July 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 18 Abstract 19 Komagataella phaffii is a yeast widely used in the pharmaceutical and biotechnology 20 industries, and is one of the two species that were formerly called Pichia pastoris. However, 21 almost all laboratory work on K. phaffii has been done on strains derived from a single 22 natural isolate, CBS7435. There is little information about the genetic properties of K. phaffii 23 or its sequence diversity. Genetic analysis is difficult because, although K. phaffii makes asci 24 with four spores, the spores are small and tend to clump together, making the asci hard to 25 dissect. Here, we sequenced the genomes of all the known isolates of this species, and find 26 that K. phaffii has only been isolated from nature four times. We analyzed the meiotic 27 recombination landscape in a cross between auxotrophically marked strains derived from two 28 isolates that differ at 44,000 single nucleotide polymorphism sites. We conducted tetrad 29 analysis by making use of the property that haploids of this species do not mate in rich 30 media, which enabled us to isolate and sequence the four types of haploid cell that are 31 present in the colony that forms when a tetratype ascus germinates. We found that 32 approximately 25 crossovers occur per meiosis, which is 3.5 times fewer than in 33 Saccharomyces cerevisiae. Recombination is suppressed, and genetic diversity among 34 natural isolates is low, in a region around centromeres that is much larger than the 35 centromeres themselves. Our method of tetrad analysis without tetrad dissection will be 36 applicable to other species whose spores do not mate spontaneously after germination. 37 38 39 Author summary 40 To better understand the basic genetics of the budding yeast Komagataella phaffii, which has 41 many applications in biotechnology, we investigated its genetic diversity and its meiotic 42 recombination landscape. We made a genetic cross between strains derived from two 43 natural isolates, and developed a method for characterizing the genomes of the four spores 44 resulting from meiosis, which were previously impossible to isolate. We found that K. phaffii 45 has a lower recombination rate than Saccharomyces cerevisiae. It shows a large zone of 46 suppressed recombination around its centromeres, which may be due to the structural 47 differences between centromeres in K. phaffii and S. cerevisiae. 48 2 bioRxiv preprint doi: https://doi.org/10.1101/704627; this version posted July 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 49 Introduction 50 Komagataella phaffii is the most widely used yeast species for production of heterologous 51 proteins, such as the expression of antibody fragments for the pharmaceutical industry. It has 52 several advantages over Saccharomyces cerevisiae as a cell factory, including 53 thermotolerance, respiratory growth to very high cell densities, and the ability to induce high 54 levels of foreign gene expression using its methanol oxidase promoter [1-3]. K. phaffii is 55 better known under its previous name Pichia pastoris, but in 2009 it was realized that the 56 name ‘P. pastoris’ had been applied to a heterogeneous group of strains that actually belong 57 to two separate species, which are now called K. phaffii and K. pastoris [4]. Their genomes 58 differ by approximately 10% DNA sequence divergence and two reciprocal translocations [5]. 59 Phylogenetically, Komagataella species are members of the methylotrophic yeasts clade 60 (family Pichiaceae) and are only distantly related to better-known yeasts such as 61 S. cerevisiae and Candida albicans [6]. 62 63 Almost all research on K. phaffii has been done using the genetic background of strain 64 CBS7435 (synonymous with NRRL Y-11430) [7]. The origin of this strain has been unclear 65 because it was deposited in the CBS and NRRL culture collections in connection with a US 66 patent granted to Phillips Petroleum (see discussion in [4]), but in this study we show that 67 CBS7435 is identical to the type strain of K. phaffii, which was isolated from an oak tree. The 68 widely-used K. phaffii strains GS115 and X-33 are derivatives of CBS7435 and are 69 components of a commercial protein expression kit marketed by Invitrogen / Life 70 Technologies / Thermo Fisher. GS115 was made from CBS7435 by random mutagenesis 71 with nitrosoguanidine and includes a his4 mutation among a few dozen point mutations [5, 8]. 72 X-33 is a derivative of GS115 in which the HIS4 gene was reverted to wildtype by site- 73 directed mutagenesis [9]. 74 75 The public culture collections include a few natural isolates of K. phaffii, but these have 76 received little attention. Most of them were isolated from exudates (slime fluxes) on trees. 77 The genetic diversity that is naturally present in populations of K. phaffii could potentially be 78 used to improve its performance in biotechnological applications. In principle, beneficial 79 alleles from natural isolates could be introduced into biotechnology strains by breeding. As a 80 first step towards this goal, in this study we surveyed the nucleotide sequence diversity that 81 is present in all six isolates of K. phaffii that are available from culture collections. 82 83 Although techniques for inserting foreign genes into the K. phaffii genome and controlling 84 their expression are well established, other aspects of the genetics and life cycle of this yeast 3 bioRxiv preprint doi: https://doi.org/10.1101/704627; this version posted July 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 85 are much less studied [10]. K. phaffii has four chromosomes and grows primarily as a 86 haploid. Mating only occurs when induced by nitrogen depletion [10-12]. Zygotes usually 87 sporulate immediately after mating, but in crosses between haploids carrying auxotrophic 88 markers, the diploid progeny can be maintained by transferring them to nitrogen-replete 89 media and selecting for prototrophy [11]. Mating occurs between MATa and MATα cells, and 90 haploids can switch their mating type by inverting a 138-kb section of chromosome 4 [12, 91 13]. The recent development of stable heterothallic strains of K. phaffii makes it possible to 92 carry out controlled genetic crosses without requiring selectable markers [13, 14]. 93 94 One of the classical techniques used for genetic studies in yeasts, particularly for analysis of 95 recombination and gene conversion, is tetrad analysis [15-18]. Yeast meiosis usually results 96 in the formation of an ascus containing four ascospores (a tetrad), which carry the four sets 97 of chromosomes produced by meiosis. In species such as S. cerevisiae or 98 Schizosaccharomyces pombe, the ascus can be dissected by micromanipulation, allowing 99 each spore to be germinated separately into a haploid colony (a segregant) and analyzed. 100 Tetrad analysis is also possible in other eukaryotes in which the four products of a meiosis 101 remain attached to each other, such as other fungi, green algae, and plants, although 102 recovery of the four meiotic products in plants for whole genome sequencing is difficult [19- 103 22]. 104 105 In the yeasts S. cerevisiae, Saccharomyces paradoxus, and Lachancea kluyveri, the meiotic 106 recombination landscape has been studied in detail by analyzing the genomes of the four 107 segregants from multiple tetrads, either by genome sequencing or by microarray analysis of 108 dissected tetrads [23-27]. However, tetrad analysis has not been achieved in K. phaffii 109 because its spores are much smaller than those of S. cerevisiae and they tend to stick 110 together and form clumps [10]. K. phaffii tetrads are 1-2 µm in diameter (Fig 1), which is 111 approximately four times smaller than tetrads in S. cerevisiae [28, 29]. Previous genetic 112 analysis in K. phaffii has relied on random spore methods, which are less powerful than 113 tetrad analysis [10]. Similar problems make tetrad dissection in the related methylotrophic 114 yeast Ogataea polymorpha difficult but not impossible [30, 31]. Here, we developed a 115 method for tetrad analysis in K. phaffii that does not require asci to be dissected. We made a 116 genetic cross between strains derived from CBS7435 and a divergent natural isolate, 117 enabling us to investigate the landscape of meiotic recombination (that is, the distribution of 118 recombination sites along chromosomes) in this species for the first time.
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