Intergeneric chromosomal transfer in yeast results in improved phenotypes and widespread transcriptional responses

Domain: chemistry
Published: 2026-01-08
Editorial outcome: major_revision

Abstract
Interspecific genetic exchanges caused by natural hybridization or horizontal gene transfer can lead to enhanced phenotypes, which are often of interest for industrial applications and evolutionary research. However, transferring genetic materials between distantly related species, such as intergeneric yeasts, present technical challenges. In this study, we establish a method to transfer individual chromosomes from Saccharomyces cerevisiae (Sc) into Kluyveromyces marxianus (Km), an emerging model for bioproduction. The Sc chromosome of interest is circularized, genetically modified to carry Km centromeres and replication origins, and transformed into Km via protoplast transformation. Using this method, we generate two synthetic strains, each containing a full set of Km chromosomes and either Sc chromosome I or III. The Sc chromosomes exhibit normal replication, segregation, and active transcription after the transfer. The synthetic strains display enhanced phenotypes in flocculation and salt tolerance, which is found to be caused by transgressive expression of FLO9 and SPS22 on the transferred Sc chromosomes, respectively. Transcriptomic analysis reveal that transgressive expression is prevalent among the transferred Sc genes, suggesting evolution of lineage-specific cis- and trans-regulatory interactions across a long evolutionary timescale. Our strategy has potential applications in optimizing cell factories, constructing synthetic genomes, and advancing evolutionary research.

Introduction
The transfer of genetic materials between species is one of the major driving forces for adaptation1,2. Chromosomes, which carry packs of genes, can introduce abundant genetic variation and contribute to evolution of adaptive traits when transferred between species. For example, horizontal transfer of plastid chromosomes between tobacco plants (Nicotiana) aided chloroplast capture3. Horizontal transfer of chromosomes between Fusarium species, a group of filamentous fungi, led to new pathogenic lineages4. Furthermore, hybridization is by nature a process of interspecific chromosome transfers. Interspecific hybridization often leads to enhanced phenotypes, giving rise to industrially important strains such as the lager yeast Saccharomyces pastorianus5 and hybrid crops like triticale6. Therefore, introduction of genetic materials from a distantly related species may hold great potential for phenotypic improvement in agricultural and industrial practices. In addition, understanding the phenotypic and molecular consequences of interspecific genetic exchanges may shed light on genome evolution, such as the molecular bases for heterosis7 and evolution of gene regulation8,9.

In this study, we explored the phenotypic and molecular consequences of chromosomal transfers between Kluyveromyces marxianus (Km) and Saccharomyces cerevisiae (Sc). K. marxianus is a yeast species that belong to the Saccharomycetaceae family but only distantly related to S. cerevisiae (Fig. 1a). It has been previously argued that it may serve as an emerging model for bioproduction, including heterologous proteins, bioethanol and bulk chemicals10,11. It has a number of traits different from S. cerevisiae, including a high growth rate, thermotolerance, and the ability to assimilate a wide variety of sugars12–14. On the other hand, S. cerevisiae is more tolerant to ethanol than K. marxianus11. It is widely used in industrial fermentation and has arguably the best-studied genome among eukaryotes15. Mixing the genetic material between the two species is expected to provide phenotypes for industrial development, as well as insights for understanding their evolution.

Stable allodiploid hybrids between K. marxianus and S. cerevisiae haven’t been made available, although partial integration of S. cerevisiae genome fragments into K. marxianus has been achieved by protoplast fusion16,17. In order to introduce S. cerevisiae genetic materials in a systematic, controllable manner, we developed a genetic-engineering strategy to artificially transfer chromosomes between the two species. Artificial chromosome transfer was first established in 1977, when murine chromosomes were transferred into human cells via microcells18. To date, artificial transfer of functional chromosomes has primarily been achieved between closely related species (dashed lines, Fig. 1b), including transfers of human chromosome 21 into mouse and chicken cells19–21, and between bacteria species Mycoplasma mycoides and M. capricolum22. S. cerevisiae (Sc) and K. marxianus (Km) diverged 114 million years ago23, significantly exceeding the evolutionary distance between the species that have been shown capable of natural or artificial chromosomal transfers (Fig. 1b). Previous efforts of protoplast fusion showed that the Sc genome was often unstable after the fusion with Km17. One possible explanation is the incompatibility of replicating elements such as centromere (CEN) and autonomously replicating sequence (ARS), as previously shown in hybrid studies24,25.

In this study, we engineer the Sc chromosome with a series of genetic modifications to resolve potential incompatibilities, including circularization of the Sc chromosome and insertion of ARS and CEN from the host species26. The engineered Sc chromosome is transformed into Km, creating a “monochromosomal hybrid”. Through phenotypic and gene expression analysis, we show that the chromosome transfer led to beneficial phenotypes associated with overexpression of genes from the Sc chromosome. The transfer triggers widespread transcriptional responses in the transferred and host genomes, suggesting cross-species regulation. The pervasive transgressive expression found among the transferred genes provides evidence for regulatory evolution across a long evolutionary timescale, as well as potential implications for synthetic biology.

Results
Compatibility of ARS and CEN between Sc and Km
It is critical to ensure proper replication and segregation of the heterologous chromosome upon chromosome transfer. Therefore, we started by investigating the compatibility of ARS and CEN between Sc and Km. In S. cerevisiae, there are 228 functional ARS sites distributed across the genome27, sharing an 11-bp consensus sequence28. The distribution of ARS in the Km genome is less characterized, and only 15 Km ARSs have been experimentally validated29–32. Different from canonical ARS in other yeasts, Km ARSs are diverse and do not share a common consensus sequence32. There is one CEN per chromosome in both Sc and Km, each consisting of three conserved centromere DNA elements (CDEI, II and III)33. The Km CDEs are similar to their Sc counterparts, with the exception that the Km CDEII was approximately twice the length of that in Sc34. We selected two ARS-CEN pairs from each species for our experiments, based on their chromosomal locations and previously-demonstrated functionality in plasmids31,32,35–38. Pairs on the same chromosome were tested together to maximize intraspecific ARS-CEN compatibility: ScARSH4 and ScCEN635,36 (Sc chrII), ScARS137 and ScCEN4 (Sc chrIV), KmARS1 and KmCEN531,38 (Km chrV), and KmARS1832 and KmCEN3 (Km chrIII).

We examined transformation success and stability of plasmids carrying different combinations of KmARS1, KmCEN5, ScARSH4 and ScCEN6. We found that KmARS1 and KmCEN5 were respectively essential for successful transformation (Fig. 1c, left) and plasmid maintenance in Km (Fig. 1d, left). Neither could be replaced by their Sc counterparts, suggesting incompatibility. Plasmids carrying different combination of ARS and CEN from Sc (blue) and Km (red). KmCEN5+KmARS1 produced transformants in Sc (Fig. 1c, right), but with poor stability (Fig. 1d, right), suggesting that KmARS1, but not KmCEN5, can function in Sc. Similar results were obtained using combinations of KmARS18, KmCEN3, ScARS1 and ScCEN4 (Supplementary Fig. 1). The observed incompatibility could explain the rapid loss of Sc chromosomes in previous protoplast fusion between Km and Sc17. Finally, the plasmid containing both ARSs and CENs from Sc and Km, known as “double CEN/ARS plasmid”, could replicate and segregate stably in both species (Fig. 1c, d, right). Therefore, the issue of Sc chromosomal instability in Km may be resolved by engineering KmCEN and KmARS into Sc chromosomes.

Using double CEN/ARS plasmids carrying different antibiotic markers, we examined the mating rate between Km and Sc. We transformed Km and Sc haploids with plasmids carrying a kanR or hygR marker, mixed them in different combinations (see Methods) and selected for zygotes on double antibiotic medium (Fig. 1e). We found that the mating efficiency between Km MATa and MATα cells, as well as between Sc MATa and MATα cells, was around 16%. However, mixing 1.6×10^7 Km cells with 1.6×10^6 Sc cells, regardless of their mating types, failed to generate any zygotes, based on both double selection (Fig. 1e) and microscopic observation (Supplementary Fig. 2). These results demonstrated a prezygotic reproductive barrier between the two species, which urges a synthetic method for generating Km-Sc hybrids.

Engineering and transferring Sc chromosomes into Km
We selected the smallest Sc chromosome, chrI (252 kb, 85 genes based W303 annotation), and the third smallest chromosome, chrIII (341 kb, 156 genes), for proof-of-principle experiments of Sc-Km chromosomal transfer. The experimental pipeline is shown in Fig. 2a. First, we circularized the chromosomes of interest to avoid potential incompatibility of telomeres. Sc telomeres (TELs) are composed of TG1−3 repeat sequences, while Km TELs consist of a long repeat motif (25 nt)39. We removed the TELs from Sc chrI and chrIII, respectively, and joined the ends with KmURA3 with CRISPR/Cas940. To avoid homologous recombination at the telomeres, we also removed the telomere-associated long repetitive sequence on chrI during the circularization40. Next, to ensure stable maintenance of Sc chromosomes in Km, we inserted KmCEN5/ARS1 adjacent to the native CENs on chrI and chrIII. Given that chrI and chrIII contain 5 and 12 ARSs, respectively27, we placed another copy of KmARS1 close to a native ScARS, which was about ∼100 kb away from the KmCEN5/ARS1 in the circular chromosomes (see Methods for details). The engineered circular chrI and chrIII were named R1 and R3, respectively. Sc cells with R1 (“Sc-R1”) or R3 (“Sc-R3”) showed no fitness defect under normal culture conditions as well as in the presence of microtubule-depolymerizing and DNA-damaging agents (Supplementary Fig. 3), indicating that the introduction of KmARS1 and KmCEN5 did not affect chromosome replication in Sc. The circularization was confirmed by pulsed-field gel electrophoresis (PFGE), which showed an absence of linear chrI or chrIII in the gel (Fig. 2b). Following a protocol developed by Noskov et al.41, we extracted and column-purified R1 and R3, with the undesired linear chromosomes removed by exonuclease. Finally, the purified R1 and R3 were separately transformed into Km using protoplast transformation42, yielding four R1 transformants (Kluyveromyces-Saccharomyces-R1, “KS-R1” hereafter) and one R3 transformant (“KS-R3”). The low number of transformants indicates a need for further optimization of the transformation protocol, especially for transferring larger chromosomes.

We next examined the integrity of the transferred chromosomes. All four KS-R1 transformants retained six Sc-specific markers in R1 (Fig. 2c, see Supplementary Data 1 for marker positions), indicating successful transfer. The transfer was further confirmed by restriction digest (Fig. 2e) and whole genome sequencing, which found no mutations in the transferred R1. In the PCR analysis of KS-R3, marker 7 was absent (Fig. 2d). PFGE showed that the size of linearized R3 was smaller than the expected 340 kb (Fig. 2e). Genome sequencing revealed a 29 kb deletion between two direct repeats of putative Ty elements in R3 (Fig. 2f & Supplementary Fig. 4). The deletion was potentially due to homologous recombination between Ty elements, the rate of which has been shown to significantly increase in circular plasmids during transformation43. The deleted region contained 13 genes (MAK32, PET18, MAK31, HTL1, HSP30, YCR022C, YCR023C, SLM5, YCR024C-B, PMP1, YCR025C, NPP1, RHB1). We introduced the deleted region, in six individual segments via plasmids, into Km cells (Supplementary Fig. 4). None of the segments affected growth in (Supplementary Fig. 4), so it was unlikely that the deletion was an adaptive response to overcome incompatibility caused by single genes in this region.

We next evaluated the Km genome integrity after transformation. The PFGE patterns of Km chromosomes in KS-R1 and KS-R3 were consistent with the parent (Supplementary Fig. 5). Genome sequencing revealed 33 mutations in 11 ORFs, as well as 82 SNPs and indels in intergenic sequences in Km chromosomes in KS-R1 (Supplementary Data 2). KS-R3 contained 4 mutations in 4 ORFs, and 47 SNPs and indels in intergenic sequences (Supplementary Data 2). These mutations might have been induced during the protoplast transformation. Overall, the transfer of R1 or R3 into Km did not result in fusions, translocations, or other large-scale rearrangements in the Km chromosomes.

Subsequently, we examined the KS strains for any fitness defects. In rich medium (YPD), the growth curves of KS-R1 and KS-R3 were indistinguishable from Km-V, the parental Km strain with an empty vector (Fig. 2g). Under non-selective conditions, R1 and R3 were highly stable in the KS strains. The loss rate per generation for R1 and R3 in YPD was 0.11% and 0.84%, respectively (Supplementary Data 3), a level comparable to that of circular YACs in Km (0.33-0.64%)44. Furthermore, KS-R1 and KS-R3 exhibited robust growth in the presence of the microtubule-depolymerizing agents benomyl (BML) and thiabendazole (TBZ), DNA replication inhibitor hydroxyurea (HU), DNA-damaging agents methyl methane sulfonate (MMS) and camptothecin (CPT)45, or protein synthesis inhibitor cycloheximide (CHX) (Fig. 2h). Taken together, the introduction of R1 and R3 did not affect growth, chromosome segregation, DNA replication, or protein synthesis in rich medium. The Km cells were tolerant to the transplantation of Sc chromosomes, reflecting cellular plasticity.

Enhanced phenotypes due to interactions between Km-encoded trans-factors and Sc cis-regulatory sequences
We next investigated if the transferred chromosomes conferred different phenotypes. We treated the KS strains and their parents with 28 environmental conditions, chosen to represent common stressors for yeast growth (Fig. 3a & Supplementary Fig. 6-7). For KS-R1, the phenotypes of the four independent transformants were generally consistent (Supplementary Fig. 7), so we primarily analyzed one representative transformant (Fig. 3a & Supplementary Fig. 7). We primarily compared the KS strains to Sc and Km strains with an empty double CEN/ARS plasmid (referred to as Sc-V and Km-V respectively), which carries the same antibiotic and KmURA3 markers as the circularized chromosomes, to account for phenotypic effects associated with the vector (see Supplementary Fig. 8 for details). Sc-R1 and Sc-R3 were also examined to account for effects of chromosomal circularization and other genetic modifications, but their phenotypes were for the most times consistent with Sc-V (Fig. 3a & Supplementary Fig. 6).

For each strain, we semi-quantified the growth on solid media under different conditions, normalized the data to YPD growth at 30°C, and then adjusted the data in reference to Km-V to reflect inter-strain differences (Fig. 3a & Supplementary Fig. 6b). Sc-V and Km-V showed significant growth differences under 17 out of the 28 conditions (2% ethanol, Ser, 1/4N, 1/2AA 1/2N, 20, 37 and 42°C, 60 mM AcOH, and all three H2O2, TM and NaCl treatments; FDR-adjusted p<0.05 based on linear model, Supplementary Data 4), reflecting phenotypic divergence between species. KS-R1 and KS-R3 exhibited comparable growth to Km-V under 27 and 17 conditions, respectively (FDR-adjusted p>0.05, linear model; Fig. 3a & Supplementary Data 4), suggesting that the small number of genes in R1 and R3 did not trigger a widespread metabolic reprogramming in Km. Under a few conditions, R1 and R3 caused increased sensitivity in KS compared to Km, including the sensitivity to 42°C for KS-R1, and to tunicamycin (TM), 1/4N, 1/2AA 1/2N, 20°C, 0.016% and 0.024% H2O2 for KS-R3 (FDR-adjusted p<0.05, linear model, Fig. 3a-b & Supplementary Data 4). Under these conditions, the KS phenotypes were intermediate to those of Km and Sc, consistent with dominant effects exerted by the introduced Sc chromosomes. The defective phenotypes were not shared by the two strains, indicating that the defects were caused by specific genes in R1 or R3, rather than a general effect caused by chromosomal transfers. Additionally, we ruled out that the mutations in the Km genome caused the phenotypes, because the strains no longer exhibited the phenotypes after losing the Sc chromosomes (Supplementary Fig. 9).

Importantly, the chromosomal transfer resulted in enhanced phenotypes that were superior to both parents. KS-R3 exhibited enhanced tolerance to 0.8M and 1M sodium chloride (NaCl) compared to both parents (FDR-adjusted p<0.05, linear model in reference to Km-V; Fig. 3a & Supplementary Fig. 6b, Supplementary Data 4). High salt is a common stress in industrial processes. Therefore, increased salt tolerance is a beneficial phenotype for industrial production46. We next investigated the molecular mechanisms by which R3 increased salt tolerance in KS. There are three genes in R3 that have been associated with salt tolerance based on previous large-scale studies: SPS2247, STE5048 and HMLALPHA247. We expressed the three candidate Sc genes in Km with a centromeric plasmid, including their coding sequences (CDSs) and 1000 bp-upstream sequences (promoters). We found that only ScSPS22 enhanced salt tolerance to the same level as KS-R3 (Supplementary Fig. 10, Fig. 3c, upper panel), indicating that it was the causal gene. ScSPS22 is involved in β-glucan synthesis and related to cell wall function49. In S. cerevisiae, overexpression of ScSPS22 was previously found to cause reduced salt tolerance47, which was recapitulated in our study (Fig. 3c, lower panel). It remains to be understood why overexpression of ScSPS22 had an opposite effect in Km than Sc, possibility related to the difference in their cell-wall composition50.

In order to understand the contribution of cis-regulatory and coding sequences (CDS) to the phenotypic effect of ScSPS22, we constructed a chimeric allele consisting of the promoter of KmSPS22 (FIM1_814) and the CDS of ScSPS22, namely Pkm-ScSPS22. There was no substantial difference between Pkm-ScSPS22 and full-length KmSPS22 when expressed with a centromeric plasmid (Fig. 3c, upper panel), suggesting a lack of functional divergence in the CDS. On the contrary, Pkm-ScSPS22 conferred a much lower level of salt tolerance than ScSPS22 driven by its endogenous promoter, indicating that the Sc promoter was causal for the increase in salt tolerance.

The different phenotypic effects of Sc and Km promoters suggest potential divergence in gene expression. Therefore, we analyzed the transcription level of ScSPS22 and KmSPS22 in KS-R3 and the parental strains by qPCR (Fig. 3d). The relative mRNA level of ScSPS22 in KS-R3 was respectively 53 and 80 times higher than those in Sc-R3 with and without NaCl (Fig. 3d, right panel), suggesting that (1) a significant difference in trans-acting regulators for ScSPS22 between Sc and KS-R3; and (2) the up-regulation of ScSPS22 in KS-R3 is constitutive. The relative mRNA level of KmSPS22 did not significantly differ between KS-R3 and Km-V (adjusted p-value=0.056, Tukey’s test; Fig. 3d, left panel), indicating little difference in the trans-acting factors for KmSPS22 between Km and KS-R3.

When transformed into Km on a centromeric plasmid, the ScSPS22 allele was expressed at the same level as ScSPS22 in KS-R3 (adjusted p-value=0.945 and 1.000 for YPD and NaCl conditions respectively, Tukey’s test, Fig. 3d), suggesting that the promoter sequence of ScSPS22 was causal for its up-regulation in KS-R3. The chimeric allele Pkm-ScSPS22 drove lower expression than ScSPS22 in Km, consistent with cis-regulatory divergence. In Sc-R3, expressing ScSPS22 with centromeric or high-copy 2μ plasmids increased its relative mRNA level, but failed to match the level of ScSPS22 in KS-R3, suggesting that the upregulation of ScSPS22 in KS-R3 resulted from induction or de-repression by Km-specific trans-acting factors, rather than a dosage effect. Taken together, the phenotypic and expression analysis showed that the increased salt resistance of KS-R3 was caused by specific interactions between Km-encoded trans-acting regulators and the Sc promoter of SPS22.

Although KS-R1 did not show any superior phenotype among the conditions assayed in Fig. 3a, we additionally found that it exhibited a flocculation phenotype surpassing that of Km-V and Sc-R1 (Fig. 3e, f). The flocculation phenotype disappeared after the loss of R1, indicating that R1 was responsible for the enhanced flocculation (Supplementary Fig. 11). Flocculation facilitates cell sedimentation, reducing separation costs in high-density fermentation51. The FLO family genes control flocculation52, among which ScFLO9 is present on R1 and has been shown to cause flocculation in Km53. Introducing ScFLO9 with its 1000 bp upstream sequence into Km via a centromeric plasmid enhanced flocculation to the same level as KS-R1 (Fig. 3e & f). This result suggests that ScFLO9 was responsible for the increased flocculation phenotype in KS-R1. Overexpressing ScFLO9 with a centromeric or 2μ plasmid in Sc slightly enhanced flocculation (Fig. 3e & f), consistent with its known role in Sc but suggesting its effect is background-dependent.

We similarly analyzed the expression level of FLO9 to understand the molecular mechanisms of the enhanced phenotype. qPCR showed that the relative mRNA level of ScFLO9 in KS-R1 was 64.5 times higher than in Sc-R1 (Fig. 3g), again suggesting a significant change in the trans-regulatory environment. The high expression in KS-R1 was recapitulated by ScFLO9 expression from a centromeric plasmid in Km (light pink bars in Fig. 3g, adjusted p-value=0.241, Tukey’s test). The expression level of KmFLO5 (FIM1_6), the ScFLO9 ortholog, was not increased in KS-R1, consistent with a lack of change in trans environment for Km genes (Fig. 3g, left panel). Overexpressing ScFLO9 in Sc-R1 with centromeric and 2μ plasmids led to an increase in the mRNA level of ScFLO9 (purple bars in Fig. 3g), with the higher level of ScFLO9 2μ correlating with faster flocculation (Fig. 3f). Taken together, the enhanced flocculation of KS-R1 was associated with ScFLO9 being exposed to an alien trans-regulatory environment encoded by the Km genome. In both cases of SPS22 and FLO9, the genetic combinations between Km-encoded trans-factors and Sc-encoded cis-factors could never be found in nature due to reproductive isolation, demonstrating the power of cross-species synthetic biology.

Pervasive transcriptional responses triggered by chromosomal transfer
The experiments with ScSPS22 and ScFLO9 revealed significant changes in Sc gene expression after being transferred to Km. To determine if this is a general pattern, we analyzed transcriptomes of KS and the parental strains in YPD and under two stress conditions, 1μg/mL tunicamycin (TM) and 1M NaCl. The genes on R1 and on R3 were actively transcribed in KS under the three conditions (read counts > 5) (Supplementary Data 11). However, their expression in KS was poorly correlated with that in Sc, with an average correlation coefficient (R²) of 0.38 for R1 genes and 0.32 for R3 genes (Fig. 4a). This correlation was substantially lower than the correlation between the expression of human genes on Hsa21 transplanted into mouse cells and their counterparts in human cells (R²=0.90)21, which aligns with the remote evolutionary distance between Km and Sc (Fig. 1b). Compared to Sc, on average, 22.7% and 18.3% of the genes in R1 and R3, respectively, were up-regulated in KS, while 27.3% and 19.3% of the genes in R1 and R3 were down-regulated in KS, respectively (FDR-adjusted p<0.05 by Wald test and absolute log2FoldChange>1; Fig. 4b, Supplementary Data 5). The median log2-fold change for up- and down-regulated genes was 1.81 and −1.57, respectively. Consistent with the case studies of SPS22 and FLO9, the high proportion and fold change of differentially expressed Sc genes reflect dramatic differences in the trans-regulatory environment between Sc and the KS strains.

The genes showing differential expression between Sc and KS were, in general, uniformly distributed across the transferred chromosomes (Supplementary Fig. 12), but we found a region around HML that showed de-repression across multiple genes in KS-R3, providing an interesting example for chromatin-level regulatory divergence. The HML locus, along with its flanking genes GEX1 and VBA3, is repressed in Sc by SIR complex54. Consistent with this previous knowledge, we found little expression of these genes in Sc (Fig. 4c, upper panel). Interestingly, GEX1, VBA3, and HML genes were highly expressed in KS-R3 (Fig. 4c, lower panel), indicating de-repression. qPCR confirmed that the relative mRNA levels of α1 and α2 were upregulated 42-fold and 39-fold, respectively, in KS-R3 compared with Sc-R3 (Fig. 4d). The differential expression might be explained by different composition of silencers between Kluyveromyces and Saccharomyces55. The silencer of HML in KS-R3 might not be able to recruit essential proteins such as Rap1 and ORC to the locus56, contributing to the de-repression of α1 and α2, and the flanking genes GEX1 and VBA3.

While the expression of Sc genes underwent significant changes in KS, R1 and R3 also affected the expression of Km genes (Fig. 4e). Out of 4,975 Km genes, 931 (18.7%) and 803 (16.1%) genes in KS-R1 and KS-R3, respectively, exhibited significant expression differences compared to Km-Vector in YPD (FDR-adjusted p<0.05 by Wald test and absolute log2FoldChange>1; Fig. 4e, Supplementary Data 5). R1 encodes a transcription factor, Oaf1, that regulates at least 97 Sc target genes57. Among the 73 Km orthologs of the known Sc target genes (see Methods for ortholog assignment), 27 (37.0%) showed differential expression in KS-R1. R3 encodes four transcription factors regulating at least 305 Sc target genes57, with 44/160 (27.2%) of their Km orthologs differentially expressed in KS-R3 (Supplementary Data 5). This suggests that transcription factors encoded by R1 and R3 might directly alter gene expression in Km, but a large fraction of the observed transcriptional changes might be explained by indirect or uncharacterized regulation. Under TM and NaCl stresses, the number of differentially expressed genes was substantially lower than in YPD (Fig. 4e, Supplementary Data 5). These results indicate that the heterologous chromosomes induced pervasive changes in expression of the endogenous genome, potentially via Sc-Km regulatory interactions. Interestingly, regulation from the Km genome became dominant under stress conditions, removing many of the effects associated with foreign chromosomes found in YPD.

Co-evolution of cis- and trans-regulatory factors
Transferring genetic materials between remotely related species provides a unique opportunity to examine the evolution of gene expression. Evolutionary divergence of gene expression can be caused by the evolution of cis- or trans-regulatory factors, or both. Cis-regulatory elements such as promoters or enhancers are physically linked to a target gene (e.g., “promA” for “Gene A” in Fig. 5a). The regulatory activity of cis-elements may evolve through changes in transcription factor binding sites or other elements that interact with the transcription machinery. Trans-acting factors often refer to regulatory proteins such as transcription factors (TFs) encoded elsewhere in the genome (e.g., transcription factor “B” in Fig. 5a). They may include any direct or indirect regulators, which may evolve through coding or non-coding changes58, even including turnover of transcription factors (e.g., “B” may be replaced by “C” over time, Fig. 5a). It has been proposed that cis-acting changes and cis-trans co-evolution plays an increasingly important role as species diverge59, but this idea has been rarely tested in species as diverged as S. cerevisiae and K. marxianus.

The KS strains allowed us to interrogate this question (Fig. 5b)60,61. If the transferred genes showed the same expression level in KS (“KSsc”) as that in S. cerevisiae (“Psc”), it means that the expression divergence is entirely encoded by the Sc cis-regulatory elements (the change in trans-environment from Sc to KS had no effect; “donor-like” in Fig. 5c). If the Sc allele was expressed at the same level as its orthologous Km allele in KS (“KSkm”), the expression divergence is entirely determined by trans-differences (the cis-regulatory elements of Sc and Km had equivalent activities when exposed to the same trans-environment; “recipient-like” in Fig. 5c). An intermediate or transgressive expression level of Sc alleles in KS indicates contributions from both factors. In particular, transgressive expression, defined by a KSsc level outside the range of Psc and KSkm, may suggest cis-trans interactions specific to the synthetic background (Fig. 5c).

In order to characterize cis- and trans- regulatory divergence between Sc and Km, we identified Km orthologs for 62/85 genes on R1 and 100/156 genes on R3, with an average amino-acid sequence similarity of 48.0% (BLOSUM62-based similarity, see Methods and Supplementary Data 6). 106 out of the 162 genes showed significant expression divergence between Sc and Km (FDR-adjusted p<0.05, Wald test). What factors determined their divergence? Focusing on the Sc allele of these genes, we classified them into Sc-like, intermediate, Km-like, transgressive and others based on the criteria in Fig. 5c, using the transcriptomic data in YPD (Fig. 5d, Supplementary Data 7). Interestingly, we found that most transferred genes showed transgressive expression (40/106 genes, 37.7%, Fig. 5d), more than those determined by cis-alone (Sc-like) or trans-alone (Km-like). SPS22 and FLO9 analyzed in our phenotypic studies (Fig. 3) were among the most prominent examples in this class (Fig. 5d), suggesting that transgressive expression might be an important source of phenotypic improvement in these engineered strains. Transgressive expression was also the most abundant class under NaCl and TM stresses (54/132 and 36/122 genes respectively, Supplementary Fig. 13, Supplementary Data 7). Principal Component Analysis (PCA) of the 162 transferred genes across treatments and strains showed that KSsc levels were distinct from Pkm, KSkm and Psc, consistent with pervasive transgressive expression of Sc alleles (Fig. 5e). The second most abundant class in YPD was Sc-like expression (25/106, Fig. 5d), supporting an important role of cis-regulatory evolution. However, there were more Km-like genes than Sc-like genes under NaCl and TM stresses (Supplementary Fig. 13), consistent with a predominant role of trans-evolution in environmental responses62.

In the analysis above, we defined “recipient-like” by comparing KSsc to KSkm, reasoning that this was a conservative estimation of cis-effects, when the two alleles were exposed to the same trans-environment in KS. This was different from previous analyses of introgression lines60 or enhancer swaps61, where the transferred allele was compared to the recipient parent (Pkm in this case). The methodological difference was largely negligible because the trans-environment in KS was highly similar to Km, as shown in PCA (Pkm and KSkm clustered together, Fig. 5e). However, to account for the minority of cases where the transferred Sc genes influenced Km allelic expression (Fig. 4e), we performed the same analysis using KSsc/Pkm instead of KSsc/KSkm (Supplementary Fig. 14). This second method was less conservative with cis-effects, but it provided a more authentic estimate for transgressive expression: in the cases where KSkm showed transgressive expression in KS, transgressive expression of KSsc might be missed or overestimated by the first method. The results still supported the prevalence of transgressive expression of the transferred genes (37/106 genes in YPD), although there were more genes classified into Sc-like, as expected (Supplementary Fig. 14).

The prevalence of transgressive expression in KS suggests that the cis- and trans-regulatory factors often evolved in opposite directions within a lineage63. For example, when an Sc allele showed transgressively high expression in KS (KSsc > KSkm and KSsc > Psc, Fig. 5c and f), it might indicate that (1) the Sc promoter accumulated more changes to increase gene expression compared to the Km promoter, via either stronger activator activity or weaker repressor activity (“+” effects for cis-changes in the Sc lineage and correspondingly “-” effects in the Km lineage, Fig. 5f), such that KSsc > KSkm; and (2) a trans-acting factor that decreased gene expression must have co-evolved with this promoter in S. cerevisiae (“-” effects for trans-changes in the Sc lineage and correspondingly “+” effects in the Km lineage, Fig. 5f), such that KSsc > Psc.

Compensatory evolution may also be identified by examination of genes showing conserved level of expression between the two species (Supplementary Fig. 15)64. For two genomes as diverged as Sc and Km, it is as important to understand the conservation of gene expression as to understand their divergence. Across 4,307 genes with identifiable orthologs between Km and Sc-R1 (see Methods for ortholog assignment), 29.7% showed no difference in expression levels between the parental strains in YPD (FDR-adjusted P>0.05, Wald test; Supplementary Fig. 15b, c, Supplementary Data 7). This ratio was 21.9% and 12.8% under NaCl and TM conditions respectively. The expression level of the 4,307 orthologs was correlated genome-wide between species (Spearman’s rank correlation rho = 0.647 for YPD data, P<2.2e-16, Supplementary Fig. 15b), suggesting a certain level of regulatory conservation despite the deep divergence. The genes showing conserved expression in YPD were significantly enriched for rRNA processing (GO:0006364, corrected P=2.07e-20) and ribosome biogenesis (GO:0042254, corrected P=2.61e-19), consistent with a housekeeping role. There was a similar proportion of genes showing conserved expression among the transferred genes (56 out of 162, 34.6% in YPD; 18.5% in NaCl and 24.7% in TM, Supplementary Fig. 15d), providing us with a small sample to peek into the driving force of regulatory conservation – did the cis and trans regulatory factors both maintained a conserved level of activity, or they evolved in a compensatory manner under stabilizing selection? In the latter case, we would expect to find cis-regulatory divergence in the KS strains. Indeed, we found that 42 out of the 56 (75%) conserved genes showed significant cis-divergence in the KS strains in YPD (23/30 in NaCl and 29/40 in TM; FDR-adjusted P<0.05, Supplementary Fig. 15d–f), consistent with compensatory cis-trans evolution. The top genes showing this trend in YPD were CDC19, TRX3, and HIS4 (Supplementary Fig. 15e). In all three cases, the Sc allele was down-regulated in KS strains, suggesting evolution of Sc-specific activators. We note that the amount of cis-divergence found in KS may be specific to the given trans environment, e.g., the cis-effects in KS may or may not manifest themselves in an F1 hybrid. However, such dependency itself indicates co-evolution of cis-trans interactions (see Discussion).

Taken together, our results support that lineage-specific co-evolution of cis- and trans-regulatory factors was predominantly involved in the evolution of gene expression between S. cerevisiae and K. marxianus. Transgressive expression was the most prevalent pattern among the transferred genes in KS. Cis-only and trans-only evolution also occurred, with the latter predominantly contributing to environmental responses62. At the same time, around one-third of the yeast genome showed conserved gene expression despite the long evolutionary distance. Compensatory cis- and trans-evolution might underlie such conservation, consistent with stabilizing selection.

Methods
Strain and media
Yeast strains used in this study are listed in Supplementary Data 8. Sc strain W303-1A (MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15) was used for chromosome engineering. The engineered Sc chromosome was transferred into a ura3Δ Km strain, FIM-1ΔU85.

The following media were used in this study: YPD (1% yeast extract, 2% peptone, 2% dextrose)86, synthetic medium lacking uracil or leucine (SD-Ura or SD-Leu) [0.17% yeast nitrogen base without amino acids and ammonium sulfate, 2% glucose, 1 g/L sodium glutamate, 2 g/L DO Supplement-Ura (630416, Takara) or DO Supplement-Leu (630414, Takara), 2% agar for plates], SD (same recipe as SD-Ura, with the addition of 20 mg/L uracil), and ME (2% malt extract, 3% agar). G418 (A600958, Sangon) and hygromycin (H8080, Solarbio) were added to YPD at final concentrations of 200 mg/L and 300 mg/L, respectively, to prepare YPDG and YPDH media. Both antibiotics were supplemented into YPD and SD-Ura to prepare YPDGH and SD-Ura +GH, respectively. Unless otherwise indicated, cells were grown at 30°C.

Plasmids
All plasmids used in this study are listed in Supplementary Data 9. All primers are listed in Supplementary Data 1. A previously published vector LHZ626 served as a control for circular chromosomes42, which contains CEN and ARS elements from both Sc and Km (double CEN/ARS plasmid), as well as KmURA3, kanMX6, and hphMX4.

To construct plasmids for diploid selection, a fragment containing ScARSH4 and ScCEN6 was amplified from LHZ626 and inserted into the HpaI and SpeI sites of a Km-centromeric plasmid, LHZ88234, yielding LHZ1493. KanMX6 (kanR) was amplified from pFA6a-KanMX6 and used to replace hphMX4 (hygR) in LHZ1493, producing LHZ1494.

To create plasmids with different combinations of ARS and CEN, a BamHI site was first introduced between KmARS1 and KmCEN5 of LHZ882 to generate LHZ1495. Then, ScCEN6, ScARSH4, ScARSH4-ScCEN6 were inserted into LHZ1495 to replace their Km counterparts, generating LHZ1496-1498, respectively. Next, KmARS18 and KmCEN3 were inserted between the HindIII and SalI sites of LHZ1495 to replace KmARS1 and KmCEN5, giving rise to LHZ1783. ScARS1-ScCEN4, ScARS1, and ScCEN4 was inserted into LHZ1783 to replace their Km counterparts, generating LHZ1784-1786, respectively (see Supplementary Data 9 for restriction sites). Finally, ScARS1 and ScCEN4 were inserted into the SalI site of LHZ1783 to obtain LHZ1787, a plasmid with ARS and CEN from both species.

In order to remove the telomeres and join the chromosomal ends, two intermediate CRISPR plasmids targeting the chromosome ends were constructed by individually cloning the gRNAs into the SapI site of pRS425-Cas9-2×SapI. The gRNA cassette from one plasmid (containing the SNR52 promoter, gRNA, and SUP4 terminator) was then inserted into the NotI site of the other plasmid to construct the double-gRNA plasmids LHZ1499 (for chr1) and LHZ1500 (for chr3). In order to insert KmARS1 and KmCEN5 into the circularized chromosomes, we cloned the respective gRNAs into the SapI sites of pRS425-Cas9-2xSapI to construct LHZ1501∼1504. The homologous repair templates (donor) were assembled into pMD-18T with In-Fusion Snap Assembly (Clontech), according to the designs in Supplementary Data 10.

For flocculation analysis, the ScFLO9 cassette, including 1000 bp upstream, the ORF of ScFLO9, and 200 bp downstream sequence, was amplified from the genome of W303-1A. This cassette was inserted into the NotI site of LHZ626 to produce LHZ1505, and into the SmaI and SpeI sites of pRS315 (centromeric) and pRS425 (2-micron) to produce LHZ1509 and LHZ1510, respectively.

For NaCl tolerance analysis, the ScSPS22 cassette, including 1000 bp upstream sequence, the ORF of ScSPS22, and 200 bp downstream sequence, was amplified from the genome of W303-1A. This cassette was inserted into the NotI site of LHZ626 to produce LHZ1506, and into the SmaI and SpeI sites of pRS315 and pRS425 to produce LHZ1511 and LHZ1512, respectively. The 1000 bp upstream sequence of ScSPS22 in LHZ1506 was replaced by the 1000 bp upstream sequence of KmSPS22 to produce LHZ1507. The KmSPS22 cassette, including 1000 bp upstream sequence, the ORF of KmSPS22, and 200 bp downstream sequence, was amplified from the genome of FIM-1ΔU and inserted into the NotI site of LHZ626 to produce LHZ1508. The full sequences of LHZ626, LHZ1495, and pRS425-Cas9-2xSapI are listed in Supplementary Data 9.

Transformation efficiency and plasmid stability
Fim-1ΔU and W303-1A were grown in liquid YPD overnight. Cells from 1 mL-culture were pelleted and transformed with LHZ626, LHZ1495∼LHZ1498, respectively, using the lithium acetate (LiAc) method87,88. Briefly, Fim-1ΔU cells were washed and mixed with 5 μL carrier DNA (Takara, 630440), 1 μg transforming DNA, 0.4 mL PEG solution (40% PEG 4000, 0.1 M LiAc, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA), and 4.2 μL 1 M DTT. The mixture was incubated at 30°C for 15 min and then heat-shocked at 47°C for 15 min. Cells were pelleted and spread onto selective plates. Transformation of W303-1A followed the same protocol with the following modifications: DTT was omitted from the mixture, the volume of PEG solution was adjusted to 0.3 mL, and the mixture was incubated at 30°C for 30 min, heat-shocked at 42°C for 25 min, then incubated at 30°C for an additional 10 min. Colony-forming units (CFUs) on YPDH plates were counted after 3 days, to evaluate transformation efficiency (CFUs/μg DNA). To measure the stability of the plasmid, transformants were grown in liquid YPD for 24 h. The cells were then diluted and spread onto YPD and YPDH plates. Stability was determined by dividing the CFU counts on YPDH plates by those on YPD plates. The experiments were replicated three times.

Mating assay
The quantitative mating assay was performed as previously described89 with modifications. Strains transformed with LHZ1493 (hygR) were used as experimental cells, and strains transformed with LHZ1494 (kanR) served as tester cells. The experimental and tester cells were cultured overnight in YPDH and YPDG liquid media, respectively. Cells were then washed and resuspended in water to an OD600 of 1. In each cross, 100 μL experimental cells were mixed with 500 μL tester cells. The mixture resulted in a 1:5 ratio of experimental to tester cells when both cell types were Km or Sc. It resulted in a 1:10 ratio of Sc experimental cells to Km tester cells, as CFU per OD600 of Km was twice that of Sc. The mixed cells were pelleted, resuspended in 20 μL ddH2O, and spotted onto ME plates. After incubation at 30°C for 24 h, the cells were washed off the ME plates with ddH2O and then pelleted and resuspended in 1 mL ddH2O. To examine the mating result, 3 μL cell suspension was spotted on YPDH, YPDG, and YPDGH (Fig. 1e). These plates were incubated at 30°C for 24 h. To quantify mating efficiency, the cells were diluted and plated onto YPD and YPDGH plates. The mating efficiency was calculated as the number of colonies on YPDGH plates divided by one-sixth of the number on YPD plates, reflecting the 1:5 ratio of experimental to tester cells. The experiments were replicated three times.

Engineering of Sc chromosome I and III
Each step of engineering Sc chromosome I (chr1) or III (chr3) involved transforming a CRISPR plasmid (Supplementary Data 9) and a donor, which is a PCR product for homologous recombination. The composition and full sequences of the donors are listed in Supplementary Data 10.

Chr1 and chr3 were circularized as previously described90. First, LHZ1499 or LHZ1500 was transformed into W303-1A to induce double-strand breaks in both telomeres in chr1 or chr3. A donor “chr1-cir” or “chr3-cir” containing KmURA3 with homologous arms to the chromosomal ends was simultaneously transformed to connect the two chromosomal ends with KmURA3. The constructs above were designed to remove the left telomere (0-1111 bp) and the right telomere along with the repetitive sequence (208917-252221 bp) of chr1, and the left telomere (0-831 bp) and the right telomere (334198-341087 bp) of chr3. The donors contain direct repeat (DR) sequences90, allowing marker recycling by popping out KmURA3 in future studies. Successful circularization was selected by the SD-Ura-Leu medium. The transformants with circularized chromosomes were named Sc-ring1 and Sc-ring3.

In order to insert KmARS1 and KmCEN5 into the circularized chromosomes, we transformed LHZ1501 and a donor “chr1-ARS1” into Sc-ring1 to insert KmARS1 and hygR at 66090 bp of chr1, resulting in a strain named Sc-ring1L. LHZ1502 and a donor “chr1-ARS1/CEN5” were transformed into Sc-ring1L to insert KmARS1/KmCEN5 and kanR at 157346 bp of chr1, resulting in a strain named Sc-R1. Similarly, LHZ1503 and a donor “chr3-ARS1/CEN5” were transformed into Sc-ring3 to insert KmARS1/KmCEN5 and hygR at 108718 bp of chr3, resulting in a strain named Sc-ring3L. LHZ1504 and a donor “chr3-ARS1” were transformed into Sc-ring3L to insert KmARS1 and kanR at 243183 bp of chr3, resulting in a strain named Sc-R3. All transformations were performed following the protocol for W303-1A described in the “Transformation efficiency and plasmid stability” section.

Extraction and protoplast transformation of R1 and R3
R1 and R3 were extracted from Sc-R1 and Sc-R3, respectively, following a previously described method41. In brief, two 200 mL cultures of Sc-R1 or Sc-R3 were grown in SD-Ura+GH medium to an OD600 of 1.5. Cells from each 200 mL culture were harvested, washed, and resuspended in 40 ml SPE buffer (1 M sorbitol, 0.1 M sodium citrate, 0.01 M EDTA, pH 7.5). Zymolyase 20T solution (10 mg/mL, 250 μL; E1005, Zymo Research) and β-mercaptoethanol (40 μL) were added, followed by incubation at 37°C for 20 min to form spheroplasts. The spheroplasts were collected and gently resuspended in 1 mL 1 M sorbitol. Cell lysis was performed by adding 20 mL lysis buffer (50 mM Tris-HCl, 20 mM EDTA, 1% SDS, pH 12.8), followed by gentle inversion and incubation at 37°C for 30 min. An equal volume (20 mL) of phenol:chloroform:isoamyl alcohol (25:24:1) was added, mixed gently, and then centrifuged. The aqueous phase (~20 mL) was collected, supplemented with 2 mL 3 M sodium acetate (pH 5.2) and 20 mL isopropanol, mixed, and centrifuged for 30 min. The DNA pellet was washed once with 70% ethanol and dissolved in 1 mL TE buffer (pH 8.0) containing 3 μL RNase A (10 mg/mL), followed by incubation at 37°C for 30 min. DNA from the two preparations was combined and treated with 10 mL Buffer EX, 200 μL ATP-dependent exonuclease, and 300 μL 100 mM ATP at 37°C for 30 min to remove linear DNA. The sample was mixed with 12 mL Buffer QS, centrifuged, and the supernatant was loaded onto a column equilibrated with Buffer QBT. The column was washed with 30 mL Buffer QC and DNA was eluted with 12 mL Buffer QF (reagents used after RNase treatment were from large-construct kit, 12462, Qiagen). GlycoBlue (15 μL; AM9515, Ambion), tRNA (15 μL; R5636, Sigma), 1.2 mL 3 M sodium acetate (pH 5.2), and 12 mL isopropanol were added to precipitate the DNA. After centrifugation for 50 min, the pellet was washed once with 70% ethanol, air-dried, and finally dissolved in 100 μL TE buffer. A total of 10 μL R1 or R3 was transformed into Km by protoplast transformation42. Briefly, 25 mL of an overnight culture of Fim-1ΔU cells was harvested, washed, and resuspended in 20 mL citrate–phosphate buffer (10 mM citrate–phosphate, 1.5 M sorbitol, pH 6.8). Zymolyase-20T solution (60 μL) and β-mercaptoethanol (40 μL) were added, followed by incubation at 30 °C for 15~20 min to form protoplasts. Protoplasts were collected, washed twice with 1.5 M sorbitol, and gently resuspended in STC solution (1.5 M sorbitol, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5) to a final concentration of 3×10^8 cells/mL, as determined with a hemocytometer. A 100 μL aliquot of the protoplast suspension was gently mixed with R1 or R3 DNA and 5 μL carrier DNA, incubated on ice for 15 min, and then supplemented with 1 mL PEG8000 solution (20% PEG8000, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5). After gentle mixing and incubation on ice for 15 min, protoplasts were pelleted, resuspended in 800 μL STC solution, and mixed with 10 mL regeneration agar (6.9 g/L yeast nitrogen base without amino acids, 2% glucose, 0.8 M sorbitol, 3% agar, 2 g/L DO Supplement-Ura) equilibrated at 47 °C. The mixture was poured onto the selective plates (the same recipe as regeneration agar except containing 2% agar). After 3~4 days, colonies were restreaked onto SD-Ura+GH plates.

Pulsed-Field Gel Electrophoresis (PFGE)
Plugs containing the genome of Sc, Km, and KS strains were prepared by using the CHEF Yeast Genomic DNA Plug Kit (170-3593, Biorad). To separate circular R1 from linear chromosomes in KS-R1, chromosomes were separated on the CHEF MAPPERTM XA System in a 1% pulsed field certified agarose gel (162-0137, Biorad) in 0.5×TBE (diluted from 10×TBE, T1051, Solarbio) at 14°C. The running time was 24 h at 6.0 V cm-1, with a 60∼120 sec switch time ramp at an included angle of 120°. The plug containing the entrapped R1 was cut out and linearized with NotI as described in the manufacturer’s protocol. Similarly, R3 in KS-R3 was separated from linear chromosomes and then linearized with AscI. Chromosomes of W303-1A, Sc-R1, Sc-R3, linearized R1 and R3 were separated in a 1% pulsed field certified agarose gel in 0.5×TBE at 14°C. The running time was 16 h at 6.7 V cm-1, with a 10∼40 sec switch time ramp at an included angle of 120°. Lambda PFG Ladder (N0341S, NEB) was used as a size marker for PFGE.

Growth curves of KS-R1 and KS-R3 and the stabilities of transferred chromosomes
As a control, Fim-1ΔU was transformed with LHZ626 to produce Km-V (Km-Vector), which confers G418 and hygromycin resistance, as well as Ura+ growth. KS-R1, KS-R3, and Km-V were cultured in YPDGH liquid medium overnight. The overnight culture (referred to as day 0 culture) was diluted into liquid YPD to an initial OD600 of 0.2 and grown at 30 °C. To monitor growth curves, the OD600 of the culture was recorded every 2 hours for the first 14 hours. To assess the stability of the transferred chromosomes, the culture was diluted into to an OD600 of 0.2 with fresh YPD every 24 hours, a period corresponding to approximately 7 generations. Cultures from day 0 and after 5 days of growth were diluted and plated onto YPD and YPDGH plates. The loss rate of transferred chromosomes per generation (x) was calculated using the following formula:
X= 1-e ( ln(N35/N0)/35 ) ×100% (1)
where N0 and N35 represent the ratios of CFU counts on YPDGH plates to those on YPD plates after 0 and 35 generations of non-selective growth in YPD, respectively34. The experiments were replicated three times.

Genome sequencing
Km-V, Sc-R1, Sc-R3, KS-R1, and KS-R3 were cultured overnight in SD-Ura+GH liquid medium. The cultures were diluted to an OD600 of 0.2 and cultured until the OD600 reached 0.6. The cells were collected and washed once with ddH2O. Genomic DNA was extracted using the E.Z.N.A. Fungal DNA Kit (D3390, OMEGA Bio-Tek) and sequenced on the Illumina NovaSeq 6000 platform using the 150-bp pair-end sequencing strategy (BIOZERON, Shanghai, China).

To construct the reference sequences for R1 and R3, sequences of chr1 and chr3 were extracted from the W303-1A genome (GenBank assembly: GCA002163515.1). We then manually modified the sequences to reflect our genome edits, including the insertions of Km elements and deletions of telomere sequences, giving rise to reference sequences R1 and R3 (provided in Supplementary Data 1). Annotations for R1 and R3 were transferred from S288C annotations (R64) with SnapGene. The whole-genome sequencing data were processed by BIOZERON biotechnology. To identify de novo variants during circular chromosome transfer, the raw sequencing data of Sc and KS strains were aligned to reference sequences for R1 or R3 using BWA91 with default settings. Picard tools were used to remove PCR duplicates. The average sequencing depth of KS-R1, Sc-R1, KS-R3, and Sc-R3 was 298, 1,183, 1,241 and 1,134 for the respective R1 or R3 chromosome. SNPs and indels were called following the best practices of GATK HaplotypeCaller92. Then, all heterozygous variants were removed from the VCF files of KS-R1 and KS-R3. All variants coexisting in both KS-R1 and Sc-R1 or KS-R3 and Sc-R3 were filtered out from the KS-R1 and KS-R3 VCF files as well. The remaining variants were considered to be de novo mutations generated during the chromosome transfer. In the case of R1, no de novo mutation was found.

To identify variants in Km, raw sequencing data of KS-R1, KS-R3, and Km-V were aligned to the reference genome of FIM1 (GenBank assembly: GCA_001854445.2)93 combined with either R1 or R3 sequence, using BWA with default parameters. Picard tools were used to remove PCR duplicates. The average sequencing depth of the Km genome for KS-R1, KS-R3, and Km-V was 1,036, 1,102, and 615, respectively. GATK HaplotypeCaller was used to identify the variants in these strains, following its best practices. VCFtools94 was used to remove variants shared between Km-V and KS-R1 or KS-R3, as well as heterozygous calls. The remaining unique, homozygous variants in KS-R1 and KS-R3 were listed in Supplementary Data 2.

Spot assay and quantification of growth phenotypes under various conditions
To investigate whether the chromosomal transfer resulted in enhanced phenotypes, W303-1A was transformed with LHZ626 to construct a control strain Sc-V (Sc-Vector). Km-V, Sc-V, Sc-R1, Sc-R3, KS-R1, and KS-R3 were cultured overnight in liquid SD-Ura+GH medium. The cultures were diluted to an OD600 of 1.0 and subjected to five serial 5-fold dilutions. These dilutions were spotted on plates using a 48-pin replicator. To evaluate the growth in different carbon sources, we used YP in combination with 2% ethanol, 3% glycerol, or different concentrations of glucose (0.02%, 1%, 3%, and 5%). To evaluate the growth in various nitrogen sources, we placed 1 g/L sodium glutamate in the SD medium with 1 g/L threonine (Thr), 1 g/L serine (Ser), or 1.25 g of ammonium sulfate (1/4N). For the condition of 1/2AA and 1/2N, the amino acids supplemented in the SD medium were halved, and 2.5 g/L ammonium sulfate was added. For chemical treatments, YPD plates were supplemented with H2O2 (0.012%, 0.016% and 0.024%), acetic acid (AcOH, 55, 57.5 and 60 mM), TM (0.2, 0.3 and 0.4 μg/mL), NaCl (0.6, 0.8 and 1 M), 20 mM DTT, 15 μg/mL 5-FU, or 1 M sorbitol. The plates were incubated at 30 °C or at others specified temperatures for 1-3 days before the pictures were taken.

Spot quantification was performed using the ‘gitter’ package95 in R. There were three biological replicates for each treatment, except for the three H2O2 treatments and the 60 mM AcOH treatment, where one biological replicate was removed due to technical problems in quantification. There were six biological replicates for YPD at 30°C. Source data are provided in Supplementary Data 4. The quantified growth (G) under a specific condition (condition_x) was normalized to YPD growth at 30°C of the same strain (strain_i), to generate a score for relative growth (RG):
RG = log2( G straini,conditionx / G straini,YPD30°C ) (2)

In order to visualize inter-strain differences, data across three replicates were averaged and the average RG for a given strain under a given condition was subtracted by that of Km-V under the same condition, to generate RG’ for visualization (Fig. 3a):
RG’ strain i,conditionx = RG straini,condition x - RG Km-V,conditionx (3)

Growth differences were tested with a linear model: RG ~ strain + treatment + treatment:strain, where the colon indicates interaction. p-values were extracted from the linear model and adjusted with the False Discovery Rate (FDR) method in R. Km-V and YPD at 30°C were used as the reference level for strain and treatment factors, respectively.

To evaluate chromosomal stability, spot dilution was performed as above on YPD supplemented with 20 μg/mL benomyl (BML), 10 μg/mL thiabendazole (TBZ), 0.05 M hydroxyurea (HU), 0.01% methyl methanesulfonate (MMS), 5 μg/mL camptothecin (CPT), or 0.05 μg/mL cycloheximide (CHX).

To perform spot assays of SPS22-expressing cells, Sc-R3 was transformed with LHZ1511 or LHZ1512, and FIM-1ΔU was transformed with LHZ1506, LHZ1507, or LHZ1508. Transformants were cultured overnight in SD-Leu medium. The culture was diluted and spotted onto plates with or without 1 M NaCl, as described above.

Flocculation analysis
Km-V, KS-R1, and Sc-R1 were cultured overnight in SD-Ura medium. Sc-R1 carrying LHZ1509 (ScFLO9CEN) or LHZ1510 (ScFLO9 2μ) and FIM-1ΔU carrying LHZ1505 (ScFLO9CEN) were cultured overnight in SD-Leu medium. Cells were harvested and washed with ddH2O and 250 mM EDTA. After two subsequent washes with ddH2O to ensure complete removal of EDTA, cells equivalent to an OD600 of 40 were pelleted and resuspended in 2 mL of ddH2O in a 15 mL tube. 100 μL 1 M Tris-HCl (pH 7.5) was added to the cells suspension, followed by 1 min of agitation. The tube was then left undisturbed, and pictures were taken every minute during a 5 minute time window. Samples of the supernatant were taken from the surface of the cultures at various time points for OD600 measurement.

RNAseq and qPCR
Three biological replicates of Km-V, Sc-R1, Sc-R3, KS-R1, and KS-R3 were cultured overnight in SD-Ura+GH liquid medium. The cultures were diluted into YPD to achieve an OD600 of 0.2. Once the OD600 of the cultures reached 0.6, cells were collected directly for the YPD treatment. For stress treatment, the culture was supplemented with tunicamycin (T8480, Solarbio) at a final concentration of 1 μg/mL, or with NaCl at a final concentration of 1 M, and cells were collected after 1 hour. Total RNA was extracted using the ZR Fungal/Bacterial RNA MiniPrep kit (R2014, ZymoResearch). Samples were reversed transcribed using TruSeq TM RNA sample preparation Kit (Illumina, California, USA) and sequenced by Illumina HiSeq X Ten (BIOZERON, Shanghai, China). Batch information was provided in Supplementary Data 11.

The reads were mapped to corresponding reference genomes with BWA (0.7.17-r1188)91 under the default settings. Different reference genomes were used for different samples: Km-V samples were mapped to the FIM1 genome (GCA_001854445.2), S. cerevisiae samples were mapped to a W303 genome96 (GCA_002163515.1, ScRAPdb97 code W303.asm02.HP0), and KS samples were mapped to a combined genome of FIM1 and chrI and chrIII of W303. They were then processed with samtools98 to remove duplicated reads. Reads with mapping quality lower than or equal to 2 were also removed with samtools, as well as those with 30 or more soft-clipped bases. The number of reads mapped to each allele (with a minimal mapping quality of 10) was counted with htseq-count99, with the setting “-idattr ID -t CDS”. Counts from exons were summed up to generate counts per gene. The gene models for the RNA-seq analysis were based on the W303.asm02.HP0 annotation downloaded from ScRAPdb. There are 5,680 annotated genes in this genome, but not all genes were annotated to a known systematic name as in the S288C genome (R64), due to known intraspecific variation96. We performed an all-vs-all BLAST search between W303 and S288C genes (R64 assembly) and selected the top S288C hit for each W303 gene, based on E-value and query coverage. 5347 out of the 5680 (94%) W303 annotations matched S288C genes unambiguously. The rest W303 gene models were kept in the analysis but flagged with “ambiguous” in the dataset. The raw read counts are provided in Supplementary Data 11.

Differential expression was analyzed with DEseq2100. To examine changes in expression levels before and after chromosomal transfers (Sc vs. KS; Km vs KS), the data were fitted to a model of count ~ group, where group represented a combination between the strain factor (Sc/KS/Km) and the treatment factor (YPD/TM/NaCl). In the Sc analysis (Fig. 4a–d), R1 and R3 data were treated separately. Data from different treatments were combined in one data frame and normalized together. In the Km analysis, data from KS-R1, KS-R3 and Km-V across three treatments were combined and normalized together. Genes with an average read count lower than 5 of any group were removed from the analysis, leaving 218 out of 241 Sc genes (chrI and chrIII) and 4975 out of 5081 Km genes in the dataset. The median of read counts across groups was 1413 for Sc genes and 1267 for Km genes, prior to normalization. Adjusted p-values for pairwise comparisons between groups (e.g., Sc-YPD vs. KS-YPD) were derived from FDR-corrected Wald significance tests, with an FDR cutoff of 0.05. Log2 fold changes were shrunken with the ashr method101. Differentially expressed genes (DEG) were defined as an adjusted p-value lower than 0.05 and an absolute log2 fold change higher than 1.

For qPCR, RNA samples were reverse-transcribed using a PrimeScript RT Reagent Kit (RR037A, Takara, China). The qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme). The mRNA level was normalized to the average of three housekeeping genes, including MPE1, TRK1, and SWC4. Primers used in qPCR are listed in Supplementary Data 1. Statistical tests were performed with ANOVA followed by Tukey’s test to account for multiple comparisons.

Cis and trans effects
Orthologs were identified with OrthoFinder (3.0.1b1)102. Animo acid sequences of six species were provided to OrthoFinder, including S. cerevisiae (W303, GenBank #GCA_002163515.1), K. marxianus (FIM1, GCA_001854445.2), Candida albicans (SC5314, GCF_000182965.3), Lachancea thermotolerans (CBS6340, GCF_000142805.1), Naumovozyma castellii (CBS4309, GCF_000237345.1), and Torulaspora delbrueckii (CBS1146, GCF_000243375.1), along with a species tree according to Shen et al. (2018)23. There was a total of 32,488 genes across the six genomes, 93.5% of which were placed into orthogroups. There was a total of 5056 orthogroups, of which 3630 were with all species present and 2847 were single-copy orthogroups. When focusing on single-copy orthologues, the median of amino acid sequence similarity between S. cerevisiae and K. marxianus genes was 51.0% (using BLOSUM62 scoring matrix), between that of S. cerevisiae-T. delbrueckii (58.7%) and S. cerevisiae-C. albicans (37.0%), consistent with expectation (Supplementary Fig. 16a). The high level of evolutionary divergence resulted in loss of synteny (Supplementary Fig. 16b), so we did not further take into account of synteny in the ortholog analysis.

Between S. cerevisiae and K. marxianus, there were 4,851 orthologs, of which 3,813 were one-to-one orthologs. In the cases where multiple orthologs were identified for one gene (one-to-many or many-to-one) and many-to-many orthologous relationships, we performed pair-wise sequence alignments within the orthogroup and selected the reciprocal best match based on amino-acid sequence similarity, identifying 4,330 orthologous pairs. The median amino acid sequence similarity was 48.4%. The length of orthologs did not differ by more than two fold for 4,149/4,330 (95.8%) orthologous pairs, ruling out partial match due to local similarity. The orthologous genes were listed in Supplementary Data 6.

Differential gene expression was identified with DESeq2100. R1 and R3 data were combined. YPD, TM and NaCl data were analyzed separately. Out of 241 genes on chrI and chrIII of the W303 genome, we identified Km orthologs for 173 genes, 162 of which had an average read count above 5 across replicates and were retained in the analysis. First, we focused on genes showing significant divergence between Sc and Km (FDR-adjusted p<0.05 by Wald test), which included 102 genes in YPD, 132 genes in NaCl and 122 genes in TM. We designated the expression level of Km alleles in Km-V as Pkm, Sc alleles in Sc-R1 or Sc-R3 as Psc, Km alleles in KS as KSkm, and Sc alleles in KS as KSsc. We used Wald test in DESeq2 to identify significant differences between (1) KSsc and KSkm (cis-effects), (2) KSsc and Psc (trans-effects), (3) Pkm and Psc (species divergence) and (4) KSsc and Pkm (similar to KSsc-KSkm comparison but accounting for trans-differences between K. marxianus and KS), using an FDR-adjusted p-value cutoff of 0.05. Effect sizes were adjusted with the ashr method101.

Genes were classified according to Fig. 5c. “Km-like” genes were defined as no significant difference between KSsc and KSkm but a significant difference between KSsc and Psc. “Sc-like” genes were defined as no significant difference between KSsc and Psc but a significant difference between KSsc and KSkm. If KSsc was significantly different from both KSkm and Psc, the gene was further classified into “intermediate” and “transgressive”: “intermediate” means that KSsc was between Psc and KSkm, and “transgressive” means that KSsc was outside the range of Psc and KSkm. If KSsc was not significantly different from either Psc or KSkm, the gene was classified as “other (conserved)”.

The above analysis of cis-regulatory differences primarily considered allelic differences in KS, where the two alleles share the same trans-environment, similar to previous studies on F1 hybrids62,84,103,104. Comparing KSsc to Pkm also reveals cis-differences, but they might be sometimes affected by the slight differences between the trans-environment of KS and Km, i.e., reflecting cis- and trans-effects for some genes (Supplementary Fig. 14a). In order to address if the trans-difference between KS and Km changed our conclusion, we performed the same classification analysis as above, but used Pkm in place of KSkm (Supplementary Fig. 14).

In order to understand the major source of variance in the transcriptomic data, we performed a PCA with data from three treatments combined and normalized together. The normalized data were transformed with the VarianceStabilizingTransformation function in DESeq2 prior to the PCA.

Genome-wide expression divergence between Km and Sc orthologs was analyzed for 4,307 genes whose average read counts across replicates were higher than 5 for every condition. Sc data were from Sc-R1. YPD, NaCl, and TM data were analyzed separately. DEG was identified by comparing Psc and Pkm with Wald test in DESeq2, with an FDR-adjusted p-value cutoff of 0.05. GO analysis was performed with SGD Gene Ontology Term Finder (Version 0.86, GO version 2025-07-22), with 4,307 genes as the background set. Cis-divergence for the transferred genes was from comparison between KSsc and KSkm described above (FDR-adjusted p-value<0.05). The results for cis-trans analysis and genome-wide divergence are provided in Supplementary Data 7.

Statistics & reproducibility
For phenotypic analysis, RNA-seq, and gene expression assessment by qPCR, each strain was analyzed with three biological replicates. For KS-R1, the three biological replicates represent three independent transformants. The spot assay was replicated three times, with the exception of the three H2O2 treatments and the 60 mM AcOH treatment, which were replicated twice; the results obtained were consistent throughout. For Fig. 2b–e, each experiment was repeated three times independently with similar results. No data was excluded. Randomization and blinding were not applicable in this study.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Hybridization—the crossing of two or more species—can generate genetic diversity and has been recognized as an evolutionary mechanism facilitating adaptation to both natural and anthropogenic environments. This concept has inspired various research groups to use hybridization as a tool to develop novel organisms capable of addressing specific industrial challenges. In yeast, for example, hybridization has been employed to produce new strains aimed at enhancing or optimizing the production of alcoholic beverages and other value-added compounds. However, traditional hybridization approaches are generally restricted to closely related species.\nTo address this limitation, Lyu and colleagues present a novel method for transferring individual chromosomes from distantly related species. In this manuscript, they investigate the transfer of the smallest chromosomes from Saccharomyces cerevisiae (chromosomes I and III) into Kluyveromyces marxianus, and assess the phenotypic consequences of this chromosomal introduction, including growth performance under various conditions, gene expression patterns, and the complete transcriptome.\nI found the methodological approach innovative and the experimental design sound. The manuscript is clearly written and easy to follow. While the number of chromosomes and species tested is admittedly limited and the genes associated to specific phenotypes are not surprisingly new, the scope is adequate for a proof-of-concept study and demonstrates the method’s potential for broader applications. I believe this work is a strong candidate for publication in Nature Communications. That said, there are a few statements and interpretations in the manuscript that I do not fully agree with. I encourage the authors to carefully consider my comments outlined below and address the concerns raised. My decision is major revision.\nMajor comments:\n#. I would like to raise a concern regarding the use of terminology such as hybridization and hybrid strains throughout the manuscript. In my view, referring to the introduction of a single chromosome as hybridization is conceptually inaccurate. Hybridization typically refers to the sexual crossing of two distinct strains or species, leading to the combination of entire genomes. In contrast, the approach used in this study involves the targeted introduction of an individual chromosome, which more closely resembles a form of horizontal gene transfer or a synthetic introgression event, rather than true hybridization.\nWhile I acknowledge that the introduction of a chromosome may mimic certain aspects of natural introgression—such as the retention of specific genomic elements following hybridization—it does not constitute hybridization per se. For this reason, I encourage the authors to use terminology that more accurately reflects their methodology, such as chromosomal engineering, strain engineering, or chromosome transformation, which are more appropriate within the context of synthetic biology.\nClarifying this distinction will help avoid potential confusion for readers and ensure that the manuscript’s language is aligned with the techniques and concepts actually employed.\n#. In line with my previous comment regarding the use of hybridization-related terminology, I would also like to address the use of the term heterosis in the manuscript. Traditionally, heterosis refers to hybrid vigor observed in offspring resulting from the cross of two genetically distinct parents. As the strain in this study is not a true hybrid but rather a recipient of a single exogenous chromosome, the use of the term heterosis may be misleading.\nThere are two issues that arise from this usage. First, the results for the Sc-V, which are essential for interpreting any claim of a superior phenotype, are not clearly presented in the main figures. This may confuse readers or compel them to search through supplementary materials to fully understand the comparisons being made. Second, and more importantly, since the strain is not the product of hybridization, it would be more accurate to state that the strain with the introduced chromosome exhibits an enhanced or superior phenotype under specific conditions, rather than attributing the effect to heterosis.\nClarifying this point will improve the scientific accuracy of the manuscript and help prevent potential misinterpretation by readers.\n#. I find the section discussing cis and trans regulatory effects to be somewhat misleading. As I understand it, proper analysis of cis and trans effects typically requires a true hybrid background, where both alleles are exposed to the same trans-regulatory environment. This setup allows for a controlled comparison of allele-specific expression, isolating cis effects from trans influences. The authors themselves acknowledge this limitation in lines 562–565, yet still proceed with the analysis in a context that does not fulfill this condition.\nGiven that the experimental design does not involve a hybrid organism but rather a strain into which an entire chromosome from another species was introduced, the interpretation of observed expression changes as cis or trans effects is potentially confounded. I encourage the authors to clarify the limitations of this analysis and, if possible, to avoid using terminology that implies mechanistic resolution between cis and trans regulation in the absence of a shared trans environment.\n#. I couldn’t check supplementary material because I did not have access to it.\nMinor comments:\n#. Including a brief overview of the number and diversity of ARS and CEN elements in each genome would provide useful context and help the reader better understand the rationale behind the experimental design. Additionally, clarifying the criteria used to select specific ARS and CEN elements—and how the combinations were constructed—would strengthen this section.\n#. Figure 1, panel B: Is the dashed arrow pointing to K. marxianus (Km) missing? If so, please clarify whether Km is considered tolerant in this context.\n#. Please specify which version of the S. cerevisiae S288C genome annotation was used in this study.\n#. Figures 3B and S7: Ensure consistency in naming across main and supplementary figures. For example, some labels read “TM,” others “0.4 µg/mL TM (2d),” and others “TM (2d).” Standardizing these will improve clarity and readability.\nSuggestions\n#. Consider formatting species abbreviations such as Km and Sc in italics throughout the manuscript, as they refer to species names.\n#. Line 210: It would be helpful to indicate the known loss rate of circular YACs in K. marxianus for readers who may not be familiar with this value.\nMinor corrections\n#. Line 140: You may want to rephrase as: “Blue ellipses show species capable of interspecific hybridization within the genus.”", "reviewer_3": "This study reports the transformation of two complete Saccharomyces cerevisiae chromosomes into the genome of Kluyveromyces marxianus, and the resulting phenotypic effects. Specifically, the authors inserted K. marxianus ARS and centromere sequences into two small S. cerevisiae chromosomes (Chr I and III), circularized the S. cerevisiae genomes, isolated the circular chromosomes and used classic transformation protocols to inserted them one-by-one into the K. marxianus genome. The resulting two hybrid yeasts each stably carried the one extra S. cerevisiae chromosome and showed relatively normal phenotypic characteristics, with a relatively minor changes in stress resistance. Remarkably, one of the hybrids showed increased resistance to NaCl, which the authors linked to overexpression of the S. cerevisiae SPS22 gene resideing on the inserted chromosome; while the other hybrid showed increased flocculation due to overexpression of the S. cerevisiae FLO9 gene.\nThe study is interesting because it shows that 1) it is possible to stably insert complete chromosomes from one yeast species into the genome of an evolutionary distant species and 2) that insertion of a foreign chromosome yields stable strains †hat show altered phenotypes and possibly even heterosis (ie superior behavior) for certain phenotypes. As such, the reported technique could promote further research into the biology underlying interspecific hybrids as well as help develop novel industrial microbes that combine traits of two species for which we do not necessarily know the underlying genes.\nAs far as I can assess, experiments are of high standard, and the text reads well.\nDespite its merit, I have some questions regarding certain aspects on the analyses, and the study also has a few shortcomings and limitations that merit careful consideration and discussion in the manuscript and/or further experimental follow-up.\nFirstly, I wonder whether it is fair to use the methods developed by Naama Barkai’s team to attribute differences in gene expression to cis and trans effects. The Barkai team used hybrids between S. cerevisiae and the closely related S. paradoxus. This is different from the present study in two important ways. Firstly, the hybrids Barkai et al. use are “full” hybrids, i.e. both complete genomes (all chromosomes of both species) are merged. In principle, this implies that the hybrid expresses all trans factors (transcription factors) from both cerevisiae and paradoxus. Second, S. cerevisiae and S. paradoxus are genetically closely related, sharing about 85% of DNA sequences. The present study only integrates one short S. cerevisiae chromosome in the distantly related K. marxianus. Thus, the “hybrid” is by no means a complete hybrid, and most of the trans factors of S. cerevsiae are expected to be missing. Moreover, because of the genetic distance, it seems unlikely that many trans factors (transcription factors) of K. marxianus would affect transcription of their homologous targets on the one S. cerevisiae genome because of a combination of divergence in the sequence of the transcription factor (trans) and the binding site (cis). I therefore wonder whether in these evolutionary very divergent one-chromosome-hybrids, it makes any sense (or is even possible) to split up the differences in gene expression between cis and trans? Take for example an imaginary geneA encoded on Chromosome I, regulated by transcription factor B, encoded by geneB on another chromosome. If the hybrid only contains the S. cerevisiae geneA and not the S cerevisiae geneB, the expression of geneA will likely differ from the parental strain; but would it be fair to say that this is due to cis or trans? The regulating geneB is not present in the hybrid, and the K. marxianus geneB is likely too divergent to bind…. Can the authors think this through, and then elaborate and discuss this in greater detail in the text? At the very least, it should be clearly pointed out that this division between cis and trans only holds in the very restricted setup of these one-chromosome hybrids and does not yield insight into general biology and physiology of transcription. In that sense, the study falls short of delivering major novel insights into the biology of interspecific hybrids – it confirms previous reports that foreign genes, promoters and chromosomes can function in distant yeast species, possibly with altered expression patterns or functionality of the genes (eg DOI: 10.1126/science.1169766, DOI: 10.3389/fgene.2020.00404; https://doi.org/10.1016/j.gde.2022.101958 ); and as argued above, it is unclear whether much can be said about the cis and trans effects.\nSecond, for the development of industrially-relevant strains, the insertion of one complete chromosome into the genome of another species is interesting and novel, but it may not be the most productive technique. Besides possibly inserting a handful of genes that confer positive effects., inserting a complete chromosome inevitably inserts many genes that may not contribute any useful phenotype, and likely even result in detrimental effects (as was also observed here). Moreover, it is in most cases not clear which chromosome needs to be inserted, and whether one chromosome is sufficient to reach the optimal effect. For closely related species, generating interspecific hybrids may be more productive (eg doi: 10.1038/s41559-019-0997-9; doi.org/10.3389/fmicb.2025.1472832). For more distant species (like in this study), previously described techniques, such as whole-genome transformation, where genomic DNA of one species is isolated and transformed into another, likely results in the generation of a whole set / library of transformants, each carrying one or a few genes that can reside on any chromosome of the donor, and is therefore more likely to yield interesting phenotypes without the disadvantages of transferring one complete chromosome (see for example the following references: https://doi.org/10.1186/1754-6834-5-46 and doi: 10.1128/mcb.00560-21).\nIn Figure 3, the heterosis effect is difficult to assess. Firstly, it would be useful to explain the strain nomenclature in the legend; especially the Km-V strain (K. marxianus with empty vector). Second, the reason of including an empty vector control is not only to compare this strain to the strain transformed with the functional/”full” vector, but also, and primarily so, to the non-transformed wild-type, in order to assess whether the empty vector by itself conferred any phenotype. Hence, it is essential to also include data for both wild-type parental species (Km and Sc). Third, using Z-scores can be highly misleading because it can hide the magnitude of the effect. Therefore, I would suggest including more detailed data on the growth of the various strains in salt (and other) stresses; and preferably also at different levels of the stress (especially for the salt).\nIt is unclear how and why the authors specifically turned their attention to the SPS22 gene, which is only one of many genes residing on Chromosome III… The gene is relatively unknown, with virtually no link to salt stress 9and mostly to phenotypes in spores rtahr tan vegetative cells?). Moreover, as far as I can see, the reference used to link SPS22 to salt stress does not mention anything about this (Ref 47 – Chakrabortee et al., DOI: 10.1016/j.cell.2016.09.017). Can you please elaborate?\nIn line 191-196: sequentially re-inserting 13 genes that have been deleted by a gross rearrangement does not really allow you to conclude that this complete region was not lost because of incompatibilities - in principle, it is possible that the incompatibility only occurs when the whole region is present (or at least a combination of genes that were not re-inserted together in the assay). A stronger indication of the absence of incompatibilities would have been to generate several independent hybrids, and show that the same or similar region is not lost in all of them. More generally, it is a pity that, as far as I can assess, the authors have not worked with such biological replicates throughout their manuscript – this would have made the conclusions stronger compared to presenting repeats of experiments on the same one hybrid clone. Please reconsider this, or, at the very least, discuss this potential pitfall in the text."}, "author_response": "Thank you for this comment. We referred to our strains as “monochromosomal hybrids” because the transferred Sc genes and their Km orthologs co-existed in the same cells, but it is true that they do not resemble a natural hybridization process where two species mate. Because multiple colleagues, including the reviewer, have brought up this issue, we agree that using the hybrid- related terminology can be misleading for readers and have replaced them in our manuscript.\nThank you for the suggestion. We have replaced “heterosis” with “enhanced phenotype” in our manuscript. We have added data for Sc-V to Fig. 3 and Fig. S6. We found that Sc-V, Sc-R1, and Sc-R3 showed consistent phenotypes under most conditions, with a few exceptions such as 37 °C and 1 M NaCl. Because the circularized chromosomes in Sc-R1 and Sc-R3 share the same genetic modification as those in KS-R1 and KS-R3, we reasoned that Sc-R1 and Sc-R3 might be better controls than Sc-V. However, we agree with the reviewer that it is necessary to include the data of Sc-V for clarity. We have revised the figures and added relevant explanation to the text (lines 237- 239).\nWe thank both reviewers for pondering over the cis-trans analysis with us. This experimental design indeed presented a challenging analytical problem for the cis-trans question. We reasoned that the definition of cis-divergence holds as long as the two alleles are exposed to the same trans environment, but it is true that the amount of cis-divergence one can find depends on the trans-environment. As both reviewers pointed out, the cis-differences revealed in our synthetic strains may or may not manifest themselves in an F1 hybrid where both genomes are present. If an Sc promoter requires an Sc-specific TF to be activated at the same level as its orthologous Km promoter, and that TF was not in the synthetic strains (referred to as “KS” strains hereafter), the cis-differences would be more pronounced in KS than in an F1 hybrid. Such dependency itself indicates lineage-specific cis-trans co-evolution, but it would be unfair to quantitatively compare our findings in KS to previous classical studies with F1 hybrids, in terms of the relative contribution of cis- and trans-evolution. Therefore, we revised our analytical framework and removed statements comparing our study to previous F1 studies.\nHowever, we found that the interesting pattern of cis-trans compensatory evolution in our data still holds, based on evidence of (1) transgressive expression and (2) cis-divergence underlying conserved expression (Fig. 5, lines 430-550). As both reviewers pointed out, the KS strains more closely resemble introgression lines than F1 hybrids. Therefore, we revised our cis- trans analysis with a logic suited for introgression lines (Guerrero et al. 2016). First, we examined the underlying factors for expression divergence between the two species, focusing on 102 out of the 162 transferred genes. The question was: which factors (cis or trans) drove the expression level of these Sc genes to diverge from their Km orthologs? We classified the genes into “Sc-like”, “Km-like”, “intermediate”, “transgressive” and “others”, based on the expression pattern of transferred Sc genes. If a transferred gene showed the same level of expression as in the Sc parent (“Sc-like”), the divergence was cis-determined, and vice versa. The most interesting category was transgressive, meaning that the Sc expression in KS was outside the range of both parents. This was the most abundant class among the 102 genes studied. We think that this finding provides new biological insights because (1) it suggests that evolution of gene expression involved both cis and trans factors, rather than cis-only or trans-only. The two types of factors often evolved in opposite directions, possibly driven by stabilizing selection. To our knowledge, this is the first study that examined cis-trans evolution for over a hundred genes over such a long evolutionary distance, so we provide new evidence for the evolutionary timescale that stabilizing selection can act on. (2) the prevalence of transgressive expression in our data was prominent and have phenotypic implications. Transgressive expression was often interpreted as misexpression and causing deleterious fitness effects. However, the most prominent examples of transgressive expression in our data happened to be FLO9 and SPS22, two genes we independently identified from our phenotypic studies, demonstrating the phenotypic potential from cross-species regulation.\nSecond, we focused on the remaining 60 genes expressed at the same level in Sc and Km. We found that most of the genes showed cis-divergence (expression difference between Sc and Km orthologs) in KS, which indicates that the trans-factors must have co-evolved in each lineage to maintain the conserved expression level. This result provided additional evidence for compensatory cis-trans evolution. The relevant results are presented now in Fig. 5, Figs S13-15 and lines 430-550.\nWe added the limitation of the cis-trans analysis to Discussion (lines 679-693). Briefly, our results were limited because (1) it’s based on a small set of transferred genes, and (2) the cis- effects can depend on the trans environment. The discussion reads:\nWe note a few limitations in our cis-trans analysis. First, our sample size of 162 genes provided a statistically sufficient examination of regulatory evolution, but it was a small subset of the yeast genome. Future chromosomal transfers with larger chromosomes may provide more data. Second, we used the differences between KSsc and KSkm as cis-effects, reasoning that the two alleles were exposed to the same trans-environment, similar to previous studies with F1 hybrids 61,86. However, we note that the estimation of cis effects might depend on trans- environment. For example, we might find more cis-divergence in our “monochromosomal hybrids” than in F1 hybrids due to unavailability of certain Sc-specific trans-factors.\nFurthermore, unlike F1 analysis, we cannot properly estimate trans-divergence by subtracting cis effects from parental differences, because Sc and Km alleles experienced different levels of trans- changes. Therefore, we did not quantitatively compare our conclusions to previous F1 studies. We reasoned that our analysis, similar to previous ones with introgression lines and enhancer swaps, still informs about cis-trans co-evolution, and the abundant transgressive expression provides novel insights for both regulatory evolution and future phenotypic studies.\nThank you for the suggestion. We included a brief overview of ARS and CEN elements in the K. marxianus and S. cerevisiae genomes. We also clarified the criteria used to select specific ARS and CEN elements (lines 94-106). The added text reads:\nIn S. cerevisiae, there are 228 functional ARS sites distributed across the genome 27, sharing an 11-bp consensus sequence 28. The distribution of ARS in the Km genome is less characterized, and only 15 KmARSs have been experimentally validated 29–32. Different from canonical ARS in other yeasts, KmARSs are diverse and do not share a common consensus sequence 32. There is one CEN per chromosome in both Sc and Km, each consisting of three conserved centromere DNA elements (CDEI, II and III) 33. The Km CDEs are similar to their Sc counterparts, with the exception that the Km CDEII was approximately twice the length of that in Sc 34. We selected two ARS-CEN pairs from each species for our experiments, based on their chromosomal locations and previously-demonstrated functionality in plasmids 31,32,35–38. Pairs on the same chromosome were tested together to maximize intraspecific ARS-CEN compatibility: ScARSH4 and ScCEN6 35,36 (Sc chrII), ScARS1 37 and ScCEN4 (Sc chrIV), KmARS1 and KmCEN5 31,38 (Km chrV), and KmARS18 32 and KmCEN3 (Km chrIII).\nThanks. We have added the dashline and indicated in the legend that Km is considered tolerant based on findings in this study.\nWe used R64 assembly in our study. We have added this information to the text.\nThanks! We have made the labels consistent.\nThank you. We have made the changes.\nThank you for the suggestion. The loss rate of circular YACs (0.33~0.64% loss per generation) was included in the revised manuscript (line 204).\nThanks. We made the change.\nThank you again for the comments regarding our cis-trans analysis. We have addressed the issue of full F1 hybrids in our responses to reviewer #1 above, acknowledging that it would not be fair to directly compare the relative cis- and trans-contributions in our studies to previous studies with full hybrids. We have revised the analysis in a framework similar to previous analysis of introgression lines (lines 430-533) and added discussion of limitation (lines 679-693). We also added illustrations of how the promoter of a focal gene A can interact with a transcription factor B, as the reviewer kindly suggested. We hope the revised text and figures clarify our line of thoughts on cis-trans evolution.\nRegarding the second point that the two species might be often too diverged to cross-regulate each other, our findings of transgressive expression partly support this point, because many Sc genes cannot be turned on or off “correctly” in Km (Fig. 5). However, we note that all the transferred Sc genes were actively transcribed (Fig. 4a), and there were roughly equal number of genes being up- and down-regulated in the synthetic strains (KS) compared to Sc (Fig. 4b). Therefore, the Sc cis-elements can be bound by Km trans-factors, just often producing a different outcome.\nThe reviewer was correct in stating that both cis- and trans-factors must have evolved since the divergence of Sc and Km, and it is indeed the right question to ask, does it still make sense to split cis and trans for the two highly diverged species? Or, as the phylogenetic distance increases, until when do we stop caring whether cis or trans contributes more? Such interspecific analysis would indeed lose its point if there was not a theoretical framework clearly presented. We argue that the biological question about regulatory evolution is deeply rooted in modern evolutionary biology, originated from the finding about lac operons (Francois Jacob \"Evolution and Tinkering\" 1977) and recently developed by evo-devo studies. These studies often found that, when a gene is transferred to another species, its gene expression was often determined by it cis-regulatory sequences (“donor-like”), suggesting that the trans environment was relatively conserved. Such evidence argues for a predominant role of cis-regulatory evolution in divergence of gene expression. However, the evo-devo findings are often based on case studies and the transgenic experiments were often restricted to a few closely related taxa. There have been few studies that have systematically examined cis-trans evolution in highly diverged species. Therefore, we think that our study does provide new biological insights on interspecific hybrids, exactly because we are studying two highly divergent genomes. As far as we know, the evolutionary distance between Sc and Km exceeded any previous species pair where interspecific exchange of genetic materials has been made possible either naturally or artificially (Fig. 1b). The Saccharomyces species examined in the references mentioned by the reviewer can actually be viewed as “closely related” from a broader evolutionary timescale. The average nucleotide identity to S. cerevisiae is 90% for S. paradoxus and 80% for S. bayanus (S. uvarum) in coding regions (Kellis et al. 2003 Nature), whereas the median of amino acid sequence similarity between S. cerevisiae and K. marxianus was around 48%. For the question of cis-trans co-evolution, the furthest phylogenetic distance so far examined by systematic studies was between S. cerevisiae and S. uvarum (diverged 20 million years ago), so it remained unknown if compensatory evolution occurred at a greater evolutionary timespan (e.g., Km and Sc which diverged 114 million years ago). As explained above (see the response to Reviewer #1), our findings with transgressive expression supported compensatory cis- trans co-evolution, providing new evidence for stabilizing selection on gene expression at a long evolutionary timescale.\nWe have added the relevant background to Discussion (lines 643-678), which we hope help elucidate the biological insights our study provides. The discussion reads:\nThe KS strains allowed for exploring patterns of regulatory evolution at an unprecedented long evolutionary timescale. It has been long debated whether evolution of gene expression primarily occurs through cis, trans, or both types of changes. A number of examples, especially from the evo-devo field, have argued for an important role of cis-regulatory changes 79,80. That is, when a gene was transferred to a different species, its expression pattern was often determined by its cis-regulatory sequences (donor-like), suggesting that the trans environment was relatively conserved 63. Recent transcriptomic and experimental studies showed that compensatory evolution of cis and trans-acting factors is common, possibly driven by stabilizing selection 9,61,63,64,81. However, most systematic studies so far were restricted to intrageneric hybrids 61,64,81,82 or introgression lines 62. The furthest phylogenetic distance examined so far by genome-wide analysis was probably between Saccharomyces cerevisiae and S. uvarum 61,83,84, two species that share 80% nucleotide identity in coding regions 85. Studies on the Saccharomyces species found that gene expression divergence reached a plateau as the phylogenetic distance increases within the genus, with the contribution from cis-regulatory differences and compensatory changes becoming increasingly important 61. Still, little is known for regulatory divergence beyond this phylogenetic scope. It is imaginable that both cis and trans-acting factors continuously accumulate nucleotide substitutions over a long evolutionary timespan, but it remains unknown whether they evolve in a compensatory manner under stabilizing selection, like what has been found within a genus, or they tend to change in the same direction to increase expression divergence. Evidence from enhancer-swaps showed a higher proportion of misregulation in cross-genus experiments than within Drosophila flies or Caenorhabditis worms, indicating that lineage- specific cis-trans co-evolution becomes abundant beyond the genus level, but the number of genes cataloged in this study was relatively small 63. Our study examined the expression pattern of over a hundred Sc genes transferred to Km, providing new evidence for long-term regulatory divergence at a relatively systematic scale. We found that transgressive expression, rather than cis-determined, was the most common pattern (37.7%) among the transferred genes. It is consistent with the model where both cis and trans changes accumulate with time, and they tend to act in opposite directions. At the same time, around one third of genes showed conserved expression between Km and Sc, with contributions from compensatory cis-trans co-evolution as well. The data together point to that evolution of gene expression is under stabilizing selection in Saccharomycetaceae yeasts, across an evolutionary timespan of 114 million years. From a practical point of view, the prevalent transgression and its association with enhanced phenotypes demonstrate the potential of cross-species regulation in generating transgressive phenotypes, providing an alternative route to previous approaches that focus on optimizing individual proteins.\nThank you for raising this point. We acknowledge that there always needs to a compromise between the “all-gene-at-once” full-hybrid approach and the “one-gene-at-a time” library/WGT approach. The main differences among these approaches are (1) the ability to screen for polygenic effects, (2) the level of incompatibility and (3) experimental throughput. The full hybrids allow for screen for all possible genetic interactions from the foreign genome, at the expense of high incompatibility. The single-gene library approach cannot screen for beneficial phenotypes caused by co-existence of multiple foreign genes, but confers minimal incompatibility. Transferring one chromosome at a time allows some level of polygenic screen, with hundreds of gene at a time. The strategy can be extended by mating strains harboring different alien chromosomes, screening for phenotypic effects caused by interactions between chromosomes. There can be a low level of incompatibility, but we argue that it might be resolved by directed evolution after the strain construction.\nThe full-hybrid approach is not possible for Sc and Km. Chromosomal transfers and the WGT technique may be complementary approaches in screening for beneficial phenotypes caused by interspecific genetic exchanges. Briefly, WGT might be low in throughput because it relies on random integration of foreign DNA fragments into the host genome, whereas chromosomal transfers are more stable and screens hundreds of genes at a time. Genomic libraries provide high- throughput, and our findings about ARS and CEN compatibility might facilitate future applications of genomic libraries in Km.\nWe have added the discussion above and the related references to the manuscript (lines 585- 627), which reads:\nThere are several advantages in interspecific chromosomal transfers for synthetic biology purposes. First, it enables combination of genetic materials from remotely related species, partly circumventing the genomic incompatibility that prevented interspecific hybridization. The synthetic “monochromosomal hybrids” were viable, providing novel materials for phenotypic screens or downstream experiments such as directed evolution 72. In particular, directed evolution may help resolve any residual incompatibility, by selecting for mutations that improve the desired traits and/or compensate for the incompatibility.\nSecond, our strategy of chromosomal transfers may provide a complementary approach to whole-genome transformation (WGT) 73,74 or genomic libraries, in terms of (1) the ability to screen for polygenic effects and (2) a medium-level of experimental throughput. Both WGT and genomic libraries may be used for genome-wide interspecific genetic screens, where foreign genes were individually introduced to the host, avoiding incompatibility. Compared to WGT and genomic libraries, the strategy of chromosomal transfers is limited in its ability to screen the entire genome but has the strength of screening for polygenic effects. Although our experiments with chr1 and chr3 mainly found single-gene effects, future transfers with larger chromosomes might potentially find beneficial phenotypes necessitated by interactions among multiple genes in the transferred chromosome. Additionally, as demonstrated in our study, cross-species regulation may be an important source for novel phenotypes (Figs. 3 & 5). Our approach enables screening for such interactions by introducing full-length cis-regulatory elements, which are often missed from WGT or genomic libraries. Furthermore, our strategy allows for possibilities of transferring multiple chromosomes into Km. This can be done either by marker recycling, i.e., popping out URA3 in the (evolved) synthetic strains and transforming another chromosome subsequently, or by mating with another (evolved) synthetic strain containing a different chromosome. Therefore, chromosomal transfer is particularly advantageous for screening for polygenic effects, including interactions within and among foreign chromosomes, as well as between host and foreign genes.\nOur strategy provides a medium-throughput screening capability, between that of WGT and genomic libraries. In WGT studies, genomic DNA of a foreign species was transformed into the host species without a stable vector 73,74. The foreign genes were expected to randomly integrate into the host genome, and only the transformants that conferred desired phenotypes were selected. The success rate of such strategy depends on the efficiency of homologous recombination and screening conditions. Also, random integration of foreign genes can disrupt the host genome, lowering the success rate even more. From this perspective, the experimental throughput of such WGT approach can be low. Compared to WGT, chromosomal transfers allow screening for hundreds of genes at a time once a stable strain is established, so it might provide a higher throughput than the WGT approach. Genomic libraries on vectors allow generation of stable, pooled transformants for high-throughput screens with a genome-wide coverage, such as the MoBY-ORF library in S. cerevisiae 75. However, direct application of S. cerevisiae-based molecular libraries in non-model yeasts such as Km requires technical optimization. In this regard, our findings about CEN and ARS compatibility may inform future studies to convert genomic libraries on Sc-based vectors into Km-compatible versions, advancing interspecific genetic screens.\nThank you. We have added the strain nomenclature to the legend. We have added a supplementary figure, Fig. S8, with all the phenotypic data for Sc, Sc-Vector, Km and Km-Vector. We found that the vector did not affect phenotypes for most conditions, but caused minor differences under 42 ℃, AcOH, TM (0.4 μg/mL), 5-FU, NaCl and sorbitol treatments. The differences were possibly related to the KmURA3 or two antibiotic markers carried by the vector, but the exact cause remains to be investigated. Because KS-R1, KS-R3, Sc-R1, and Sc-R3 all contain the same markers as the vector, we used Km-V and Sc-V, rather than the parental strains, as references when evaluating phenotypes.\nThank you for this suggestion. We have replaced the Z-scores and now use log2 fold change relative to YPD growth to quantify growth. In order to visualize the differences between strains, we compared the growth scores of all strains to that of Km-V. We have updated the heatmap in Fig. 3a and provided detailed data in Fig. S6. We also added treatments of different concentrations of NaCl, TM, H₂O₂, and AcOH.\nThank you for this question. That is a very sharp observation. We actually surveyed all R3 genes in Saccharomyces genome database (SGD) for relevant phenotypes and identified three genes associated with NaCl resistance: ScSPS22, ScSTE50, and ScHMLALPHA2. The phenotype data in SGD often come from large-scale studies using the yeast deletion or overexpression collections, so the findings about individual genes need to be experimentally validated. Here, the salt phenotype of SPS22 and HMLALPHA2 came from a large-scale overexpression study (Chakrabortee et al. 2016 Cell), described respectively in line 1604 and line 1605 of Table S1 accompanying this paper. The phenotype of STE50 came from a large-scale deletion study (Giaever et al. 2002 Nature), where its deletion caused reduced tolerance to NaCl.\nWe actually experimentally examined all three candidate genes in K. marxianus. Only expression of ScSPS22 enhanced K. marxianus’ tolerance to NaCl (Fig. S10), suggesting that ScSPS22 is the gene responsible for the salt-resistant phenotype of KS-R3. Therefore, we carried out further studies on SPS22. We have clarified the above reasoning in our manuscript (lines 265-270) and added the figure as Fig. S10.\nWe agree with the reviewer that incompatibility may arise when the entire region is present, or from specific combinations of genes that were not reinserted together in the assay. We have revised the sentence, which now reads: “None of the segments affected growth (Fig. S4), so it was unlikely that the deletion was an adaptive response to overcome incompatibility caused by single genes in this region.”\nThank you for the advice with independent transformants. We were able to repeat our phenotypic experiments with three additional transformants of KS-R1 and added the data to our manuscript (Fig. S7). The four KS-R1 clones exhibited highly similar phenotypes under 25/28 treatments. Under acetic acid treatments, KS-R1-3 showed slightly weaker growth than the other three transformants, but the phenotypes from the three other transformants were consistent and were used as representative data for our conclusion. These data indeed strengthened our study and ensured reproducibility of our conclusion. Unfortunately, we were only able to obtain a single KS-R3 clone, so we could not perform the experiments with more biological replicates. We have added discussion of this pitfall to the manuscript (lines 580-584), which reads: “Finally, we note that one potential technical pitfall was the difficulty in acquiring independent transformants, as exemplified by R3. Only one transformant of Km-R3 was acquired, which contained a deletion potentially mediated by Ty elements during transformation (Fig. 2). Future efforts are needed to optimize the transformation efficiency to ensure scalability.”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors addressed all my previous comments. I have just two additional observations:\n#. Table Format: I recommend uploading the tables in Excel format rather than PDF. This facilitates easier data inspection and reuse. If the submission system does not support Excel files, the authors could alternatively upload the tables to a data repository that provides temporary peer-review links.\n#. Statistical Analysis: I have concerns regarding the use of t-tests and the reported p-values. The current approach may not be appropriate, especially if multiple comparisons are involved. I suggest applying a more suitable statistical test and including corrections for multiple testing (e.g., Bonferroni, Benjamini-Hochberg) to ensure the robustness of the results.", "reviewer_3": "The authors have addressed all my concerns and suggestions."}, "author_response": "Thank you. We have uploaded the tables in Excel format.\nThank you for this suggestion. We have addressed the issue of multiple testing by (1) replacing the t-tests in the qPCR analysis with ANOVA followed by Tukey’s test; (2) replacing the t-tests in the phenotypic screen (Fig. 3a) with p-values extracted from a linear model followed by False Discovery Rate (FDR) adjustment. The latter resulted in a few changes in significance for conditions that were marginally significant but did not change our conclusion (Sc and Km diverged in many conditions; KS strains showed growth defect compared to Km in a few conditions; KS-R3 was significantly enhanced for salt resistance).\nWe have revised our Results and Methods accordingly (lines 242-357, 899-902, 970-971)."}, {"round": 3, "reviewer_comments": {"reviewer_1": "All good now."}, "author_response": ""}]

Decision letter