Inversion of the chromosomal region between two mating type loci switches the mating type in Hansenula polymorpha.

Yeast mating type is determined by the genotype at the mating type locus (MAT). In homothallic (self-fertile) Saccharomycotina such as Saccharomyces cerevisiae and Kluveromyces lactis, high-efficiency switching between a and α mating types enables mating. Two silent mating type cassettes, in addition to an active MAT locus, are essential components of the mating type switching mechanism. In this study, we investigated the structure and functions of mating type genes in H. polymorpha (also designated as Ogataea polymorpha). The H. polymorpha genome was found to harbor two MAT loci, MAT1 and MAT2, that are ∼18 kb apart on the same chromosome. MAT1-encoded α1 specifies α cell identity, whereas none of the mating type genes were required for a identity and mating. MAT1-encoded α2 and MAT2-encoded a1 were, however, essential for meiosis. When present in the location next to SLA2 and SUI1 genes, MAT1 or MAT2 was transcriptionally active, while the other was repressed. An inversion of the MAT intervening region was induced by nutrient limitation, resulting in the swapping of the chromosomal locations of two MAT loci, and hence switching of mating type identity. Inversion-deficient mutants exhibited severe defects only in mating with each other, suggesting that this inversion is the mechanism of mating type switching and homothallism. This chromosomal inversion-based mechanism represents a novel form of mating type switching that requires only two MAT loci.

Core regulatory components of the PHO pathway are conserved in the methylotrophic yeast Hansenula polymorpha.

To gain better understanding of the diversity and evolution of the gene regulation system in eukaryotes, the phosphate signal transduction (PHO) pathway in non-conventional yeasts has been studied in recent years. Here we characterized the PHO pathway of Hansenula polymorpha, which is genetically tractable and distantly related to Saccharomyces cerevisiae and Schizosaccharomyces pombe, in order to get more information for the diversity and evolution of the PHO pathway in yeasts. We generated several pho gene-deficient mutants based on the annotated draft genome of H. polymorpha BY4329. Except for the Hppho2-deficient mutant, these mutants exhibited the same phenotype of repressible acid phosphatase (APase) production as their S. cerevisiae counterparts. Subsequently, Hppho80 and Hppho85 mutants were isolated as suppressors of the Hppho81 mutation and Hppho4 was isolated from Hppho80 and Hppho85 mutants as the sole suppressor of the Hppho80 and Hppho85 mutations. To gain more complete delineation of the PHO pathway in H. polymorpha, we screened for UV-irradiated mutants that expressed APase constitutively. As a result, three classes of recessive constitutive mutations and one dominant constitutive mutation were isolated. Genetic analysis showed that one group of recessive constitutive mutations was allelic to HpPHO80 and that the dominant mutation occurred in the HpPHO81 gene. Epistasis analysis between Hppho81 and the other two classes of recessive constitutive mutations suggested that the corresponding new genes, named PHO51 and PHO53, function upstream of HpPHO81 in the PHO pathway. Taking these findings together, we conclude that the main components of the PHO pathway identified in S. cerevisiae are conserved in the methylotrophic yeast H. polymorpha, even though these organisms separated from each other before duplication of the whole genome. This finding is useful information for the study of evolution of the PHO regulatory system in yeasts.

Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network.

Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

Nuclear localization of Haa1, which is linked to its phosphorylation status, mediates lactic acid tolerance in Saccharomyces cerevisiae.

Improvement of the lactic acid resistance of the yeast Saccharomyces cerevisiae is important for the application of the yeast in industrial production of lactic acid from renewable resources. However, we still do not know the precise mechanisms of the lactic acid adaptation response in yeast and, consequently, lack effective approaches for improving its lactic acid tolerance. To enhance our understanding of the adaptation response, we screened for S. cerevisiae genes that confer enhanced lactic acid resistance when present in multiple copies and identified the transcriptional factor Haa1 as conferring resistance to toxic levels of lactic acid when overexpressed. The enhanced tolerance probably results from increased expression of its target genes. When cells that expressed Haa1 only from the endogenous promoter were exposed to lactic acid stress, the main subcellular localization of Haa1 changed from the cytoplasm to the nucleus within 5 min. This nuclear accumulation induced upregulation of the Haa1 target genes YGP1, GPG1, and SPI1, while the degree of Haa1 phosphorylation observed under lactic acid-free conditions decreased. Disruption of the exportin gene MSN5 led to accumulation of Haa1 in the nucleus even when no lactic acid was present. Since Msn5 was reported to interact with Haa1 and preferentially exports phosphorylated cargo proteins, our results suggest that regulation of the subcellular localization of Haa1, together with alteration of its phosphorylation status, mediates the adaptation to lactic acid stress in yeast.

Ketoacyl synthase domain is a major determinant for fatty acyl chain length in Saccharomyces cerevisiae.

Yeast fatty acid synthase (Fas) comprises two subunits, α6 and ß6, encoded by FAS2 and FAS1, respectively. To determine features of yeast Fas that control fatty acyl chain length, chimeric genes were constructed by combining FAS sequences from Saccharomyces cerevisiae (ScFAS) and Hansenula polymorpha (HpFAS), which mostly produces C16 and C18 fatty acids, respectively. The C16/C18 ratios decreased from 2.2 ± 0.1 in wild-type S. cerevisiae to 1.0 ± 0.1, 0.5 ± 0.2 and 0.8 ± 0.1 by replacement of ScFAS1, ScFAS2 and ScFAS1 ScFAS2 with HpFAS1, HpFAS2 and HpFAS1 HpFAS2, respectively, suggesting that the α, but not ß subunits play a major role in determining fatty acyl chain length. Replacement of phosphopantetheinyl transferase (PPT) domain with the equivalent region from HpFAS2 did not affect C16/C18 ratio. Chimeric Fas2 containing half N-terminal ScFas2 and half C-terminal HpFas2 carrying H. polymorpha ketoacyl synthase (KS) and PPT gave a remarkable decrease in C16/C18 ratio (0.6 ± 0.1), indicating that KS plays a major role in determining chain length.

Alterations in growth and fatty acid profiles under stress conditions of Hansenula polymorpha defective in polyunsaturated fatty acid synthesis.

Using chemical mutagenesis, mutants of Hansenula polymorpha that were defective in fatty acid synthesis were selected based on their growth requirements on saturated fatty acid mixtures. One mutant (S7) was incapable of synthesizing polyunsaturated fatty acids (PUFA), linoleic and α-linolenic acids. A genetic analysis demonstrated that the S7 strain had a double lesion affecting fatty acid synthesis and Δ(12)-desaturation. A segregant with a defect in PUFA synthesis (H69-2C) displayed normal growth characteristics in the temperature range of 20-42 °C through a modulation of the cellular fatty acid composition. Compared with the parental strain, this yeast mutant had increased sensitivity at low and high temperatures (15 and 48 °C, respectively) with an increased tolerance to oxidative stress. The responses to ethanol stress were similar for the parental and PUFA-defective strains. Myristic acid was also determined to play an essential role in the cell growth of H. polymorpha. These findings suggest that both the type of cellular fatty acids and the composition of fatty acids might be involved in the stress responsive mechanisms in this industrially important yeast.

Increase in rRNA content in a Saccharomyces cerevisiae suppressor strain from rrn10 disruptant by rDNA cluster duplication.

Breeding of yeast strains with higher RNA content is important because yeast RNA is a significant source of 5'-ribonucleotides, which have considerable use in both the food and pharmaceutical industries. Ribosomal RNA (rRNA) is an important source of yeast RNA as it accounts for about 80 % of total RNA content. We previously reported a dominant suppressor mutant of an rrn10 disruptant named SupE, which displays the ability not only to restore diminished RNA content caused by rrn10 disruption but also to increase the transcription level of ribosomal protein (RP) genes on an ∆rrn10 background in Saccharomyces cerevisiae. Here, to construct an S. cerevisiae strain with higher RNA content, we investigated the effect of increasing the copy number of the rDNA gene on a ∆rrn10 SUPE background. We successfully constructed a SupE strain with two copies of the rDNA cluster (ca. 300 rDNA genes) by using chromosome-splitting technology. The RNA content of this strain was 61 % higher than that of the SupE strain with a single copy of the rDNA cluster (ca. 150 rDNA genes), and 40 % higher than that of the wild-type strain with two copies of the rDNA cluster. A further increase in RNA content of 47 % was achieved by multicopy expression of the RPL40A gene in the SupE strain with two copies of the rDNA cluster. These observations suggest that we have constructed an S. cerevisiae strain with two copies of the rDNA cluster, which has achieved a considerably higher RNA content. Furthermore, the strategy taken in this study provides an effective approach to constructing S. cerevisiae strains with high potential for yeast food biotechnology.

Random Transfer of Ogataea polymorpha Genes into Saccharomyces cerevisiae Reveals a Complex Background of Heat Tolerance.

Horizontal gene transfer, a process through which an organism acquires genes from other organisms, is a rare evolutionary event in yeasts. Artificial random gene transfer can emerge as a valuable tool in yeast bioengineering to investigate the background of complex phenotypes, such as heat tolerance. In this study, a cDNA library was constructed from the mRNA of a methylotrophic yeast, Ogataea polymorpha, and then introduced into Saccharomyces cerevisiae. Ogataea polymorpha was selected because it is one of the most heat-tolerant species among yeasts. Screening of S. cerevisiae populations expressing O. polymorpha genes at high temperatures identified 59 O. polymorpha genes that contribute to heat tolerance. Gene enrichment analysis indicated that certain S. cerevisiae functions, including protein synthesis, were highly temperature-sensitive. Additionally, the results confirmed that heat tolerance in yeast is a complex phenotype dependent on multiple quantitative loci. Random gene transfer would be a useful tool for future bioengineering studies on yeasts.

Structure-function studies of ultrahigh molecular weight isoprenes provide key insights into their biosynthesis.

Some plant trans-1,4-prenyltransferases (TPTs) produce ultrahigh molecular weight trans-1,4-polyisoprene (TPI) with a molecular weight of over 1.0 million. Although plant-derived TPI has been utilized in various industries, its biosynthesis and physiological function(s) are unclear. Here, we identified three novel Eucommia ulmoides TPT isoforms-EuTPT1, 3, and 5, which synthesized TPI in vitro without other components. Crystal structure analysis of EuTPT3 revealed a dimeric architecture with a central hydrophobic tunnel. Mutation of Cys94 and Ala95 on the central hydrophobic tunnel no longer synthesizd TPI, indicating that Cys94 and Ala95 were essential for forming the dimeric architecture of ultralong-chain TPTs and TPI biosynthesis. A spatiotemporal analysis of the physiological function of TPI in E. ulmoides suggested that it is involved in seed development and maturation. Thus, our analysis provides functional and mechanistic insights into TPI biosynthesis and uncovers biological roles of TPI in plants.

Identification of protein kinase disruptions as suppressors of the calcium sensitivity of S. cerevisiae Deltaptp2 Deltamsg5 protein phosphatase double disruptant.

The double disruptant of the S. cerevisiae protein phosphatase (PPase) genes, PTP2 (phosphotyrosine-specific PPase) and MSG5 (phosphotyrosine and phosphothreonine/serine-PPase) causes calcium-sensitive growth (Cas). Previous study using Fluorescent-activated cell sorting (FACS) analysis showed that this growth defect with calcium occurs at G1-S transition in the cell cycle. We discovered that six non-essential protein kinase (PKase) disruptions (Deltabck1, Deltamkk1, Deltaslt2/Deltampk1, Deltamck1, Deltassk2 and Deltayak1) suppressed the Cas-phenotype of the Deltaptp2 Deltamsg5 double disruptant. Bck1p, Mkk1p and Slt2p are components of the mitogen-activated protein kinase (MAPK) cascade of cell wall integrity pathway (Slt2 pathway), and Mck1p is its down regulator. Ssk2p is the MAPK kinase kinase of the high-osmolarity glycerol (HOG) pathway, while Yak1p is a negative regulator for the cAMP-dependent PKA pathway. FACS analysis revealed that only the disruption of Deltassk2 and Deltayak1 but not Deltabck1, Deltamkk1, Deltaslt2 and Deltamck1 was able to suppress the delayed G1-S transition, suggesting that suppression of the growth defect is not always accompanied by suppression of the G1-S transition delay. The discovery of these PKases as suppressors revealed that in addition to the previously anticipated Slt2 pathway, HOG, Yak1p and Mck1p regulatory pathways may also be involved in the calcium sensitivity of the Deltaptp2 Deltamsg5 double disruptant.

Ethanol production from biomass by repetitive solid-state fed-batch fermentation with continuous recovery of ethanol.

To save cost and input energy for bioethanol production, a consolidated continuous solid-state fermentation system composed of a rotating drum reactor, a humidifier, and a condenser was developed. Biomass, saccharifying enzymes, yeast, and a minimum amount of water are introduced into the system. Ethanol produced by simultaneous saccharification and fermentation is continuously recovered as vapor from the headspace of the reactor, while the humidifier compensates for the water loss. From raw corn starch as a biomass model, 95 +/- 3, 226 +/- 9, 458 +/- 26, and 509 +/- 64 g l(-1) of ethanol solutions were recovered continuously when the ethanol content in reactor was controlled at 10-20, 30-50, 50-70 and 75-85 g kg-mixture(-1), respectively. The residue showed a lesser volume and higher solid content than that obtained by conventional liquid fermentation. The cost and energy for intensive waste water treatment are decreased, and the continuous fermentation enabled the sustainability of enzyme activity and yeast in the system.

Mating-type switching and mating-type gene array expression in the methylotrophic yeast Ogataea thermomethanolica TBRC656.

The methylotrophic yeast, Ogataea thermomethanolica TBRC656, is an attractive host organism for heterologous protein production owing to the availability of protein expression vectors and a genome-editing tool. In this study, we focused on mating-type switching and gene expression in order to elucidate its sexual life cycle and establish genetic approaches applicable for the strain. A putative mating-type gene cluster was identified in TBRC656 that is syntenic to the cluster in Ogataea parapolymorpha DL-1 (previously named Hansenula polymorpha). Like DL-1, TBRC656 possesses two mating loci, namely MATa and MATα, and also shows flip-flop mating-type switching. Interestingly, unlike any other methylotrophic yeast, TBRC656 robustly switched mating type during late growth in rich medium (YPD). Under nutrient depletion, mating-type switching was observed within one hour. Transcription from both MATa and MATα mating loci was detected during growth in YPD, and possibly induced upon nitrogen depletion. Gene expression from MATα was detected as a single co-transcript from a three-gene array (α2-α1-a1S). Deletion of a putative a1S ORF at the MATα locus had no observed effect on mating-type switching but demonstrated significant effect on mating-type gene expression at both MATa and MATα loci.

Yeast protein phosphatases Ptp2p and Msg5p are involved in G1-S transition, CLN2 transcription, and vacuole morphogenesis.

We previously reported that double disruption of protein phosphatase (PPase) genes PTP2 (phosphotyrosine-specific PPase) and MSG5 (phosphotyrosine and phosphothreonine/serine-PPase) causes Ca(2+) sensitive growth, whereas the single disruptions do not. This finding suggests that Ptp2p and Msg5p are involved in Ca(2+)-induced stress response in a redundant manner. To gain insight into the molecular mechanism causing calcium sensitivity of the ptp2 msg5 double disruptant, we performed fluorescence-activated cell sorting analysis and found a delayed G1 phase. This delayed G1 was consistent with the defect in bud emergence, and reduced CLN2 transcription upon addition of CaCl(2). We also found that Slt2p is hyper-phosphorylated in the Deltaptp2 Deltamsg5 double disruptant and that the vacuole of the Deltaptp2 Deltamsg5 double disruptant is fragmented even in the absence of Ca(2+). These findings suggest that both Ptp2p and Msg5p are involved in the G1 to S transition and vacuole morphogenesis possibly through their regulation of Slt2 pathway.

Advances in molecular methods to alter chromosomes and genome in the yeast Saccharomyces cerevisiae.

A fundamental issue in biotechnology is how to breed useful strains of microorganisms for efficient production of valuable biomaterials. On-going and more recent developments in gene manipulation technologies and chromosomal and genomic modifications in particular have facilitated important contributions in this area. "Chromosome manipulation technology" as an outgrowth of "gene manipulation technology" may provide opportunities for creating novel strains of organisms with a variety of genomic constitutions. A simple and rapid chromosome splitting technology called "PCR-mediated chromosome splitting" (PCS) that we recently developed has made it possible to manipulate chromosomes and genomes on a large scale in an industrially important microorganism, Saccharomyces cerevisiae. This paper focuses on recent advances in molecular methods for altering chromosomes and genome in S. cerevisiae featuring chromosome splitting technology. These advances in introducing large-scale genomic modifications are expected to accelerate the breeding of novel strains for biotechnological purposes, and to reveal functions of presently uncharacterized chromosomal regions in S. cerevisiae and other organisms.

Protein phosphatase Siw14 controls intracellular localization of Gln3 in cooperation with Npr1 kinase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Deltasiw14 disruptant exhibits caffeine sensitivity. To understand the function of Siw14, double disruptants for SIW14 and each of 102 viable protein kinases (PKase) genes were constructed and examined for suppression of caffeine sensitivity based on the premise that the sensitivity was caused either by accumulation of an unknown phosphorylated Siw14 substrate(s) or by depletion of an unphosphorylated substrate(s) of Siw14 in the Deltasiw14 disruptant. Among 102 pkase disruptions, only one, Deltanpr1, suppressed the caffeine sensitivity of the Deltasiw14 disruptant. Because Gln3 (a phosphorylated transcriptional activator)-dependent transcription is induced by disruption of NPR1, we further examined the effect of disruption and overexpression of GLN3 on the caffeine sensitivity of the Deltasiw14 disruptant. Disruption of GLN3 was found to partially suppress the caffeine sensitivity of the Deltasiw14 disruptant, while overexpression of GLN3 in wild-type cells caused caffeine sensitivity, providing the first evidence that Siw14 functions in the Gln3 regulatory network. We also found that, unlike in a wild-type background, Gln3 accumulates in the nucleus whether cells are exposed or not to caffeine in the Deltasiw14 disruptant, and that this nuclear localization was abolished by disruption of NPR1. Interestingly, the level of Gln3 phosphorylation in both the Deltasiw14 and Deltanpr1 disruptants decreased relative to wild type, independent of exposure to caffeine. We conclude that Siw14 controls the intracellular localization of Gln3 in combination with Npr1, and one of the causes for the caffeine sensitivity of the Deltasiw14 disruptant was an accumulation of dephosphorylated Gln3 in the nucleus.

Ddi1p and Rad23p play a cooperative role as negative regulators in the PHO pathway in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the PHO pathway regulates expression of phosphate-responsive genes such as PHO5, which encodes repressible acid phosphatase (rAPase). In this pathway, Pho81p functions as an inhibitor of the cyclin-cyclin-dependent kinase (CDK) complex Pho80p-Pho85p. However, the mechanism regulating the inhibitory activity of Pho81p is poorly understood. Through use of the yeast two-hybrid system, we identified the UbL-UbA protein Ddi1p as a Pho81p-binding protein. Further, Pho81p levels were found to be low under high-phosphate condition and high during phosphate starvation, indicating that Pho81p is regulated by phosphate concentration. However, our results revealed that Ddi1p and its associated protein Rad23p are not involved in the decrease in Pho81p level under high-phosphate condition. Significantly, the Deltaddi1Deltarad23 strain exhibited a remarkable increase in rAPase activity at an intermediate-phosphate concentration of 0.4mM, suggesting that Ddi1p and Rad23p play a cooperative role as negative regulators in the PHO pathway.

PCR-mediated one-step deletion of targeted chromosomal regions in haploid Saccharomyces cerevisiae.

Chromosome rearrangements, especially chromosomal deletions, have been exploited as important resources for functional analysis of genomes. To facilitate this analysis, we applied a previously developed method for chromosome splitting for the direct deletion of a designed internal or terminal chromosomal region carrying many nonessential genes in haploid Saccharomyces cerevisiae. The method, polymerase chain reaction (PCR)-mediated chromosomal deletion (PCD), consists of a two-step PCR and one transformation per deletion event. In this paper, we show that the PCD method efficiently deletes internal regions in a single transformation. Of the six chromosomal regions targeted for deletion by this method, five regions (16 to 38 kb in length) containing 10 to 19 nonessential genes were successfully eliminated at high efficiency. The one targeted region on chromosome XIII that was not deleted was subsequently found to contain sequences essential for yeast growth. While 14 individual genes in this region have been reported to be nonessential, synthetic lethal interactions may occur among these nonessential genes. Phenotypic analysis showed that four deletion strains still exhibited normal growth while possible synthetic growth defects were observed in another strain harboring a 19-gene deletion on chromosome XV. These results demonstrate that the PCD method is a useful tool for deleting genes and for analyzing their functions in defined chromosomal regions.

Chromosome XII context is important for rDNA function in yeast.

The rDNA cluster in Saccharomyces cerevisiae is located 450 kb from the left end and 610 kb from the right end of chromosome XII and consists of approximately 150 tandemly repeated copies of a 9.1 kb rDNA unit. To explore the biological significance of this specific chromosomal context, chromosome XII was split at both sides of the rDNA cluster and strains harboring deleted variants of chromosome XII consisting of 450 kb, 1500 kb (rDNA cluster only) and 610 kb were created. In the strain harboring the 1500 kb variant of chromosome XII consisting solely of rDNA, the size of the rDNA cluster was found to decrease as a result of a decrease in rDNA copy number. The frequency of silencing of URA3 inserted within the rDNA locus was found to be greater than in a wild-type strain. The localization and morphology of the nucleolus was also affected such that a single and occasionally (6-12% frequency) two foci for Nop1p and a rounded nucleolus were observed, whereas a typical crescent-shaped nucleolar structure was seen in the wild-type strain. Notably, strains harboring the 450 kb chromosome XII variant and/or the 1500 kb variant consisting solely of rDNA had shorter life spans than wild type and also accumulated extrachromosomal rDNA circles. These observations suggest that the context of chromosome XII plays an important role in maintaining a constant rDNA copy number and in physiological processes related to rDNA function in S.cerevisiae.

Genetic analysis of suppressor mutants of a pho84 disruptant in the search for genes involved in intracellular inorganic phosphate sensing in Saccharomyces cerevisiae.

To achieve inorganic phosphate (Pi) homeostasis, cells must be able to sense intracellular and extracellular Pi concentrations. In the Pi signaling (PHO) pathway in Saccharomyces cerevisiae, high Pi represses genes involved in Pi uptake (e.g., PHO84) and Pi utilization (PHO5); conversely, the cyclin-dependent kinase inhibitor Pho81 inhibits the activity of the Pho80-Pho85 cyclin-cyclin dependent kinase complex in low-Pi conditions, leading to induction of these genes. However, how yeast senses Pi availability remains unresolved. To identify factors involved in Pi sensing upstream of the Pho81-Pho80-Pho85 complex, we generated and screened suppressor mutants of a Δpho84 strain that shows constitutive PHO5 expression. By a series of genetic tests, including dominance-recessiveness, complementation and tetrad analyses, three sef (suppressor of pho84 [pho eighty-four]) mutants (sef8, sef9 and sef10) were shown to contain a novel single mutation. The sef mutants suppressed the phenotype of constitutive PHO5 expression at the transcriptional level, but did not show restored Pi uptake capacity. An epistasis-hypostasis test revealed that the sef mutations were hypostatic to pho80 mutation, indicating that their gene products function upstream of the Pho81-Pho80-Pho85 complex in the PHO pathway. The sef mutations identified are associated with gene(s) that may be involved in the homeostasis of an intracellular Pi level-sensing mechanism in S. cerevisiae.

Identification and characterization of a very long-chain fatty acid elongase gene in the methylotrophic yeast, Hansenula polymorpha.

To understand the biosynthetic network of fatty acids in the methylotrophic yeast Hansenula polymorpha, which is able to produce poly-unsaturated fatty acids, we have attempted to identify genes encoding fatty acid elongase. Here we have characterized HpELO1, a fatty acid elongase gene encoding a 319-amino-acid protein containing five predicted membrane-spanning regions that is conserved throughout the yeast Elo protein family. Phylogenetic analysis of the deduced amino acid sequence suggests that HpELO1 is an ortholog of the Saccharomyces cerevisiae ELO3 gene that is involved in the elongation of very long-chain fatty acids (VLCFAs). In the fatty acid profile of the Hpelo1Delta disruptant by gas chromatography/mass spectrometry, the amount of C24:0 and C26:0 decreased to undetectable levels, whereas there was a large accumulation of C22:0, suggesting that the HpELO1 is involved in the elongation of VLCFAs and is essential for the production of C24:0. Expression of HpELO1 suppressed the lethality of the S. cerevisiae elo2Delta elo3Delta double disruptant and recovered the synthesis of VLCFAs. Similar to the S. cerevisiae elo3Delta strain, the Hpelo1Delta disruptant exhibited the extraordinary growth sensitivity to fumonisin B(1), a ceramide synthase inhibitor. Furthermore, cells of the Hpelo1Delta disruptant were more sensitive to Zymolyase and more flocculent than the wild-type cells, clumping together and falling rapidly out of suspension, suggesting that the Hpelo1Delta mutation causes changes in cell wall composition and structure.