Functional characterization of the Hansenula polymorpha HOC1, OCH1, and OCR1 genes as members of the yeast OCH1 mannosyltransferase family involved in protein glycosylation

The alpha-1,6-mannosyltransferase encoded by Saccharomyces cerevisiae OCH1 (ScOCH1) is responsible for the outer chain initiation of N-linked oligosaccharides. To identify the genes involved in the first step of outer chain biosynthesis in the methylotrophic yeast Hansenula polymorpha, we undertook the functional analysis of three H. polymorpha genes, HpHOC1, HpOCH1, and HpOCR1, that belong to the OCH1 family containing seven members with significant sequence identities to ScOCH1. The deletions of these H. polymorpha genes individually resulted in several phenotypes suggestive of cell wall defects. Whereas the deletion of HpHOC1 (Hphoc1Delta) did not generate any detectable changes in N-glycosylation, the null mutant strains of HpOCH1 (Hpoch1Delta) and HpOCR1 (Hpocr1Delta) displayed a remarkable reduction in hypermannosylation. Although the apparent phenotypes of Hpocr1Delta were most similar to those of S. cerevisiae och1 mutants, the detailed structural analysis of N-glycans revealed that the major defect of Hpocr1Delta is not in the initiation step but rather in the subsequent step of outer chain elongation by alpha-1,2-mannose addition. Most interestingly, Hpocr1Delta showed a severe defect in the O-linked glycosylation of extracellular chitinase, representing HpOCR1 as a novel member of the OCH1 family implicated in both N- and O-linked glycosylation. In contrast, addition of the first alpha-1,6-mannose residue onto the core oligosaccharide Man8GlcNAc2 was completely blocked in Hpoch1Delta despite the comparable growth of its wild type under normal growth conditions. The complementation of the S. cerevisiae och1 null mutation by the expression of HpOCH1 and the lack of in vitro alpha-1,6-mannosyltransferase activity in Hpoch1Delta provided supportive evidence that HpOCH1 is the functional orthologue of ScOCH1. The engineered Hpoch1Delta strain with the targeted expression of Aspergillus saitoi alpha-1,2-mannosidase in the endoplasmic reticulum was shown to produce human-compatible high mannose-type Man5GlcNAc2 oligosaccharide as a major N-glycan.     

Engineering of the yeast Yarrowia lipolytica for the production of glycoproteins lacking the outer-chain mannose residues of N-glycans

In an attempt to engineer a Yarrowia lipolytica strain to produce glycoproteins lacking the outer-chain mannose residues of N-linked oligosaccharides, we investigated the functions of the OCH1 gene encoding a putative alpha-1,6-mannosyltransferase in Y. lipolytica. The complementation of the Saccharomyces cerevisiae och1 mutation by the expression of YlOCH1 and the lack of in vitro alpha-1,6-mannosyltransferase activity in the Yloch1 null mutant indicated that YlOCH1 is a functional ortholog of S. cerevisiae OCH1. The oligosaccharides assembled on two secretory glycoproteins, the Trichoderma reesei endoglucanase I and the endogenous Y. lipolytica lipase, from the Yloch1 null mutant contained a single predominant species, the core oligosaccharide Man8GlcNAc2, whereas those from the wild-type strain consisted of oligosaccharides with heterogeneous sizes, Man8GlcNAc2 to Man12GlcNAc2. Digestion with alpha-1,2- and alpha-1,6-mannosidase of the oligosaccharides from the wild-type and Yloch1 mutant strains strongly supported the possibility that the Yloch1 mutant strain has a defect in adding the first alpha-1,6-linked mannose to the core oligosaccharide. Taken together, these results indicate that YlOCH1 plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in Y. lipolytica. Therefore, the Yloch1 mutant strain can be used as a host to produce glycoproteins lacking the outer-chain mannoses and further developed for the production of therapeutic glycoproteins containing human-compatible oligosaccharides.     

Functional analysis of the ALG3 gene encoding the Dol-P-Man: Man5GlcNAc2-PP-Dol mannosyltransferase enzyme of P. pastoris

N-glycans are synthesized in both yeast and mammals through the ordered assembly of a lipid-linked core Glc(3)Man(9)GlcNAc(2) structure that is subsequently transferred to a nascent protein in the endoplasmic reticulum. Once folded, glycoproteins are then shuttled to the Golgi, where additional but divergent processing occurs in mammals and fungi. We cloned the Pichia pastoris homolog of the ALG3 gene, which encodes the enzyme that converts Man(5)GlcNAc(2)-Dol-PP to Man(6)GlcNAc(2)-Dol-PP. Deletion of this gene in an och1 mutant background resulted in the secretion of glycoproteins with a predicted Man(5)GlcNAc(2) structure that could be trimmed to Man(3)GlcNAc(2) by in vitro alpha-1,2-mannosidase treatment. However, several larger glycans ranging from Hex(6)GlcNAc(2) to Hex(12)GlcNAc(2) were also observed that were recalcitrant to an array of mannosidase digests. These results contrast the far simpler glycan profile found in Saccharomyces cerevisiae alg3-1 och1, indicating diverging Golgi processing in these two closely related yeasts. Finally, analysis of the P. pastoris alg3 deletion mutant in the presence and absence of the outer chain initiating Och1p alpha-1,6-mannosyltransferase activity suggests that the PpOch1p has a broader substrate specificity compared to its S. cerevisiae counterpart.     

OCH1 encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine-linked oligosaccharides.

The Saccharomyces cerevisiae och1 mutant shows a deficiency in the mannose outer chain elongation at the non-permissive temperature. We have cloned the OCH1 gene by complementation of temperature sensitive (ts) phenotype for growth. The integrant of OCH1 gene in the yeast chromosome can complement the ts phenotype and shows the same mapping position as that of the och1 mutation, indicating that the cloned gene is the true gene for mutation. The OCH1 gene disruptant is not lethal but ts for cell growth, and lacks mannose outer chains. The OCH1 gene sequence predicts a 55 kDa protein consisting of 480 amino acids. It contains four potential asparagine-linked (N-linked) glycosylation sites and a single transmembrane region near the N-terminus. In vitro translation/translocation analysis revealed that the large C-terminal region of the OCH1 protein is located at the lumenal side of microsomal membranes with some sugar modification, indicating a type II membrane topology. The OCH1 protein was detected in yeast membrane fractions as four forms of 58-66 kDa, which correspond to the size of a glycoprotein containing four N-linked sugar chains the length of which is almost the same or slightly larger than the inner core (Man8GlcNAc2) formed in the endoplasmic reticulum (ER). Finally, the OCH1 gene was found to encode a novel mannosyltransferase which specifically transfers [14C]mannose to the unique acceptor, the core-like oligosaccharide of cell wall mannan accumulated in the och1 disruptant.     

Coflocculation of Escherichia coli and Schizosaccharomyces pombe

Several yeasts, such as Candida utilis, Dekkera bruxellensis, Hanseniaspora guilliermondii, Kloeckera apiculata, Saccharomyces cerevisiae and Schizosaccharomyces pombe, were found to coaggregate with Escherichia coli, but S. pombe showed much less coflocculation than the other yeasts (Peng et al. 2001)). S. pombe is known to have galactose-rich cell walls and we investigated whether this might be responsible for its different behavior by studying the wild-type TP4-1D, with a mannose to galactose ratio of 1 to 1.2, and the glycosylation mutant gms1delta (Man:Gal=1:0). The wild-type induced very low levels of coflocculation (3%) while gms1delta induced a remarkable amount of coflocculation (48%). Coflocculation of the mutant was inhibited by mannose but not affected by galactose or glucose. The S. cerevisiae mnn2 mutant, with a mannan structure similar to gms1delta, also showed a high degree of coflocculation (40%). However, S. cerevisiae mutant mnn9, with a mature core similar to S. pombe, showed decreased coflocculation (21.3%). Both these S. cerevisae mutants were sensitive to mannose inhibition. Coflocculation of E. coli and gms1delta also could be inhibited by gms1delta mannan and plant lectins, such as HHA, GNA and NPA, specific to either alpha-1-3- or alpha-1-6-linked mannosyl units. From these results we conclude that the E. coli lectins may have specificity for alpha-1-6- and alpha-1-3-linked mannose residues either in the outer chain or in the core of S. pombe, but in wild-type strains these mannose residues are shielded by galactose residues.     

Engineering of the Yeast Yarrowia lipolytica for the Production of Glycoproteins Lacking the Outer-Chain Mannose Residues of N-Glycans

In an attempt to engineer a Yarrowia lipolytica strain to produce glycoproteins lacking the outer-chain mannose residues of N-linked oligosaccharides, we investigated the functions of the OCH1 gene encoding a putative α-1,6-mannosyltransferase in Y. lipolytica. The complementation of the Saccharomyces cerevisiae och1 mutation by the expression of YlOCH1 and the lack of in vitro α-1,6-mannosyltransferase activity in the Yloch1 null mutant indicated that YlOCH1 is a functional ortholog of S. cerevisiae OCH1. The oligosaccharides assembled on two secretory glycoproteins, the Trichoderma reesei endoglucanase I and the endogenous Y. lipolytica lipase, from the Yloch1 null mutant contained a single predominant species, the core oligosaccharide Man₈GlcNAc₂, whereas those from the wild-type strain consisted of oligosaccharides with heterogeneous sizes, Man₈GlcNAc₂ to Man₁₂GlcNAc₂. Digestion with α-1,2- and α-1,6-mannosidase of the oligosaccharides from the wild-type and Yloch1 mutant strains strongly supported the possibility that the Yloch1 mutant strain has a defect in adding the first α-1,6-linked mannose to the core oligosaccharide. Taken together, these results indicate that YlOCH1 plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in Y. lipolytica. Therefore, the Yloch1 mutant strain can be used as a host to produce glycoproteins lacking the outer-chain mannoses and further developed for the production of therapeutic glycoproteins containing human-compatible oligosaccharides.     

Glycoprotein biosynthesis in Saccharomyces cerevisiae. Characterization of alpha-1,6-mannosyltransferase which initiates outer chain formation

A particulate fraction from the Saccharomyces cerevisiae mnn1 mutant was obtained after extracting a 115,000 x g pellet with 0.75% Triton X-100. Incubation of this preparation with labeled Man8GlcNAc and Man9GlcNAc in the presence of GDP-mannose followed by high pressure liquid chromatography showed the formation of Man9GlcNAc and Man10GlcNAc, respectively. Analysis by high resolution 1H NMR of the products indicates that, in each case, the mannose residue added is alpha-1,6-linked to the alpha-1,6-mannose residue of the substrate as follows (where M represents mannose and Gn represents N-acetylglucosamine): (Formula: see text). The mannosyltransferase therefore catalyzes the first step specific to the biosynthesis of the outer chain of yeast mannoproteins. The apparent Km values for both substrates are similar: 0.39 mM for Man8GlcNAc and 0.35 mM for Man9GlcNAc. The alpha-1,6-mannosyltransferase exhibits maximum activity between pH 7.1 and 7.6 in Tris maleate buffer, has an absolute requirement for Mn2+, and also requires Triton X-100. These results indicate that removal of the alpha-1,2-linked mannose residue from Man9GlcNAc is not essential for the alpha-1,6-mannosyltransferase which initiates outer chain synthesis, at least when oligosaccharides are used as substrates in a cell-free system.     

Decrease in cell surface galactose residues of Schizosaccharomyces pombe enhances its coflocculation with Pediococcus damnosus

Pediococcus damnosus can coflocculate with Saccharomyces cerevisiae and cause beer acidification that may or may not be desired. Similar coflocculations occur with other yeasts except for Schizosaccharomyces pombe which has galactose-rich cell walls. We compared coflocculation rates of S. pombe wild-type species TP4-1D, having a mannose-to-galactose ratio (Man:Gal) of 5 to 6 in the cell wall, with its glycosylation mutants gms1-1 (Man:Gal = 5:1) and gms1Delta (Man:Gal = 1:0). These mutants coflocculated at a much higher level (30 to 45%) than that of the wild type (5%). Coflocculation of the mutants was inhibited by exogenous mannose but not by galactose. The S. cerevisiae mnn2 mutant, with a mannan content similar to that of gms1Delta, also showed high coflocculation (35%) and was sensitive to mannose inhibition. Coflocculation of P. damnosus and gms1Delta (or mnn2) also could be inhibited by gms1Delta mannan (with unbranched alpha-1,6-linked mannose residues), concanavalin A (mannose and glucose specific), or NPA lectin (specific for alpha-1,6-linked mannosyl units). Protease treatment of the bacterial cells completely abolished coflocculation. From these results we conclude that mannose residues on the cell surface of S. pombe serve as receptors for a P. damnosus lectin but that these receptors are shielded by galactose residues in wild-type strains. Such interactions are important in the production of Belgian acid types of beers in which mixed cultures are used to improve flavor.     

Functional characterization of alanine racemase from Schizosaccharomyces pombe: a eucaryotic counterpart to bacterial alanine racemase

Schizosaccharomyces pombe has an open reading frame, which we named alr1(+), encoding a putative protein similar to bacterial alanine racemase. We cloned the alr1(+) gene in Escherichia coli and purified the gene product (Alr1p), with an M(r) of 41,590, to homogeneity. Alr1p contains pyridoxal 5'-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent K(m) and V(max) values as follows: for L-alanine, 5.0 mM and 670 micromol/min/mg, respectively, and for D-alanine, 2.4 mM and 350 micromol/min/mg, respectively. The enzyme is almost specific to alanine, but L-serine and L-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of L-alanine, respectively. S. pombe uses D-alanine as a sole nitrogen source, but deletion of the alr1(+) gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway for L-alanine coupled with racemization plays a major role in the catabolism of D-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses L-alanine but not D-alanine as a sole nitrogen source. Moreover, D-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1(+) gene enabled S. cerevisiae to grow efficiently on D-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of D-alanine.     

Man8GlcNAc2糖基化的酿酒酵母菌株的构建

酿酒酵母糖蛋白的N-糖基化经过高尔基体的修饰后形成聚合度约150-200的甘露寡糖,高尔基体N-糖基化的糖基转移酶Mnn1p和Och1p在甘露寡糖的形成过程中起关键作用。通过同源重组置换敲除了酵母中的MNN1和OCH1基因阻断高尔基体N-糖基化修饰,分离纯化了mnn1 och1突变株中的N-糖蛋白,糖酰胺酶PNGaseF酶解释放的N-糖链经过2-氨基吡啶衍生后,利用HPLC和MALDITOF/MS结合的方法分析了突变株糖蛋白上的N-糖链。结果显示mnn1 och1突变株中的糖蛋白的N-糖链为结构单一的糖链,分子量为1794.66,推测为Man₈GlcNAc₂。     

Schizosaccharomyces pombe possesses two plasma membrane alkali metal cation/H antiporters differing in their substrate specificity

The Schizosaccharomyces pombe plasma membrane Na(+)/H(+) antiporter, SpSod2p, has been shown to belong to the subfamily of yeast Na(+)/H(+) antiporters that only recognize Na(+) and Li(+) as substrates. Nevertheless, most of the studied plasma membrane alkali metal cation/H(+) antiporters from other yeasts have broader substrate specificities, exporting K(+) and Rb(+) as well. Such antiporters probably play two roles in the physiology of cells: the elimination of surplus toxic cations, and the regulation of stable intracellular K(+) content, pH and cell volume. The systematic sequencing of the Sch. pombe genome revealed the presence of an as-yet uncharacterized homolog of the Spsod2 gene (designated Spsod22). Spsod22 and Spsod2 were expressed in Saccharomyces cerevisiae cells lacking their own alkali metal cation efflux systems, and the transport properties of both Sch. pombe antiporters were compared to those of the Sac. cerevisiae Nha1 antiporter expressed under the same conditions. Here we show that SpSod22p has broad substrate specificity upon heterologous expression in Sac. cerevisiae cells and contributes to cell tolerance to high external levels of K(+). Thus, the Sch. pombe genome encodes two plasma membrane alkali metal cation/H(+) antiporters that play different roles in the physiology of the yeast.     

Characterization of glycosylated variants of beta-lactoglobulin expressed in Pichia pastoris

Glycosylated variants of beta-lactoglobulin (BLG) were produced in the methylotrophic yeast Pichia pastoris to mimic the glycosylation pattern of glycodelin, a homologue of BLG found in humans. Glycodelin has three sites for glycosylation, corresponding to amino acids 63-65 (S1), 85-87 (S2) and 28-30 (S3) of BLG. These three sites were engineered into BLG to produce the variants S2, S12 and S123, which carried one, two and three glycosylation sites, respectively. The oligosaccharides on these BLG variants ranged from (mannose)(9)(N-acetylglucosamine)(2) (Man(9)GN(2)) to Man(15)GN(2) and were of the alpha-linked high mannose type. The variant S123 exhibited highest levels of glycosylation, with the range of glycans being Man(9-14)GN(2). Digestion of S123 with alpha-1,2 linkage specific mannosidase resulted in a single product corresponding to Man(6)GN(2). These results indicated a glycosylation pattern consisting of a Man(5)GN(2) structure extended by 4-9 mannose residues attached mainly by alpha-1,2 linkages. The results also indicated extension of the Man(5)GN(2) structure by a single alpha-1,6-linked mannose. The N-linked glycosylation pathway in P.pastoris is significantly different from that in Saccharomyces cerevisiae, with the addition of shorter outer chains to the core and no alpha-1,3 outer extensions.     

Fission yeast Mog1p homologue, which interacts with the small GTPase Ran, is required for mitosis-to-interphase transition and poly(A)(+) RNA metabolism

We have cloned and characterized the Schizosaccharomyces pombe gene mog1(+), which encodes a protein with homology to the Saccharomyces cerevisiae Mog1p participating in the Ran-GTPase system. The S. pombe Mog1p is predominantly localized in the nucleus. In contrast to the S. cerevisiae MOG1 gene, the S. pombe mog1(+) gene is essential for cell viability. mog1(+) is required for the mitosis-to-interphase transition, as the mog1-1 mutant arrests at restrictive temperatures as septated, binucleated cells with highly condensed chromosomes and an aberrant nuclear envelope. FACS analysis showed that these cells do not undergo a subsequent round of DNA replication. Surprisingly, also unlike the Delta mog1 mutation in S. cerevisiae, the mog1-1 mutation causes nucleolar accumulation of poly(A)(+) RNA at the restrictive temperature in S. pombe, but the signals do not overlap with the fibrillarin-rich region of the nucleolus. Thus, we found that mog1(+) is required for the mitosis-to-interphase transition and a class of RNA metabolism. In our attempt to identify suppressors of mog1-1, we isolated the spi1(+) gene, which encodes the fission yeast homologue of Ran. We found that overexpression of Spi1p rescues the S. pombe Delta mog1 cells from death. On the basis of these results, we conclude that mog1(+) is involved in the Ran-GTPase system.     

Isolation and characterization of Nrf1p, a novel negative regulator of the Cdc42p GTPase in Schizosaccharomyces pombe

The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction pathways in eukaryotic cells leading to actin rearrangements. A cross-species genetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24(ts) mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One cDNA, nrf1(+), encoded an approximately 15 kD protein with three potential transmembrane domains and 78% amino-acid identity to a S. cerevisiae gene, designated NRF1. A S. pombe Deltanrf1 mutant was viable but overexpression of nrf1(+) in S. pombe resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology indicative of loss of polarized cell growth along with partially delocalized cortical actin and large vacuoles. nrf1(+) also displayed synthetic overdose phenotypes with cdc42 and pak1 alleles. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to intracellular membranes, including vacuolar membranes, and to sites of septum formation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S. pombe Cdc24p homolog Scd1p. Taken together, these data are consistent with Nrf1p functioning as a negative regulator of Cdc42p within the cell polarity pathway.     

α-1,6-甘露糖转移酶基因敲除的毕赤酵母菌株构建及其用于融合蛋白HSA/GM-CSF 表达的研究

酵母对蛋白的糖基化修饰过程不同于哺乳动物,其特点为产生高甘露糖型糖基且易发生过度糖基化。本研究通过两步基因重组敲除目标基因的方法成功敲除了毕赤酵母中的α-1,6-甘露糖转移酶(och1p)基因,获得了och1敲除的菌株。以此为基础,构建了高效表达人血清白蛋白与粒细胞-巨噬细胞集落刺激因子融合蛋白(HSA/GM-CSF)的工程酵母,与野生型毕赤酵母表达的过度糖基化HSA/GM-CSF不同,och1敲除菌表达的该融合蛋白糖基化程度明显降低,这为该融合蛋白的开发提供了重要基础。och1敲除菌株的构建不仅提供了一个对糖蛋白进行低糖基化修饰的毕赤酵母表达系统,而且为进一步的酵母糖基工程改造提供了基础。     

A functional homologue of the RNA1 gene product in Schizosaccharomyces pombe: purification, biochemical characterization, and identification of a leucine-rich repeat motif.

The RNA1 gene from Saccharomyces cerevisiae is defined by the temperature-sensitive rna1-1 mutation that interferes with the maturation and/or nucleocytoplasmic transport of RNA. We describe the purification of a 44-kDa protein from the evolutionary distant fission yeast Schizosaccharomyces pombe and the cloning and sequence analysis of the corresponding gene. Although this protein shares only 42% sequence identity with the RNA1 gene product, it represents a functional homologue because the expression of the S. pombe gene in S. cerevisiae complements the rna1-1 defect. Disruption in S. pombe of the gene encoding the 44-kDa protein, for which we propose the name S. pombe rna1p, reveals that it is essential for growth. Our analysis of purified S. pombe rna1p represents the first biochemical characterization of an RNA1 gene product and reveals that it is a monomeric protein of globular shape. Cell fractionation and immunofluorescence microscopy indicate that rna1p is a cytoplasmic protein possibly enriched in the nuclear periphery. We identify a sequence motif of 29 residues, which is rich in leucine and repeated eight times both in S. pombe and in S. cerevisiae rna1p. Similar leucine-rich repeats present in a series of other proteins, e.g., the mammalian ribonuclease/angiogenin inhibitor, adenylyl cyclase from S. cerevisiae, the toll protein from Drosophila melanogaster, and the sds22 protein phosphatase regulatory subunit from S. pombe, are thought to be involved in protein-protein interactions. Thus rna1p may act as a scaffold protein possibly interacting in the nuclear periphery with a protein ligand that could be associated with exported RNA.     

Dolichol phosphate mannose synthase from the filamentous fungus Trichoderma reesei belongs to the human and Schizosaccharomyces pombe class of the enzyme

Dolichol phosphate mannose (DPM) synthase activity, which is required in N:-glycosylation, O-mannosylation, and glycosylphosphatidylinositol membrane anchoring of protein, has been postulated to regulate the Trichoderma reesei secretory pathway. We have cloned a T.reesei cDNA that encodes a 243 amino acid protein whose amino acid sequence shows 67% and 65% identity, respectively, to the Schizosaccharomyces pombe and human DPM synthases, and which lacks the COOH-terminal hydrophobic domain characteristic of the Saccharomyces cerevisiae class of synthase. The Trichoderma dpm1 (Trdpm1) gene complements a lethal null mutation in the S.pombe dpm1(+) gene, but neither restores viability of a S.cerevisiae dpm1-disruptant nor complements the temperature-sensitivity of the S. cerevisiae dpm1-6 mutant. The T.reesei DPM synthase is therefore a member of the "human" class of enzyme. Overexpression of Trdpm1 in a dpm1(+)::his7/dpm1(+) S.pombe diploid resulted in a 4-fold increase in specific DPM synthase activity. However, neither the wild type T. reesei DPM synthase, nor a chimera consisting of this protein and the hydrophobic COOH terminus of the S.cerevisiae DPM synthase, complemented an S.cerevisiae dpm1 null mutant or gave active enzyme when expressed in E.coli. The level of the Trdpm1 mRNA in T.reesei QM9414 strain was dependent on the composition of the culture medium. Expression levels of Trdpm1 were directly correlated with the protein secretory capacity of the fungus.