Toshihiro Komeda

Efficient Antibody Production upon Suppression of O Mannosylation in the Yeast Ogataea minuta

ABSTRACT When antibodies were expressed in the methylotrophic yeast Ogataea minuta, we found that abnormal O mannosylation occurred in the secreted antibody. Yeast-specific O mannosylation is initiated by the addition of mannose at serine (Ser) or threonine (Thr) residues in the endoplasmic reticulum via protein O mannosyltransferase (Pmt) activity. To suppress the addition of O-linked sugar chains on antibodies, we examined the possibility of inhibiting Pmt activity by the addition of a Pmt inhibitor during cultivation. The Pmt inhibitor was found to partially suppress the O mannosylation on the antibodies. Surprisingly, the suppression of O mannosylation was associated with an increased amount of assembled antibody (H2L2) and enhanced the antigen-binding activity of the secreted antibody. In this study, we demonstrated the expression of human antibody in O. minuta and elucidated the relationship between O mannosylation and antibody production in yeast.

Regulation and evaluation of five methanol-inducible promoters in the methylotrophic yeast Candida boidinii.

We isolated the promoter regions of five methanol-inducible genes (P(AOD1), alcohol oxidase; P(DAS1), dihydroxyacetone synthase; P(FDH1), formate dehydrogenase; P(PMP20), Pmp20; and P(PMP47), Pmp47) from the Candida boidinii genome, and evaluated their strength and studied their regulation using the acid phosphatase gene of Saccharomyces cerevisiae (ScPHO5) as the reporter. Of the five promoters, P(DAS1) was the strongest methanol-inducible promoter whose strength was approximately 1.5 times higher than that of the commonly used P(AOD1) in methanol-induced cells. Although the expression of P(AOD1) and P(DAS1) was completely repressed by the presence of glucose, formate-induced expression of P(FDH1) was not repressed by glucose. Expression under P(PMP47), another methanol-inducible promoter, was highly induced by oleate. The induction kinetics of P(PMP47) and P(DAS1) revealed that methanol induces the expression of peroxisome membrane protein Pmp47, earlier than the expression of matrix enzyme dihydroxyacetone synthase (Das1p), and that this information is contained in the promoter region of the respective gene. This is the first report which evaluates several methanol-inducible promoters in parallel in the methylotrophic yeast.

Efficient production of L-lactic acid by Crabtree-negative yeast Candida boidinii

Industrial production of L‐lactic acid, which in polymerized form as poly‐lactic acid is widely used as a biodegradable plastic, has been attracting world‐wide attention. By genetic engineering we constructed a strain of the Crabtree‐negative yeast Candida boidinii that efficiently produced a large amount of L‐lactic acid. The alcohol fermentation pathway of C. boidinii was altered by disruption of the PDC1 gene encoding pyruvate decarboxylase, resulting in an ethanol production that was reduced to 17% of the wild‐type strain. The alcohol fermentation pathway of the PDC1 deletion strain was then successfully utilized for the synthesis of L‐lactic acid by placing the bovine L‐lactate dehydrogenase‐encoding gene under the control of the PDC1 promoter by targeted integration. Optimizing the conditions for batch culture in a 5 l jar‐fermenter resulted in an L‐lactic acid production reaching 85.9 g/l within 48 h. This productivity (1.79 g/l/h) is the highest thus far reported for L‐lactic acid‐producing yeasts. DDBJ/EMBL/GenBank nucleotide database with Accession Nos. AB440630 and AB440631. Copyright

Construction of Protease-deficient Candida boidinii Strains Useful for Recombinant Protein Production: Cloning and Disruption of Proteinase A Gene (PEP4) and Proteinase B Gene…

The yeast Candida boidinii PEP4 and PRB1 genes, encoding proteinase A (PrA) and proteinase B (PrB), respectively, have been cloned and their primary structures were analyzed. The open reading frames of the PEP4 gene (1263 bp encoding a protein of 420 amino acids) and the PRB1 gene (1683 bp encoding a protein of 560 amino acids) were found. The deduced amino acid sequences of PrA and PrB are very similar to Saccharomyces cerevisiae PrA and PrB (64% and 61% identities, respectively). Both PEP4 and PRB1 genes were disrupted in the C. boidinii genome by one-step gene disruption. The resultant pep4Δ and the pep4Δ prb1Δ strains lost protease activity when compared with the wild-type original strain. The constructed C. boidinii strains are expected to be useful hosts for heterologous protein production.

Production of trehalose from starch by novel trehalose-producing enzymes from Sulfolobus solfataricus KM1

A new process for trehalose production from starch was developed using a novel glycosyltransferase and a novel α-amylase from Sulfolobus solfataricus KM1. The yield of trehalose from starch was 81.5% using the two enzymes and a thermostable debranching enzyme. Trehalose production was carried out at high temperature over 60°C and at a high concentration of starch with no risk of contamination by microorganisms or retrogradation of starch.

Cis-acting elements sufficient for induction of FDH1 expression by formate in the methylotrophic yeast Candida boidinii

The FDH1 gene of Candida boidinii encodes an NAD+-dependent formate dehydrogenase, which catalyzes the last reaction in the methanol dissimilation pathway. FDH1 expression is strongly induced by methanol, as are the promoters of the genes AOD1 (alcohol oxidase) and DAS1 (dihydroxyacetone synthase). FDH1 expression can be induced by formate when cells are grown on a medium containing glucose as a carbon source, whereas expression of AOD1 and DAS1 is completely repressed in the presence of glucose. Using deletion analyses, we identified two cis-acting regulatory elements, termed UAS-FM and UAS-M, respectively, in the 5′ non-coding region of the FDH1 gene. Both elements were necessary for full induction of the FDH1 promoter by methanol, while only the UAS-FM element was required for full induction by formate. Irrespective of whether induction was achieved with methanol or formate, the UAS-FM element enhanced the level of induction of the FDH1 promoter in a manner dependent on the number of copies, but independent of their orientation, and also converted the ACT1 promoter from a constitutive into an inducible element. Our results not only provide a powerful promoter for heterologous gene expression, but also yield insights into the mechanism of regulation of FDH1 expression at the molecular level.

Production of active bovine cathepsin C (dipeptidyl aminopeptidase I) in the methylotrophic yeast Candida boidinii

Abstract. The heterologous production of active bovine cathepsin C (CTC; dipeptidyl aminopeptidase I) was investigated. Attempts to express CTC in Escherichia coli were hampered by formation of inclusion bodies that were partially degraded. To overcome this impediment, secretion of recombinant CTC was attempted in the methylotrophic yeast Candida boidinii. A DNA fragment encoding bovine procathepsin C was synthesized based on preferred codon usage in C. boidinii and placed downstream of the C. boidinii proteinase A signal sequence resulting in secretion of active CTC into the culture medium. The gene was expressed under the control of the methanol-inducible formate dehydrogenase gene promoter. Production levels were significantly improved by using a protease-deficient strain, changing medium composition, and by lowering the temperature of induction. When the recombinant C. boidinii was grown for 90xa0h in a jar-fermenter, active CTC was secreted with a yield of up to approximately 12xa0mg/l.

Cloning and sequence analysis of the Candida boidinii ADE2 gene.

Candida boidinii ADE2 gene (phosphoribosyl‐5‐aminoimidazole carboxylase; AIRC, EC 4. 1. 1. 21) has been cloned by homology to the Saccharomyces cerevisiae ADE2 gene. The cloned C. boidinii ADE2 gene complemented the ade2 mutation of S. cerevisiae. Sequence analysis showed a single open reading frame of 1719 nucleotides coding for a polypeptide of 573 residues. Comparison of the deduced amino acid sequence with those of AIRC enzymes from other yeasts showed marked homology among yeast AIRCs. The sequence has already been submitted to DDBJ/EBML/GenBank under Accession No. AB034950. Copyright

Substrate recognition mechanism of a glycosyltrehalose trehalohydrolase from Sulfolobus solfataricus KM1.

Glycosyltrehalose trehalohydrolase (GTHase) is an α‐amylase that cleaves the α‐1,4 bond adjacent to the α‐1,1 bond of maltooligosyltrehalose to release trehalose. To investigate the catalytic and substrate recognition mechanisms of GTHase, two residues, Asp252 (nucleophile) and Glu283 (general acid/base), located at the catalytic site of GTHase were mutated (Asp252→Ser (D252S), Glu (D252E) and Glu283→Gln (E283Q)), and the activity and structure of the enzyme were investigated. The E283Q, D252E, and D252S mutants showed only 0.04, 0.03, and 0.6% of enzymatic activity against the wild‐type, respectively. The crystal structure of the E283Q mutant GTHase in complex with the substrate, maltotriosyltrehalose (G3‐Tre), was determined to 2.6‐Å resolution. The structure with G3‐Tre indicated that GTHase has at least five substrate binding subsites and that Glu283 is the catalytic acid, and Asp252 is the nucleophile that attacks the C1 carbon in the glycosidic linkage of G3‐Tre. The complex structure also revealed a scheme for substrate recognition by GTHase. Substrate recognition involves two unique interactions: stacking of Tyr325 with the terminal glucose ring of the trehalose moiety and perpendicularly placement of Trp215 to the pyranose rings at the subsites −1 and +1 glucose.

Antibody production by a protease-deficient strain of methylotrophic yeast, Ogataea minuta

Background At present the expression system of mammalian cells such as CHO has been adopted as the conventional method to produce antibody for pharmaceuticals. However a novel method of producing antibody has been sought after as a substitute for the costly production of antibody by mammalian cells. Therefore, we tried to construct a novel antibody production system by using methylotrophic yeast, O. minuta. Results When human antibody genes were introduced in the methylotrophic yeast O. minuta to produce an antibody, the heavy chain of the antibody was partially degraded (see Figure 1, lane 1). Peptide sequencing revealed that degradation occurred in the CH1 region (see Figure 2). In order to inhibit this degradation, the YPS1 gene coding Aspartic protease attached to the plasma membrane, a homologue of Saccharomyces cerevisiae, was cloned from O. minuta, and we constructed the Δyps1 strain. As a result, the Δyps1 strain repressed the partial degradation of the antibody (see Figure 1, lane2). from The 4th Recombinant Protein Production Meeting: a comparative view on host physiology Barcelona, Spain. 21–23 September 2006