The effect of nitrogen source on yield and glycosylation of a human cystatin C mutant expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Human cystatin C is a cysteine proteinase inhibitor with potential applications as an anti-viral agent, cancer tumor growth inhibitor, and in prevention of proteolysis during food processing. A glycosylated cystatin C mutant with increased temperature stability was developed for the latter application [Nakamura et al. (1998) FEBS Lett 427:252–254]. A recombinant variant of cystatin C [Nakamura et al. (2000) International patent no. PLTCA99/00717] with two potential sites for N-linked glycosylation was expressed in Pichia pastoris Mut^s . Little of the cystatin C produced was in the glycosylated form under fermentation conditions of pH 6, temperature 28°C, methanol only feed, and ammonium hydroxide as a nitrogen source. Thus, the effect of addition of complex nitrogen sources, peptone and amino acid supplements, on the yield and glycosylation of this mutant cystatin C were investigated. A full factorial design experiment using 2-l fermenters was performed with three factors: ammonium hydroxide, peptone, and an amino acid mix, at two levels, absent or present. Peptone addition was found to have a positive, and the most significant, effect on cell specific cystatin C yield. A maximum mutant cystatin C yield of 0.82 mol (g-dry cell weight)^–1 min^–1 was obtained when all three nitrogen sources were used together. However, under these conditions only 16% of protein was in the glycosylated form since ammonia was found to have a significant negative effect on glycosylation extent. The maximum extent of glycosylation was 30% when peptone and amino acid mix were the only nitrogen sources added.     

Pichia surface display: display of proteins on the surface of glycoengineered Pichia pastoris strains

Expression of proteins on the surface of yeasts has a wide range of applications in biotechnology, such as directed evolution of proteins for increased affinity and thermal stability, screening of antibody libraries, epitope mapping, and use as whole-cell biocatalysts. However, hyperglycosylation can interfere with overall protein accessibility on the surface. Therefore, the less elaborate hyperglycosylation in wild type Pichia pastoris and the availability of glycoengineered strains make this yeast an excellent alternative for surface display of glycoproteins. Here, we report the implementation of the well-established a-agglutinin-based yeast surface display technology in P. pastoris. Four heterologous proteins were expressed on the surface of a wild type and a glycoengineered strain. Surface display levels were monitored by Western blot, immunofluorescence microscopy, and FACS analysis. The availability of glycoengineered strains makes P. pastoris an excellent alternative for surface display of glycoproteins and paves the way for new applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Efficient lipid production with Trichosporon fermentans and its use for biodiesel preparation

Effects of medium components and culture conditions on biomass and lipid production of Trichosporon fermentans were studied. The optimal nitrogen source, carbon source and C/N molar ratio were peptone, glucose and 163, respectively. The favorable initial pH of the medium and temperature were 6.5 and 25 degrees C. Under the optimized conditions, a biomass of 28.1 g/l and a lipid content of 62.4% could be achieved after culture for 7 days, which were much higher than the original values (19.4 g/l and 50.8%) and the results reported by other groups. T. fermentans could grow well in pretreated waste molasses and a lipid yield of 12.8 g/l could be achieved with waste molasses of 15% total sugar concentration (w/v) at pH 6.0, representing the best result with oleaginous microorganisms on agro-industrial residues. Addition of various sugars to the pretreated molasses could efficiently enhance the accumulation of lipid and the lipid content reached as high as above 50%. Similar to vegetable oils, the lipid mainly contains palmitic acid, stearic acid, oleic acid and linoleic acid and the unsaturated fatty acids amount to about 64% of the total fatty acids. The microbial oil with an acid value of 5.6 mg KOH/g was transesterified to biodiesel by base catalysis after removal of free fatty acids and a high methyl ester yield of 92% was obtained.     

Improved secretory production of glucoamylase in Pichia pastoris by combination of genetic manipulations

Rhizopus oryzae glucoamylase (GA) has been genetically engineered with modified signal peptide (MSP), increased copy number of the gene, and coexpression of SEC4, a gene encoding a Rab protein associated with secretory vesicles, and its secretion level has been successfully raised up to 100-fold in Pichia pastoris. The MSP was designed to contain the signal peptide of mouse salivary alpha-amylase (S8L) fused to the pro-region of the signal peptide of Saccharomyces cerevisiae alpha-mating factor to replace the wild type signal peptide (WTSP) of GA. The P. pastoris transformant MSPGA-1 containing a single copy of MSPGA gene showed a 3.6-fold increase in GA secretion as compared to that of WTSPGA-1. Moreover, the P. pastoris transformant MSPGA-7 harboring seven copies of the MSPGA inserts was identified and showed 56-fold higher secreted GA than WTSPGA-1. In addition, we found that overexpression of SEC4 further doubled the secretion level of GA in each MSPGA/P. pastoris transformant. Taken together, the MSPGA-7-SEC4 clone showed as much as 100-fold secretion level of GA when compared to WTSPGA-1. In summary, we have demonstrated that combination of the aforementioned genetic manipulations resulted in high level secretion of R. oryzae GA in P. pastoris.     

Recombinant human fibrinogen expressed in the yeast Pichia pastoris was assembled and biologically active

Fibrinogen is a large plasma glycoprotein with a molecular mass of 340 kDa that plays a critical role in the final stage of blood coagulation. Human plasma fibrinogen is a dimeric molecule comprising two sets of three different polypeptides (Aα, 66 kDa; Bβ, 55 kDa; γ, 48 kDa). To express recombinant human fibrinogen in the methylotrophic yeast Pichia pastoris, we constructed an expression vector containing three individual fibrinogen chain cDNAs under the control of the mutated AOX2 (mAOX2) promoter. First, P. pastoris GTS115 was transformed with the vector, but the expressed recombinant fibrinogen suffered severe degradation by yeast-derived proteases under conventional nutrient culture conditions. Fibrinogen degradation was prevented by using the protease A-deficient strain SMD1168 as a host strain and regulating the pH of the culture to between 5.5 and 7.0. Western blot analysis revealed that the Aα, Bβ and γ chains of recombinant fibrinogen were assembled and secreted as a complete molecule. The Bβ chain of the recombinant fibrinogen was N-glycosylated but the Aα chain, as in plasma fibrinogen, was not. The γ chains however were heterologous, one being N-glycosylated and the other not. The recombinant fibrinogen was capable of forming a thrombin-induced clot in the presence of factor XIIIa and both the glycosylated and the non-glycosylated γ chains were involved in the formation of cross-linking fibrin. The present study indicates that the recombinant fibrinogen expressed in P. pastoris, although different from plasma fibrinogen in post-translational modification, is correctly assembled and biologically active.     

Optimized production of scygonadin in Pichia pastoris and analysis of its antimicrobial and antiviral activities

The crab antimicrobial peptide scygonadin is confirmed to have antimicrobial activity against bacteria and it is probably associated with the reproductive immunity in Scylla paramamosain. To obtain large quantity of scygonadin for further biological assays, a 306 bp cDNA sequence encoding the mature peptide of scygonadin was cloned into a secretion vector of pPIC9K, and a high-level of the recombinant scygonadin was achieved in Pichia pastoris. The optimal expression condition was determined as incubation with 0.5% methanol for 48 h at 28 °C under pH 6.0, and a total of 70 mg scygonadin was expressed in 1 L culture medium. The recombinant product was purified and 97% pure scygonadin was obtained using immobilized metal affinity chromatography with a yield of 46 mg/L. The recombinant scygonadin was confirmed using SDS-PAGE analysis and MS-fingerprinting. P. pastoris-derived scygonadin exhibited relatively higher antimicrobial activities against bacteria than Escherichia coli-derived scygonadin. The antimicrobial activity of the recombinant scygonadin against pathogenic Aeromonas hydrophila showed salt resistant and the killing kinetics of A. hydrophila was time dependent. Besides, the antiviral assay demonstrated that scygonadin could interfere with white spot syndrome virus (WSSV) replication in vitro-cultured crayfish haematopoietic (Hpt) cells. Taken together, this is the first report on the heterologous expression of scygonadin in P. pastoris, and P. pastoris is an effective expression system for producing large quantities of biological active scygonadin for both research and agricultural application.     

Mycoplasma fermentans glycolipid-antigen as a pathogen of rheumatoid arthritis

Mycoplasma fermentans has been suspected as one of the causative pathogenic microorganisms of rheumatoid arthritis (RA) however, the pathogenic mechanism is still unclear. We, previously, reported that glycolipid-antigens (GGPL-I and III) are the major antigens of M. fermentans. Monoclonal antibody against the GGPL-III could detect the existence of the GGPL-III antigens in synovial tissues from RA patients. GGPL-III antigens were detected in 38.1% (32/84) of RA patient’s tissues, but not in osteoarthritis (OA) and normal synovial tissues. Immunoelectron microscopy revealed that a part of GGPL-III antigens are located at endoplasmic reticulum. GGPL-III significantly induced TNF-α and IL-6 production from peripheral blood mononulear cells, and also proliferation of synovial fibroblasts. Further study is necessary to prove that M. fermentans is a causative microorganism of RA; however, the new mechanisms of disease pathogenesis provides hope for the development of effective and safe immunotherapeutic strategies based on the lipid-antigen, GGPL-III, in the near future.     

Toxoplasma gondii: serological characterization and immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichia pastoris

The full length surface antigen 2 (SAG2) gene of the protozoan parasite Toxoplasma gondii was cloned and intracellularly expressed in the Pichia pastoris expression system. The molecular weight of the expressed recombinant SAG2 (36 kDa) was much larger than the native SAG2 (22 kDa). This discrepancy in size was due to hyperglycosylation, as deglycosylation assay reduced the size of the recombinant SAG2 to 22 kDa. Despite being hyperglycosylated, the recombinant SAG2 reacted strongly with pooled anti-Toxoplasma human serum, pooled anti-Toxoplasma mouse serum and a SAG2-specific monoclonal antibody. The glycosylated recombinant SAG2 was further evaluated in Western blot and in-house enzyme-linked immunosorbent assay (ELISA) using 80 human serum samples, including confirmed early acute (IgM positive, IgG negative; n = 20), acute (IgM positive, IgG positive; n = 20) and chronic (IgM negative, IgG positive; n = 20) toxoplasmosis patients, and toxoplasmosis negative control patients (n = 20). Results of the Western blot showed that the recombinant SAG2 reacted with all 60 samples of the toxoplasmosis cases but not with the Toxoplasma-negative samples. The sensitivity of in-house ELISA was 80%, 95% and 100% for early acute, acute and chronic patients’ serum samples, respectively. Vaccination study showed that serum from mice immunised with the glycosylated recombinant SAG2 reacted specifically with the native SAG2 of T. gondii. The mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P < 0.01) and their survival time was increased compared to controls. Therefore, the present study shows that the P. pastoris-derived recombinant SAG2 was specific and suitable for use as antigen for detecting anti-Toxoplasma IgG and IgM antibodies. The vaccination study showed that recombinant SAG2 protein was immunoprotective in mice against lethal challenge.     

Biochemical and functional properties of the full-length cation-dependent mannose 6-phosphate receptor expressed in Pichia pastoris

A glycosylation-deficient, full-length cation-dependent mannose 6-phosphate receptor (CD-MPR) containing a yeast signal sequence was expressed in Pichia pastoris using the constitutive promoter of the PGAP gene. The membrane-bound receptor was solubilized using detergents and purified by pentamannosyl phosphate-agarose affinity chromatography. Equilibrium binding studies identified a binding affinity of 2 nM for the lysosomal enzyme, beta-glucuronidase. To probe the linkage specificity of the recombinant CD-MPR, inhibition binding studies were conducted using non-phosphorylated oligomannoses which demonstrated that Manalpha1,2Man exhibits a 4-fold higher inhibition than Manalpha1,3Man and Manalpha1,6Man. The receptor was capable of associating into oligomeric forms and enzymatic deglycosylation revealed the presence of high-mannose sugars at the single potential N-glycosylation site. Mass spectrometric analysis revealed that the receptor was palmitoylated at the two potential cysteines in its cytoplasmic domain. In conclusion, the full-length CD-MPR produced in P. pastoris is structurally and functionally suitable for crystallization studies.     

A putative phospholipase C is involved in Pichia fermentans dimorphic transition

Background

Pichia fermentans DiSAABA 726 is a dimorphic yeast that reversibly shifts from yeast-like to pseudohyphal morphology. This yeast behaves as a promising antagonist of Monilia spp. in the yeast-like form, but becomes a destructive plant pathogen in the pseudohyphal form thus raising the problem of the biological risk associated with the use of dimorphic yeasts as microbial antagonists in the biocontrol of phytopathogenic fungi.

Methods

Pichia fermentans DiSAABA 726 was grown in urea- and methionine-containing media in order to induce and separate yeast-like and pseudohyphal morphologies. Total RNA was extracted from yeast-like cells and pseudohyphae and retro-transcribed into cDNA. A rapid subtraction hybridization approach was utilized to obtain the cDNA sequences putatively over-expressed during growth on methionine-containing medium and involved in pseudohyphal transition.

Results

Five genes that are over-expressed during yeast-like/pseudohyphal dimorphic transition were isolated. One of these, encoding a putative phospholipase C, is involved in P. fermentans filamentation. In fact, while the inhibition of phospholipase C, by means of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (Et-18), is accompanied by a significant reduction of pseudohyphae formation in P. fermentans, the addition of exogenous cAMP fully restores pseudohyphal growth also in the presence of Et-18.

Conclusion

Phospholipase C is part of a putative “methionine sensing machinery” that activates cAMP-PKA signal transduction pathway and controls P. fermentans yeast-like/pseudohyphal dimorphic transition.

General significance

Phospholipase C is a promising molecular target for further investigations into the link between pseudohyphae formation and pathogenicity in P. fermentans.     

Functional expression of the human breast cancer resistance protein in Pichia pastoris

We report functional expression of BCRP in Pichia pastoris in which BCRP was produced as a 62 kDa underglycosylated protein. BCRP expression level in P. pastoris was comparable to that in HEK cells. The basal BCRP ATPase activity in the yeast membranes was approximately 40-80 nmol Pi/min/mg protein, which can be modulated by known BCRP substrates and inhibitors. Photolabeling of BCRP with 8-azido[alpha-32P]ATP was dependent preferentially on the presence of Co2+ than Mg2+ and could be inhibited by a molar excess of ATP. Vanadate-induced trapping of 8-azido[alpha-32P]ADP by BCRP was much more significant in the presence of Co2+ than that with Mg2+. The Km and Vmax values of BCRP for [3H]E1S transport were 3.6+/-0.3 microM and 55.2+/-1.6 pmol/min/mg protein, respectively. This efficient and cost-effective expression system should facilitate large scale production and purification of BCRP for further structural and functional analyses.     

Molecular genetic manipulation of Pichia pastoris SEC4 governs cell growth and glucoamylase secretion

We have previously engineered a recombinant Pichia pastoris GS115 transformant, MSPGA-7, harboring seven copies of glucoamylase (GA) fused with modified signal peptide. High yield secretion of GA was achieved as an extra copy of SEC4 was integrated to the transformant. To elucidate the physiological role of SEC4, a dominant-negative mutant of SEC4, SEC4(S28N), was overexpressed under the control of alchohol oxidase 1 (AOX1) promoter in P. pastoris strain MSPGA-7 as well as a set of host cells harboring multi-copy of wild type SEC4. We found that SEC4(S28N) mutation in the key guanine nucleotide binding domain reduced guanine nucleotide binding affinity, hence it blocked the transport of vesicles required for targeting and fusion to the plasma membrane. The inhibitory levels of cell growth and GA secretion were correlated with the dosage of SEC4(S28N) gene. In addition, overexpression of SEC4 driven by AOX1 promoter in MSPGA-7 improved the secretory production of GA, but demonstrated the delay of cell growth by increased gene dosage of SEC4. Interestingly, a limited level of Sec4p did not disturb the cell growth. It was because expression of only one copy of SEC4 resulted in delay of cell growth at an early stage while still maintaining high level Sec4p at long-term incubation. Accordingly, as glyceraldehyde-3-phosphate dehydrogenase promoter was used to substitute AOX1 promoter to drive the SEC4 expression, enhanced GA secretion but not inhibition of cell growth was achieved. Taken together, our results demonstrate that SEC4 is essential for P. pastoris in regulating cell growth and heterologous protein secretion in a dosage-dependent manner.     

Abdominal-B is essential for proper sexually dimorphic development of the Drosophila gonad

Sexual dimorphism requires the integration of positional information in the embryo with the sex determination pathway. Homeotic genes are a major source of positional information responsible for patterning along the anterior-posterior axis in embryonic development, and are likely to play a critical role in sexual dimorphism. Here, we investigate the role of homeotic genes in the sexually dimorphic development of the gonad in Drosophila. We have found that Abdominal-B (ABD-B) is expressed in a sexually dimorphic manner in the embryonic gonad. Furthermore, Abd-B is necessary and sufficient for specification of a sexually dimorphic cell type, the male-specific somatic gonadal precursors (msSGPs). In Abd-B mutants, the msSGPs are not specified and male gonads now resemble female gonads with respect to these cells. Ectopic expression of Abd-B is sufficient to induce formation of extra msSGPs in additional segments of the embryo. Abd-B works together with abdominal-A to pattern the non-sexually dimorphic somatic gonad in both sexes, while Abd-B alone specifies the msSGPs. Our results indicate that Abd-B acts at multiple levels to regulate gonad development and that Abd-B class homeotic genes are conserved factors in establishing gonad sexual dimorphism in diverse species.     

Dorso-ventral asymmetric functions of teashirt in Drosophila eye development depend on spatial cues provided by early DV patterning genes

The teashirt (tsh) gene has dorso-ventral (DV) asymmetric functions in Drosophila eye development: promoting eye development in dorsal and suppressing eye development in ventral by Wingless mediated Homothorax (HTH) induction [Development 129 (2002) 4271]. We looked for DV spatial cues required by tsh for its asymmetric functions. The dorsal Iroquois-Complex (Iro-C) genes and Delta (Dl) are required and sufficient for the tsh dorsal functions. The ventral Serrate (Ser), but not fringe (fng) or Lobe (L), is required and sufficient for the tsh ventral function. We propose that DV asymmetric function of tsh represents a novel tier of DV pattern regulation, which takes place after the spatial expression patterns of early DV patterning genes are established in the eye.     

High-quality genome sequence of Pichia pastoris CBS7435

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) CBS7435 is the parental strain of commonly used P. pastoris recombinant protein production hosts making it well suited for improving the understanding of associated genomic features. Here, we present a 9.35 Mbp high-quality genome sequence of P. pastoris CBS7435 established by a combination of 454 and Illumina sequencing. An automatic annotation of the genome sequence yielded 5007 protein-coding genes, 124 tRNAs and 29 rRNAs. Moreover, we report the complete DNA sequence of the first mitochondrial genome of a methylotrophic yeast. Fifteen genes encoding proteins, 2 rRNA and 25 tRNA loci were identified on the 35.7 kbp circular, mitochondrial DNA. Furthermore, the architecture of the putative alpha mating factor protein of P. pastoris CBS7435 turned out to be more complex than the corresponding protein of Saccharomyces cerevisiae.     

Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an alpha-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4x10(4) U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions.     

Intracellular expression of Vitreoscilla hemoglobin improves production of Yarrowia lipolytica lipase LIP2 in a recombinant Pichia pastoris

The Yarrowia lipolytica lipase LIP2 (YlLIP2) gene lip2 and Vitreoscilla hemoglobin gene vgb were co-expressed in Pichia pastoris, both under the control of AOX1 promoter, in order to alleviate respiration limitation under conditions of high cell-density fermentation and enhance YlLIP2 production. The results showed that recombinant P. pastoris strains harboring the lip2 and vgb genes (VHb⁺) displayed higher biomass and YlLIP2 activity than control strains (VHb⁻). Compared with VHb⁻ cells, the expression levels of YlLIP2 in VHb-expressing cells when oxygen was not a limiting factor were improved 31.5% in shake-flask culture and 22% in a 10-L fermentor. Under non-limiting dissolved oxygen (DO) conditions, the maximum YlLIP2 activity of VHb⁺ in a 10-L fermentor reached 33,000 U/mL. Oxygen limitation had a more negative effect on YlLIP2 productivity in VHb⁻ cells than in VHb⁺ cells. The highest YlLIP2 activity of VHb⁺ cells was approximately 1.84-fold higher than that of VHb⁻ cells at lower DO levels. Moreover, the recombinant strain VHb⁺ exhibited a higher specific oxygen uptake rate and achieved higher cell viability under oxygen limiting and non-limiting conditions compared with VHb⁻ cells. Therefore, the above results suggest that intracellular expression of VHb in recombinant P. pastoris has the potential to improve cell growth and industrial enzyme production.     

Ethanol production from acid hydrolysates based on the construction and demolition wood waste using Pichia stipitis

The feasibility of ethanol production from the construction and demolition (C&D) wood waste acid hydrolysates was investigated. The chemical compositions of the classified C&D wood waste were analyzed. Concentrated sulfuric acid hydrolysis was used to obtain the saccharide hydrolysates and the inhibitors in the hydrolysates were also analyzed. The C&D wood waste composed of lumber, plywood, particleboard, and medium density fiberboard (MDF) had polysaccharide (cellulose, xylan, and glucomannan) fractions of 60.7-67.9%. The sugar composition (glucose, xylose, and mannose) of the C&D wood wastes varied according to the type of wood. The additives used in the wood processing did not appear to be released into the saccharide solution under acid hydrolysis. Although some fermentation inhibitors were detected in the hydrolysates, they did not affect the ethanol production by Pichia stipitis. The hexose sugar-based ethanol yield and ethanol yield efficiency were 0.42-0.46 g ethanol/g substrate and 84.7-90.7%, respectively. Therefore, the C&D wood wastes dumped in landfill sites could be used as a raw material feedstock for the production of bioethanol.