YaliCMulti and YaliHMulti: Stable, efficient multi-copy integration tools for engineering Yarrowia lipolytica.

Yarrowia lipolytica is widely used in biotechnology to produce recombinant proteins, food ingredients and diverse natural products. However, unstable expression of plasmids, difficult and time-consuming integration of single and low-copy-number plasmids hampers the construction of efficient production pathways and application to industrial production. Here, by exploiting sequence diversity in the long terminal repeats (LTRs) of retrotransposons and ribosomal DNA (rDNA) sequences, a set of vectors and methods that can recycle multiple and high-copy-number plasmids was developed that can achieve stable integration of long-pathway genes in Y. lipolytica. By combining these sequences, amino acids and antibiotic tags with the Cre-LoxP system, a series of multi-copy site integration recyclable vectors were constructed and assessed using the green fluorescent protein (HrGFP) reporter system. Furthermore, by combining the consensus sequence with the vector backbone of a rapidly degrading selective marker and a weak promoter, multiple integrated high-copy-number vectors were obtained and high levels of stable HrGFP expression were achieved. To validate the universality of the tools, simple integration of essential biosynthesis modules was explored, and 7.3 g/L of L-ergothioneine and 8.3 g/L of (2S)-naringenin were achieved in a 5 L fermenter, the highest titres reported to date for Y. lipolytica. These novel multi-copy genome integration strategies provide convenient and effective tools for further metabolic engineering of Y. lipolytica.

Specific detection of Phytophthora parasitica by recombinase polymerase amplification (RPA) assays based on a unique multi-copy genomic sequence.

Phytophthora parasitica is a highly destructive oomycete plant pathogen that is capable of infecting a wide range of hosts including many agricultural cash crops, fruit trees, and ornamental garden plants. One of the most important diseases caused by P. parasitica worldwide is black shank of tobacco. Rapid, sensitive, and specific pathogen detection is crucial for early rapid diagnosis which can facilitate effective disease management. In this study, we used a genomics approach to identify repeated sequences in the genome of P. parasitica by genome sequence alignment, and identified a 203 bp P. parasitica-specific sequence, PpM34, that is present in 31-60 copies in the genome. The P. parasitica genome-specificity of PpM34 was supported by PCR amplification of 24 genetically diverse strains of P. parasitica, 32 strains representing twelve other Phytophthora species, one Pythium specie, six fungal species and three bacterial species, all of which are plant pathogens. Our PCR and real-time PCR assays showed that the PpM34 sequence was highly sensitive in specifically detecting P. parasitica. Finally, we developed a PpM34-based high-efficiency Recombinase Polymerase Amplification (RPA) assay, which allowed us to specifically detect as little as 1 pg of P. parasitica total DNA from both pure cultures and infected Nicotiana benthamiana at 39°C using a fluorometric thermal cycler. The sensitivity, specificity, convenience and rapidity of this assay represents a major improvement for early diagnosis of P. parasitica infection.

Expression and Surface Display of an Acidic Cold-Active Chitosanase in Pichia pastoris Using Multi-Copy Expression and High-Density Cultivation.

Chitosanase hydrolyzes ß-(1,4)-linked glycosidic bonds are used in chitosan chains to release oligosaccharide mixtures. Here, we cloned and expressed a cold-adapted chitosanase (CDA, Genbank: MW094131) using multi-copy expression plasmids (CDA1/2/3/4) in Pichia pastoris. We identified elevated CDA expression levels in multi-copy strains, with strain PCDA4 selected for high-density fermentation and enzyme-activity studies. The high-density fermentation approach generated a CDA yield of 20014.8 U/mL, with temperature and pH optimization experiments revealing the highest CDA activity at 20 °C and 5.0, respectively. CDA was stable at 10 °C and 20 °C. Thus, CDA could be used at low temperatures. CDA was then displayed on P. pastoris using multi-copy expression plasmids. Then, multi-copy strains were constructed and labelled as PCDA(1-3)-AGα1. Further studies showed that the expression of CDA(1-3)-AGα1 in multi-copy strains was increased, and that strain PCDA3-AGα1 was chosen for high-density fermentation and enzyme activity studies. By using a multi-copy expression and high-density fermentation approach, we observed CDA-AGα1 expression yields of 102415 U/g dry cell weight. These data showed that the displayed CDA exhibited improved thermostability and was more stable over wider temperature and pH ranges than free CDA. In addition, displayed CDA could be reused. Thus, the data showed that displaying enzymes on P. pastoris may have applications in industrial settings.

Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity.

BACKGROUND: Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. RESULTS: This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1-7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. CONCLUSIONS: This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.

High-level extracellular production and immobilisation of methyl parathion hydrolase from Plesiomonas sp. M6 expressed in Pichia pastoris.

Methyl parathion hydrolase (MPH) hydrolyses methyl parathion efficiently and specifically. Herein, we produced MPH from Plesiomonas sp. M6 using a Pichia pastoris multi-copy expression system. The original signal peptide sequence of the target gene was removed, and a modified coding sequence was synthesised. Multi-copy expression plasmids containing MPH were constructed using pHBM905BDM, and used to generate recombinant strains containing 1, 2, 3 or 4 copies of the MPH gene. The results showed that a higher target gene copy number increased the production of recombinant MPH (MPH-R), as anticipated. The expression level of the recombinant strain containing four copies of the MPH gene was increased to 1.9 U/ml using 500 ml shake flasks, and the specific activity was 15.8 U/mg. High-density fermentation further increased the target protein yield to 18.4 U/ml. Several metal ions were tested as additives, and Ni2+, Co2+ and Mg2+ at a concentration of 1 mM enhanced MPH-R activity by 196%, 201% and 154%, respectively. Enzyme immobilisation was then applied to overcome the difficulties in recovery, recycling and long-term stability associated with the free enzyme. Immobilised MPH-R exhibited significantly enhanced thermal and long-term stability, as well as broad pH adaptability. In the presence of inhibitors and chelating agents such as sodium dodecyl sulphate (SDS), immobilised MPH-R displayed 2-fold higher activity than free MPH-R, demonstrating its potential for industrial application.

A Bioinformatic Workflow for InDel Analysis in the Wheat Multi-Copy α-Gliadin Gene Family Engineered with CRISPR/Cas9.

The α-gliadins of wheat, along with other gluten components, are responsible for bread viscoelastic properties. However, they are also related to human pathologies as celiac disease or non-celiac wheat sensitivity. CRISPR/Cas was successfully used to knockout α-gliadin genes in bread and durum wheat, therefore, obtaining low gluten wheat lines. Nevertheless, the mutation analysis of these genes is complex as they present multiple and high homology copies arranged in tandem in A, B, and D subgenomes. In this work, we present a bioinformatic pipeline based on NGS amplicon sequencing for the analysis of insertions and deletions (InDels) in α-gliadin genes targeted with two single guides RNA (sgRNA). This approach allows the identification of mutated amplicons and the analysis of InDels through comparison to the most similar wild type parental sequence. TMM normalization was performed for inter-sample comparisons; being able to study the abundance of each InDel throughout generations and observe the effects of the segregation of Cas9 coding sequence in different lines. The usefulness of the workflow is relevant to identify possible genomic rearrangements such as large deletions due to Cas9 cleavage activity. This pipeline enables a fast characterization of mutations in multiple samples for a multi-copy gene family.

Improving expression of thermostable trehalase from Myceliophthora sepedonium in Aspergillus niger mediated by the CRISPR/Cas9 tool and its purification, characterization.

Trehalase catalyzes the conversion of one molecule of trehalose into two glucose molecules. The trehalase TreM from thermophilic fungus Myceliophthora sepedonium was expressed in Aspergillus niger via traditional homologous recombination with trehalase activity of 406.44 U/mL. The multi-copy knock-in expression strategy mediated by the CRISPR/Cas9 tool was used to improve the production of the TreM trehalase in Aspergillus niger, which was up to 1943.06 U/mL with a low-background of secreted proteins, 4.8-fold than the transformant obtained via the traditional method. The highest recombinant trehalase activity of the shake fermentation supernatant achieved 4268.29 U/mL when 1.5% glucose was added. Activity assaying showed that the recombinant TreM possessed a specific activity of 679.09 U/mg after gel filtration chromatography purification. The recombinant TreM displayed optimal activity at pH 5.6 and 60 °C and exhibited prominent stability under the conditions of 45-50 °C and pH 4.0-7.5. The activity of recombinant TreM was strongly enhanced by Co2+ (1, 5 mM), Cu2+ (1 mM), Mn2+ (1, 5 mM) and ATP (5 mM), and was greatly inhibited by Cu2+ (10 mM), EDTA (10 mM) and SDS (10 mM).

Optimized expression of classical swine fever virus E2 protein via combined strategy in Pichia pastoris.

Precaution of classical swine fever (CSF) is an important mission for the worldwide swine industry. Glycoprotein E2 is the leading antigen candidate for subunit vaccine of classical swine fever virus (CSFV). In this study, two Spy-tagged E2 genes were synthesized in vitro and subcloned into pMCO-AOX vector for intracellular expression in Pichia pastoris after methanol induction. Western blot analysis and semi-quantitative analysis showed that the yield of recombinant E2 protein was improved 17.87 folds by using co-translocational signal peptide cSIG. After the construction of the tandem multiple copy expression vectors, further increase of E2 production was observed by repetitive transforming expression vectors into P. pastoris genome. Finally, the yeast transformants harboring 8 or 16 copies of cSIG-E2-Spy increased the E2 expression level by 27.01-fold or 30.72-fold, respectively. These results demonstrate that utilizing co-translocational signal peptide together with multi-copy integration strategy can increase the production of recombinant E2 protein efficiently.

High-level expression of highly active and thermostable trehalase from Myceliophthora thermophila in Aspergillus niger by using the CRISPR/Cas9 tool and its application in ethanol fermentation.

Trehalase catalyzes the hydrolysis of the non-reducing disaccharide trehalose. The highly active trehalase MthT from Myceliophthora thermophila was screened from the trehalase genes of six species of filamentous fungi. An ingenious multi-copy knock-in expression strategy mediated by the CRISPR/Cas9 tool and medium optimization were used to improve MthT production in Aspergillus niger, up to 1698.83 U/mL. The protein background was dramatically abated due to insertion. The recombinant MthT showed optimal activity at pH 5.5 and 60 °C, and exhibited prominent thermal stability between 50 and 60 °C under acid conditions (pH 4.5-6.5). The ethanol conversion rate (ethanol yield/total glucose) was significantly improved by addition of MthT (51.88%) compared with MthT absence (34.38%), using 30% starch saccharification liquid. The results of this study provided an effective strategy, established a convenient platform for heterologous expression in A. niger and showed a potential strategy to decrease production costs in industrial ethanol production.

aMSGE: advanced multiplex site-specific genome engineering with orthogonal modular recombinases in actinomycetes.

Chromosomal integration of genes and pathways is of particular importance for large-scale and long-term fermentation in industrial biotechnology. However, stable, multi-copy integration of long DNA segments (e.g., large gene clusters) remains challenging. Here, we describe a plug-and-play toolkit that allows for high-efficiency, single-step, multi-locus integration of natural product (NP) biosynthetic gene clusters (BGCs) in actinomycetes, based on the innovative concept of "multiple integrases-multiple attB sites". This toolkit consists of 27 synthetic modular plasmids, which contain single- or multi-integration modules (from two to four) derived from five orthogonal site-specific recombination (SSR) systems. The multi-integration modules can be readily ligated into plasmids containing large BGCs by Gibson assembly, which can be simultaneously inserted into multiple native attB sites in a single step. We demonstrated the applicability of this toolkit by performing stabilized amplification of acetyl-CoA carboxylase genes to facilitate actinorhodin biosynthesis in Streptomyces coelicolor. Furthermore, using this toolkit, we achieved a 185.6% increase in 5-oxomilbemycin titers (from 2.23 to 6.37 g/L) in Streptomyces hygroscopicus via the multi-locus integration of the entire 5-oxomilbemycin BGC (72 kb) (up to four copies). Compared with previously reported methods, the advanced multiplex site-specific genome engineering (aMSGE) method does not require the introduction of any modifications into host genomes before the amplification of target genes or BGCs, which will drastically simplify and accelerate efforts to improve NP production. Considering that SSR systems are widely distributed in a variety of industrial microbes, this novel technique also promises to be a valuable tool for the enhanced biosynthesis of other high-value bioproducts.

Toward the construction of a technology platform for chemicals production from methanol: D-lactic acid production from methanol by an engineered yeast Pichia pastoris.

With the reduction in oil reserves and steady increases in the price of oil, alternative carbon sources like methanol are promising, but an efficient conversion process to fuels and other chemicals is still desired. In this study, we demonstrated for the first time the production of lactic acid from methanol using a lactate dehydrogenase copy number amplifying strategy in Pichia pastoris. We engineered methylotrophic yeast (Pichia pastoris) producing D-lactic acid by D-lactate dehydrogenase gene (d-LDH) integration into the non-transcribed spacer of the ribosomal DNA (rDNA) locus and post-transformational amplification. The resultant engineered strains GS115/S8/Z3 and GS115/S16/Z3 produced 3.48 and 3.26 g/L of D-lactic acid from methanol, respectively, in a 96-h test tube fermentation. To our knowledge, this is the first report about D-lactic acid production from methanol by an engineered P. pastoris strain. The technique of gene integration into the rDNA locus and post-transformational gene amplification could be useful for metabolic engineering in P. pastoris, and the chemical production from methanol by engineered P. pastoris represents a promising industrial technology.

Constitutive expression of the active fragment of human vasostatin Vs30 in Pichia pastoris SMD1168H.

Vasostatin 30 (Vs30) is an active fragment derived from the N-terminal region (135-164 aa) of human calreticulin and has the ability to inhibit angiogenesis. In this work, the expression of Vs30 was performed using a protease-deficient strain of the methylotrophic yeast Pichia pastoris. The vs30 gene was optimized for P. pastoris preferential codon usage and inserted into constitutive expression vector pGAPZαA. In addition, a plasmid with four copies of the expression cassette was obtained and transformed into P. pastoris. The flask fermentation conditions were: culture volume of 25 mL in 250 mL baffled flasks at 28 °C, pH 6 and harvest time of 48 h. Up to 21.07 mg/L Vs30 were attained and purified by ultrafiltration with a 30-kDa cut-off membrane and the recovery was 49.7%. Bioactivity of Vs30 was confirmed by the inhibition of cell proliferation, as well as the inhibition of the capillary-like structures formation of EA.hy926 cells in vitro. This work constitutes the first report on the expression of Vs30 in Pichia pastoris using a constitutive promoter and multi-copy approach such as strategies to improve the recombinant Vs30 expression.

Multi-copy single-stranded DNA in Escherichia coli.

Multi-copy single-stranded DNA (msDNA) is composed of covalently bound single-stranded DNA and RNA, and synthesized by retron-encoded reverse transcriptase. msDNA-synthesizing systems are thought to be a recent acquisition by Escherichia coli because, to date, only seven types of msDNA, which differ markedly in their primary nucleotide sequences, have been found in a small subset of E. coli strains. The wide use of E. coli in molecular research means that it is important to understand more about these stable, covalently bound, single-stranded DNA or RNA compounds. The present review provides insights into the molecular biosynthesis, distribution and function of E. coli msDNA to raise awareness about these special molecules.

High-level expression of two thermophilic ß-mannanases in Yarrowialipolytica.

Two thermophilic ß-mannanases (ManA and ManB)were successfully expressed in Yarrowialipolytica using vector pINA1296I. The sequences of manA from Aspergillus niger CBS 513.88 and manB from Bacillus subtilis BCC41051 were optimized based on codon-usage bias in Y.lipolytica and synthesized by overlapping polymerase chain reaction (PCR). We utilized the pINA1296I vector, which allows inserting and expression of multiple copies of an expression cassette, to engineer recombinant strains containing multiple copies of manA or manB. Following verification of target-gene expression by quantitative PCR, fermentation experiments indicated that recombinant protein levels and enzyme activity increased along with increasing manA/manB copy number.After production in a 10 l fermenter, we obtained maximum enzyme activity from strains YLA6 and YLB6 of3024 U/mL and 1024 U/mL, respectively. Additionally, purification and characterization results revealed that the optimum pH and temperature for manA activity were pH∼5 and ∼70 °C, and for manB activity were pH∼7 and 60 °C, respectively. These results indicated that the thermo stabilities of these two enzymes were higher than most other mannanases, making them potentially useful for industrial applications.

Overproduction of Rummeliibacillus pycnus arginase with multi-copy insertion of the arg R.pyc cassette into the Bacillus subtilis chromosome.

A plasmid-less and marker-less strain with multi-copy integration of the arginase gene from Rummeliibacillus pycnus was constructed using Bacillus subtilis 168 as a host. A total of nine copies of the arg R.pyc cassettes, in which the R. pycnus arginase gene was fused with the strong promoter P43, were inserted into the recipient chromosome. These multiple insertions were completed via step-by-step integrations into designed (2 copies) and random (9 copies) sites, respectively. A strategy for random site integration was developed based on the construction of the arg R.pyc cassette sandwiched between "front" and "back" homologous arms which were randomly restricted from chromosomal DNA. An antibiotic resistance marker was applied in transformant selection and was eliminated via the Cre/lox system. Performance showed that the highest enzyme activity (14.5 U/mL) was obtained after culture in flasks, and this segregation stable strain could efficiently hydrolyze L-arginine with a 97.2% molar yield, showing potential application in the food industry.

HSFY and ZNF280BY show copy number variations within 17 water buffalo populations.

Recent transcriptomic analysis of the bovine Y chromosome revealed abundant presence of multi-copy protein coding gene families on the male-specific region of the Y chromosome (MSY). Copy number variations (CNVs) of several MSY genes are closely related to semen quality and male reproduction in cattle. However, the CNVs of MSY genes in water buffalo are largely unknown. Therefore, this study aimed to investigate the CNVs of HSFY and ZNF280BY of 298 buffaloes from 17 populations distributed in China, Vietnam and Laos using quantitative PCR. Our results revealed that the median copy numbers of the HSFY and ZNF280BY genes were 47 (ranging from 20 to 145) and 269 (ranging from 73 to 974) respectively. In conclusion, this study indicated that HSFY and ZNF280BY showed abundant CNVs within swamp buffalo populations.

A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae.

Despite recent advances in genome editing capabilities for the model organism Saccharomyces cerevisiae, the chromosomal integration of large biochemical pathways for stable industrial production remains challenging. In this work, we developed a simple platform for high-efficiency, single-step, markerless, multi-copy chromosomal integration of full biochemical pathways in Saccharomyces cerevisiae. In this Di-CRISPR (delta integration CRISPR-Cas) platform based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas), we specifically designed guide RNA sequences to target multiple delta sites in the yeast genome. The generation of double stranded breaks at the delta sites allowed simultaneous integration of multiple copies of linearized donor DNA containing large biochemical pathways. With our newly developed Di-CRISPR platform, we were able to attain highly efficient and markerless integration of large biochemical pathways and achieve an unprecedented 18-copy genomic integration of a 24 kb combined xylose utilization and (R,R)-2,3-butanediol (BDO) production pathway in a single step, thus generating a strain that was able to produce BDO directly from xylose. The simplicity and high efficiency of the Di-CRISPR platform could provide a superior alternative to high copy plasmids and would render this platform an invaluable tool for genome editing and metabolic engineering in S. cerevisiae.

High-level expression of Proteinase K from Tritirachium album Limber in Pichia pastoris using multi-copy expression strains.

Proteinase K is widely used in scientific research and industries. This report was aimed to achieve high-level expression of proteinase K using Pichia pastoris GS115 as the host strain. The coding sequence of a variant of proteinase K that has higher activity than the wild type protein was chosen and optimized based on the codon usage preference of P. pastoris. The novel open reading frame was synthesized and a series of multi-copy expression vectors were constructed based on the pHBM905BDM plasmid, allowing for the tandem integration of multiple copies of the target gene into the genome of P. pastoris with a single recombination. These strains were used to study the correlation between the gene copy number and the expression level of proteinase K. The results of quantitative polymerase chain reaction (qPCR) indicated that the tandem expression cassettes were integrated into the host genome stably. Meanwhile, the results of qPCR and enzyme activity assays indicated that the mRNA and protein expression levels of the target gene increased as the gene copy number increased. Moreover, the effect of gene dosage on the expression level of the recombinant protein was more obvious using high-density fermentation. The maximum expression level and enzyme activity of proteinase K, which were obtained from the recombinant yeast strain bearing 5 copies of the target gene after an 84-h induction, were approximately 8.069 mg/mL and 108,295 U/mL, respectively. The recombinant proteinase was purified and characterized. The optimum pH and temperature for the activity of this protease were approximately pH 11 and 55 °C, respectively.

Expression of a chimeric human/salmon calcitonin gene integrated into the Saccharomyces cerevisiae genome using rDNA sequences as recombination sites.

Calcitonin participates in controlling homeostasis of calcium and phosphorus and plays an important role in bone metabolism. The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to express chimeric human/salmon calcitonin (hsCT) without the use of antibiotics. To do so, a homologous recombination plasmid pUC18-rDNA2-ura3-P pgk -5hsCT-rDNA1 was constructed, which contains two segments of ribosomal DNA of 1.1 kb (rDNA1) and 1.4 kb (rDNA2), to integrate the heterologous gene into host rDNA. A DNA fragment containing five copies of a chimeric human/salmon calcitonin gene (5hsCT) under the control of the promoter for phosphoglycerate kinase (P pgk ) was constructed to express 5hsCT in S. cerevisiae using ura3 as a selectable auxotrophic marker gene. After digestion by restriction endonuclease HpaI, a linear fragment, rDNA2-ura3-P pgk -5hsCT-rDNA1, was obtained and transformed into the △ura3 mutant of S. cerevisiae by the lithium acetate method. The ura3-P pgk -5hsCT sequence was introduced into the genome at rDNA sites by homologous recombination, and the recombinant strain YS-5hsCT was obtained. Southern blot analysis revealed that the 5hsCT had been integrated successfully into the genome of S. cerevisiae. The results of Western blot and ELISA confirmed that the 5hsCT protein had been expressed in the recombinant strain YS-5hsCT. The expression level reached 2.04 % of total proteins. S. cerevisiae YS-5hsCT decreased serum calcium in mice by oral administration and even 0.01 g lyophilized S. cerevisiae YS-5hsCT/kg decreased serum calcium by 0.498 mM. This work has produced a commercial yeast strain potentially useful for the treatment of osteoporosis.

A simple strategy for the generation of multi-copy Pichia pastoris with the efficient expression of mannanase.

Pichia pastoris expression system was widely used for producing heterologous proteins. Some researches revealed that the increase in the copy number could improve the expression of foreign genes in P. pastoris. Hundreds or thousands of antibiotic-resistance recombinants need to be screened because the frequency of multiple gene insertion events is very low in P. pastoris. The traditional method of constructing multi-copy gene is complicated, tedious, and full of randomness. Here, we developed a rapid method for constructing multi-copy Pichia expression vectors harboring mannanase gene. The developed strategy is easy to manipulate genetically and can precisely generate plasmids with a certain copy number of heterologous gene. The average mannanase activities of recombinants randomly chosen from two-, four-, and six-copy recombinant libraries were 1.7-, 2.2-, and 1.3-folds, respectively, of that from single-copy recombinant library. The result revealed that the strategy could effectively improve the expression of foreign proteins in P. pastoris.