Methanol-inducible Promoter Research Articles

Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii

BackgroundThe β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly.ResultsFirstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).ConclusionThis study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

Transcriptome analysis reveals methanol metabolism variations for the growth damage caused by overexpression of chimeric transactivators in Pichia pastoris

Methanol is a promising substrate for sustainable biomanufacturing, and Pichia pastoris has become a commonly used yeast for methanol utilization due to its powerful methanol metabolic pathways and methanol inducible promoter. Previous reconstruction of gene circuits highly improved transcriptional activity, but excessive expression of chimeric transactivator damaged cell growth on methanol. Here we employed transcriptome analysis to investigate the effects of chimeric transactivator overexpression on cellular metabolism and regulatory networks. The results showed that strong expression of chimeric transactivator unexpectedly downregulated methanol metabolism, especially the alcohol oxidase 1 (AOX1), but without remarkable changes in expression of transcriptional factors. Meanwhile, the synthesis of peroxisomes also varied with chimeric transactivator expression. In addition, the enrichment analysis of differentially expressed genes revealed their impact on cellular metabolism. The gene expression patterns caused by different expression levels of chimeric transactivators have also been clarified. This work provides useful information to understand the transcriptional regulation of the AOX1 promoter and methanol signaling. It revealed the importance of balancing transcription factor expression for the host improvement.

Design and construction of light-regulated gene transcription and protein translation systems in yeast P. Pastoris

IntroductionP. pastoris is a common host for effective biosynthesis of heterologous proteins as well as small molecules. Accurate regulation of gene transcription and protein synthesis is necessary to coordinate synthetic gene circuits and optimize cellular energy distribution. Traditional methanol or other inducible promoters, natural or engineered, have defects in either fermentation safety or expression capacity. The utilization of chemical inducers typically adds complexity to the product purification process, but there is no other well-controlled protein synthesis system than promoters yet. ObjectiveThe study aimed to address the aforementioned challenges by constructing light-regulated gene transcription and protein translation systems with excellent expression capacity and light sensitivity. MethodsTrans-acting factors were designed by linking the N. crassa blue-light sensor WC-1 with the activation domain of endogenous transcription factors. Light inducible or repressive promoters were then constructed through chimeric design of cis-elements (light-responsive elements, LREs) and endogenous promoters. Various configurations of trans-acting factor/LRE pairs, along with different LRE positions and copy numbers were tested for optimal promoter performance. In addition to transcription, a light-repressive translation system was constructed through the “rare codon brake” design. Rare codons were deliberately utilized to serve as brakes during protein synthesis, which were switched on and off through the light-regulated changes in the expression of the corresponding pLRE-tRNA. ResultsAs demonstrated with GFP, the light-inducible promoter 4pLRE-cPAOX1 was 70 % stronger than the constitutive promoter PGAP, with L/D ratio = 77. The light-repressive promoter PGAP-pLRE was strictly suppressed by light, with expression capacity comparable with PGAP in darkness. As for the light-repressive translation system, the “triple brake” design successfully eliminated leakage and achieved light repression on protein synthesis without any impact on mRNA expression. ConclusionThe newly designed light-regulated transcription and translation systems offer innovative tools that optimize the application of P. pastoris in biotechnology and synthetic biology.

Engineering the methylotrophic yeast Ogataea polymorpha for lactate production from methanol.

Introduction: Lactate has gained increasing attention as a platform chemical, particularly for the production of the bioplastic poly-lactic acid (PLA). While current microbial lactate production processes primarily rely on the use of sugars as carbon sources, it is possible to envision a future where lactate can be produced from sustainable, non-food substrates. Methanol could be such a potential substrate, as it can be produced by (electro)chemical hydrogenation from CO2. Methods: In this study, the use of the methylotrophic yeast Ogataea polymorpha as a host organism for lactate production from methanol was explored. To enable lactate production in Ogataea polymorpha, four different lactate dehydrogenases were expressed under the control of the methanol-inducible MOX promoter. The L-lactate dehydrogenase of Lactobacillus helveticus performed well in the yeast, and the lactate production of this engineered strain could additionally be improved by conducting methanol fed-batch experiments in shake flasks. Further, the impact of different nitrogen sources and the resulting pH levels on production was examined more closely. In order to increase methanol assimilation of the lactate-producing strain, an adaptive laboratory evolution experiment was performed. Results and Discussion: The growth rate of the lactate-producing strain on methanol was increased by 55%, while at the same time lactate production was preserved. The highest lactate titer of 3.8g/L in this study was obtained by cultivating this evolved strain in a methanol fed-batch experiment in shake flasks with urea as nitrogen source. This study provides a proof of principle that Ogataea polymorpha is a suitable host organism for the production of lactate using methanol as carbon source. In addition, it offers guidance for the engineering of methylotrophic organisms that produce platform chemicals from CO2-derived substrates. With reduced land use, this technology will promote the development of a sustainable industrial biotechnology in the future.

Improving AOX1 promoter efficiency by overexpression of Mit1 transcription factor.

Reprogramming in transcriptional regulation provides an effective tool for adjusting cellular metabolic activities. The strong methanol-inducible alcohol oxidase-1 promoter (pAOX1) is commonly used for heterologous gene expression in the yeast Pichia pastoris. Here, we present a novel Pichia pastoris strain engineered to co-express methanol-induced transcription factor 1 (Mit1) and the target protein. Mit1 upregulates pAOX1 in response to methanol. Two model proteins (VEGF and eGFP) have been used as the target proteins under the control of pAOX1. The sequence of Mit1 had obtained from the yeast genome and likewise cloned under the control of pAOX1. The results indicated a 1.9 and 2.2 fold increase in the detected VEGF and eGFP, respectively, when co-expressed with Mit1. Furthermore, the double-recombinant cells, containing Mit-1 and eGFP, produced 1.3 fold more eGFP when the methanol feeding concentration was doubled. The real-time PCR indicated a slight increase in the Mit1 expression, probably due to the negative regulatory feedback loop that exists for the intrinsic yeast Mit1. Overexpression of Mit1 also led to duplication of AOX1 enzyme activity, which may enhance the yeast cells' capacity for methanol detoxification. Overexpression of Mit1 could be considered a promising strategy for upregulation of target recombinant proteins in Pichia pastoris. Intracellular overexpression of Mit1 upregulates the heterologous target gene (eGFP) production, which is expressed under the control of pAOX1.

Effect of N-glycosylation on secretion, stability, and biological activity of recombinant human interleukin-3 (hIL-3) in Pichia pastoris.

Human interleukin-3 (hIL-3) is a clinically important cytokine used to treat hematological malignancies, bone marrow transplantation, cytopenias, and immunological disorders. The cloning of hIL-3 gene was previously reported by our group, where its expression was optimized under methanol-inducible AOX1 promoter having N-terminal α mating factor signal sequence from Saccharomyces cerevisiae. This study investigated the role of glycosylation pattern on its molecular stability, secretion efficiency, and biological activity using the mutagenesis approach. The two N-linked glycosylation positions at N15th (Asn15) and N70th (Asn70) were sequentially mutated to generate three recombinant hIL-3 variants, i.e., N15A, N70A, and N15/70A. Asparagine at these positions was replaced with non-polar alanine amino acid (Ala, A). The alteration of N-linked glycosylation sites was disadvantageous to its efficient secretion in Pichia pastoris, where a 52.32%, 36.48%, 71.41%lower production was observed in N15A, N70A, and N15/70A mutants, respectively, as compared to native control. The fully glycosylated native hIL-3 protein showed higher thermal stability over its deglycosylated counterparts. The biological activity of native, N15A, N70A, and N15/70A hIL-3 protein was evaluated, where N15/70A mutant showed slightly higher proliferation efficacy than other combinations.

Predicting high recombinant protein producer strains of Pichia pastoris MutS using the oxygen transfer rate as an indicator of metabolic burden

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a widely used host for recombinant protein production. In this study, a clonal library of P. pastoris MutS strains (S indicates slow methanol utilization) was screened for high green fluorescent protein (GFP) production. The expression cassette was under the control of the methanol inducible AOX promoter. The growth behavior was online-monitored in 48-well and 96-well microtiter plates by measuring the oxygen transfer rate (OTR). By comparing the different GFP producing strains, a correlation was established between the slope of the cumulative oxygen transfer during the methanol metabolization phase and the strain’s production performance. The correlation corresponds to metabolic burden during methanol induction. The findings were validated using a pre-selected strain library (7 strains) of high, medium, and low GFP producers. For those strains, the gene copy number was determined via Whole Genome Sequencing. The results were consistent with the described OTR correlation. Additionally, a larger clone library (45 strains) was tested to validate the applicability of the proposed method. The results from this study suggest that the cumulative oxygen transfer can be used as a screening criterion for protein production performance that allows for a simple primary screening process, facilitating the pre-selection of high producing strains.

Active Expression of Human Hyaluronidase PH20 and Characterization of Its Hydrolysis Pattern.

Hyaluronidases are a group of glycosidases catalyzing the degradation of hyaluronic acid (HA). Because of the advantages of effectively hydrolyzing the HA-rich matrix and low immunogenicity, human hyaluronidase PH20 (hPH20) is widely used in the medical field. Here, we realized the active expression of recombinant hPH20 by Pichia pastoris under a methanol-induced promoter PAOX1. By optimizing the composition of the C-terminal domain and fusing protein tags, we constructed a fusion mutant AP2-△491C with the extracellular hyaluronidase activity of 258.1 U·L−1 in a 3-L bioreactor, the highest expression level of recombinant hPH20 produced by microbes. Furthermore, we found recombinant hPH20 hydrolyzed the β-1,4 glycosidic bonds sequentially from the reducing end of o-HAs, with HA6 NA as the smallest substrate. The result will provide important theoretical guidance for the directed evolution of the enzyme to prepare multifunctional o-HAs with specific molecular weights.

Synthetic Biology Toolkit for Marker-Less Integration of Multigene Pathways into Pichia pastoris via CRISPR/Cas9.

Pichia pastoris, an important methylotrophic yeast, is currently mainly used for the expression of recombinant proteins and has great potential applications in the production of value-added compounds (e.g., chemical and natural products). However, the construction of P. pastoris cell factories is largely hindered by the lack of genetic tools for the manipulation of multigene biosynthetic pathways. Therefore, the present study aimed to establish a CRISPR-based synthetic biology toolkit for the integration and assembly of multigene biosynthetic pathways into the chromosome of P. pastoris. First, 23 intergenic regions were selected and characterized as potential integration sites, with a focus on the integration efficiency and heterologous gene expression levels. In addition, a panel of constitutive and methanol-inducible promoters with different strengths (weak, medium, and strong promoters) were characterized to control the expression of biosynthetic pathway genes to the desirable levels. With a series of gRNA plasmids (for single-locus, two-loci, and three-loci integration) and donor plasmids (containing homology arms for integration and promoters and terminators for driving heterologous gene expression) as major components, a CRISPR-based synthetic biology toolkit was established, which enabled the integration of one locus, two loci, and three loci with efficiencies as high as ∼100, ∼93, and ∼75%, respectively, in P. pastoris GS115 strain. Finally, the application of the toolkit was demonstrated by the construction of a series of P. pastoris cell factories, which could produce 2,3-butanediol, β-carotene, zeaxanthin, and astaxanthin with methanol as the sole carbon and energy source. The P. pastoris synthetic biology toolkit is highly standardized and can be employed to construct P. pastoris cell factories with high efficiency.

Engineering of Promoters for Gene Expression in Pichia pastoris.

The availability of exceptionally strong and tightly regulated promoters is a key feature of Komagataella phaffii (syn. Pichia pastoris), a widely applied yeast expression system for heterologous protein production. Most commonly, the methanol-inducible promoter of the alcohol oxidase 1 gene (PAOX1) and the constitutive promoter of the glyceraldehyde 3 phosphate dehydrogenase gene (PGAP) have been used. Recently, also promising novel constitutive (PGCW14), regulated (PGTH1, PCAT1), and bidirectional promoters (histone promoters and synthetic hybrid variants) have been reported.As natural promoters showed so far limited tunability of expression levels and regulatory profiles, various promoter engineering efforts have been undertaken for P. pastoris . PAOX1, PDAS2, PGAP, and PGCW14 have been engineered by systematic deletion studies or random mutagenesis of upstream regulatory sequences. New engineering strategies have focused on PAOX1 core promoter modifications by random or rational approaches and transcriptional regulatory circuits to render PAOX1 independent of methanol induction. These promoter engineering efforts in P. pastoris have resulted in improved, sequence-diversified synthetic promoter variants allowing coordinated fine-tuning of gene expression for a multitude of biotechnological applications.

Heterologous Expression of Histidine Acid Phytase From Pantoea sp. 3.5.1 in Methylotrophic Yeast Pichia Pastoris

Background and Objective:The major storage form of phosphorus in plant-derived feed is presented by phytates and not digested by animals. Phytases are able to hydrolyze phytates and successfully used as feed additives. Nevertheless, nowadays, there is a constant search of new phytases and expression systems for better production of these enzymes. In this study, we describe cloning and expression of gene encoding histidine acid phytase fromPantoeasp. 3.5.1 using methylotrophic yeastPichia pastorisas the host.Methods:The phytase gene was placed under the control of the methanol-inducible AOX1 promoter and expressed inP. pastoris. Experiments of small-scale phytase expression and activity assays were used to test recombinant colonies. Four different signal peptides were screened for better secretion of phytase byP. pastoris. After 36 h of methanol induction in shake flasks, the maximum extracellular phytase activity (3.2 U/ml) was observed inP. pastorisstrain with integrated construct based on pPINK-HC vector andKluyveromyces maxianusinulinase gene signal sequence. This phytase was isolated and purified using affinity chromatography.Results:Recombinant phytase was a glycosylated protein, had a molecular weight of around 90 kDa and showed maximum activity at pH 4.0 and at 50°C. Recombinant phytase had excellent thermal stability – it retained high residual activity (100% ± 2%) after 1 hour of heat treatment at 70°C.Conclusion:The enhanced thermostability of the recombinant phytase, its expression provided by strong inducible promotor and the effectively designed expression cassette, the simple purification procedure of the secreted enzyme, and the possibility of large-scale expression make the foundation for further production of this bacterial phytase inP. pastorisat an industrial scale.

Enhancement of docosahexaenoic acid production by overexpression of ATP-citrate lyase and acetyl-CoA carboxylase in Schizochytrium sp.

BackgroundDocosahexaenoic acid (DHA) is an important omega-3 long-chain polyunsaturated fatty acid that has a variety of physiological functions for infant development and human health. Although metabolic engineering was previously demonstrated to be a highly efficient way to rapidly increase lipid production, metabolic engineering has seldom been previously used to increase DHA accumulation in Schizochytrium spp.ResultsHere, a sensitive β-galactosidase reporter system was established to screen for strong promoters in Schizochytrium sp. Four constitutive promoters (EF-1αp, TEF-1p, ccg1p, and ubiquitinp) and one methanol-induced AOX1 promoter were characterized by the reporter system with the promoter activity ccg1p> TEF-1p > AOX1p (induced) > EF-1αp > ubiquitinp. With the strong constitutive promoter ccg1p, Schizochytrium ATP-citrate lyase (ACL) and acetyl-CoA carboxylase (ACC) were overexpressed in Schizochytrium sp. ATCC 20888. The cells were cultivated at 28 °C and 250 rpm for 120 h with glucose as the carbon source. Shake-flask fermentation results showed that the overexpression strains exhibited growth curves and biomass similar to those of the wild-type strain. The lipid contents of the wild-type strain and of the OACL, OACC, and OACL-ACC strains were 53.8, 68.8, 69.8, and 73.0%, respectively, and the lipid yields of the overexpression strains were increased by 21.9, 30.5, and 38.3%, respectively. DHA yields of the wild-type strain and of the corresponding overexpression strains were 4.3, 5.3, 6.1, and 6.4 g/L, i.e., DHA yields of the overexpression strains were increased by 23.3, 41.9, and 48.8%, respectively.ConclusionsAcetyl-CoA and malonyl-CoA are precursors for fatty acid synthesis. ACL catalyzes the conversion of citrate in the cytoplasm into acetyl-CoA, and ACC catalyzes the synthesis of malonyl-CoA from acetyl-CoA. The results demonstrate that overexpression of ACL and ACC enhances lipid accumulation and DHA production in Schizochytrium sp.

Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications.

The methanol utilization (Mut) phenotype in the yeast Pichia pastoris (syn. Komagataella spp.) is defined by the deletion of the genes AOX1 and AOX2. The Mut− phenotype cannot grow on methanol as a single carbon source. We assessed the Mut− phenotype for secreted recombinant protein production. The methanol inducible AOX1 promoter (PAOX1) was active in the Mut− phenotype and showed adequate eGFP fluorescence levels and protein yields (YP/X) in small‐scale screenings. Different bioreactor cultivation scenarios with methanol excess concentrations were tested using PAOX1HSA and PAOX1vHH expression constructs. Scenario B comprising a glucose‐methanol phase and a 72‐hr‐long methanol only phase was the best performing, producing 531 mg/L HSA and 1631 mg/L vHH. 61% of the HSA was produced in the methanol only phase where no biomass growth was observed, representing a special case of growth independent production. By using the Mut− phenotype, the oxygen demand, heat output, and specific methanol uptake (q methanol) in the methanol phase were reduced by more than 80% compared with the MutS phenotype. The highlighted improved process parameters coupled with growth independent protein production are overlooked benefits of the Mut− strain for current and future applications in the field of recombinant protein production.

Coexpression of Kex2 Endoproteinase and Hac1 Transcription Factor to Improve the Secretory Expression of Bovine Lactoferrin in Pichia pastoris

The large-scale production of functional recombinant lactoferrin has become a major goal because of its medicinal value and global demand. Secreting recombinant proteins into a culture medium offers a way to simplify protein purification and avoid toxicity from intracellularly accumulated materials. In this study, after 84 h of induction with methanol in a shaking flask, the recombinant bovine lactoferrin (rbLf) titer in the culture supernatant of the strain that integrated two copies of the rbLf gene was only 121.6 μg/L. A bottleneck might have existed in the folding and secretion pathways of rbLf. We then attempted to further improve the rbLf titer by overexpressing the transcription factor Haclp and α-signal peptide-cutting protease Kex2p with different promoters. Results showed that the inducible coexpression of Haclp and Kex2p linked with the 2A sequence improved the rbLf titer 5.0-fold (735.8 μg/L) after 84 h of induction with methanol. The maximal titer in a shaking flask was 1,150.5 μg/L after 120 h of induction. The rbLf titer achieved 35.6 mg/L in a 5 L fed-batch fermenter. Thus, Kex2 and Hacl overexpression driven by methanol-induced promoter alleviated the bottleneck in the folding and secretion pathways and greatly improved the secretory expression of rbLf in Pichia pastoris.

Engineering the expression system for Komagataella phaffii (Pichia pastoris): an attempt to develop a methanol-free expression system.

ABSTRACTThe construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) was attempted by engineering a strong methanol-inducible DAS1 promoter using Citrobacter braakii phytase production as a model case. Constitutive expression of KpTRM1, formerly PRM1—a positive transcription regulator for methanol-utilization (MUT) genes of K. phaffii,was demonstrated to produce phytase without addition of methanol, especially when a DAS1 promoter was used but not an AOX1 promoter. Another positive regulator, Mxr1p, did not have the same effect on the DAS1 promoter, while it was more effective than KpTrmp1 on the AOX1 promoter. Removing a potential upstream repression sequence (URS) and multiplying UAS1DAS1 in the DAS1 promoter significantly enhanced the yield of C. braakii phytase with methanol-feeding, which surpassed the native AOX1 promoter by 80%. However, multiplying UAS1DAS1 did not affect the yield of methanol-free expression by constitutive KpTrm1p. Another important region to enhance the effect of KpTrm1p under a methanol-free condition was identified in the DAS1 promoter, and was termed ESPDAS1. Nevertheless, methanol-free phytase production using an engineered DAS1 promoter outperformed phytase production with the GAP promoter by 25%. Difference in regulation by known transcription factors on the AOX1 promoter and the DAS1 promoter was also illustrated.

Expression of recombinant enhanced green fluorescent protein provides insight into foreign gene-expression differences between Mut+ and MutS strains of Pichia pastoris.

Pichia pastoris is a very popular yeast for recombinant protein production, mainly due to the strong, methanol-inducible PAOX1 promoter. Methanol induction however poses several drawbacks. One approach to improve processes is to use MutS strains with reduced methanol catabolic ability. Various reports claim that MutS allows higher recombinant protein production levels than Mut+ but scarcely elaborate on reasons for differences. In this study, enhanced green fluorescent protein was used as a PAOX1 -driven reporter for the investigation of expression differences between Mut+ and MutS strains. Mut+ exhibited higher responses to methanol, with faster growth (0.07 vs. 0.01hr-1 ) and higher consumption of methanol (2.25 vs. 1.81mmol/gDCW .hr) and oxygen (2.2 vs. 0.66mmol/gDCW .hr) than MutS. Mut+ yielded more biomass than MutS (2.3 vs. 1.3gDCW /L), and carbon dioxide analysis of bioreactor off-gas suggested that considerable amounts of methanol were consumed by Mut+ via the dissimilatory pathway. In contrast, it was demonstrated that the MutS population switched to an induced state more rapidly than Mut+. In addition, MutS exhibited 3.4-fold higher fluorescence levels per cell (77,509 vs. 23,783SFU) indicative of higher recombinant protein production. The findings were verified by similar results obtained during the expression of a lipase. Based on the differences in response to methanol versus recombinant protein production, it was proposed that higher energy availability occurs in MutS for recombinant protein synthesis, contrary to Mut+ that uses the energy to maintain high levels of methanol catabolic pathways and biomass production.

Recombinant O-mannosylated protein production (PstS-1) from Mycobacterium tuberculosis in Pichia pastoris (Komagataella phaffii) as a tool to study tuberculosis infection

BackgroundPichia pastoris (syn. Komagataella phaffii) is one of the most highly utilized eukaryotic expression systems for the production of heterologous glycoproteins, being able to perform both N- and O-mannosylation. In this study, we present the expression in P. pastoris of an O-mannosylated recombinant version of the 38 kDa glycolipoprotein PstS-1 from Mycobacterium tuberculosis (Mtb), that is similar in primary structure to the native secreted protein.ResultsThe recombinant PstS-1 (rPstS-1) was produced without the native lipidation signal. Glycoprotein expression was under the control of the methanol-inducible promoter pAOX1, with secretion being directed by the α-mating factor secretion signal. Production of rPstS-1 was carried out in baffled shake flasks (BSFs) and controlled bioreactors. A production up to ~ 46 mg/L of the recombinant protein was achieved in both the BSFs and the bioreactors. The recombinant protein was recovered from the supernatant and purified in three steps, achieving a preparation with 98% electrophoretic purity. The primary and secondary structures of the recombinant protein were characterized, as well as its O-mannosylation pattern. Furthermore, a cross-reactivity analysis using serum antibodies from patients with active tuberculosis demonstrated recognition of the recombinant glycoprotein, indirectly indicating the similarity between the recombinant PstS-1 and the native protein from Mtb.ConclusionsrPstS-1 (98.9% sequence identity, O-mannosylated, and without tags) was produced and secreted by P. pastoris, demonstrating that this yeast is a useful cell factory that could also be used to produce other glycosylated Mtb antigens. The rPstS-1 could be used as a tool for studying the role of this molecule during Mtb infection, and to develop and improve vaccines or kits based on the recombinant protein for serodiagnosis.

Methylotrophic Yeasts: Current Understanding of Their C1-Metabolism and its Regulation by Sensing Methanol for Survival on Plant Leaves.

Methylotrophic yeasts, which are able to utilize methanol as the sole carbon and energy source, have been intensively studied in terms of physiological function and practical applications. When these yeasts grow on methanol, the genes encoding enzymes and proteins involved in methanol metabolism are strongly induced. Simultaneously, peroxisomes, organelles that contain the key enzymes for methanol metabolism, massively proliferate. These characteristics have made methylotrophic yeasts efficient hosts for heterologous protein production using strong and methanol-inducible gene promoters and also model organisms for the study of peroxisome dynamics. Much attention has been paid to the interaction between methylotrophic microorganisms and plants. In this chapter, we describe how methylotrophic yeasts proliferate and survive on plant leaves, focusing on their physiological functions and lifestyle in the phyllosphere. Our current understanding of the molecular basis of methanol-inducible gene expression, including methanol-sensing and its applications, is also summarized.

High-level expression of Thermomyces dupontii thermo-alkaline lipase in Pichia pastoris under the control of different promoters.

In this study, 15 methanol-inducible and 9 constitutive promoters were used to drive the expression of Thermomyces dupontii lipase (TDL) in Pichia pastoris. Of the 15 methanol-inducible promoters, formaldehyde dehydrogenase promoter (PFLD1) showed the highest efficiency in driving lipase production, followed by alcohol oxidase 1 (PAOX1) and dihydroxyacetone synthase (PDAS1) promoters. The maximum lipase activity of transformants with PFLD1, PAOX1 and PDAS1 promoters in 5-l bioreactor was 27,076, 24,159 and 22,342U/ml, respectively. For the nine constitutive promoters, glycosyl phosphatidyl inositol-anchored protein promoter (PGCW14) produced the highest amount of lipases in a medium containing glucose or glycerol as the only carbon source, followed by mitochondrial alcohol dehydrogenase isozyme (P0472) and glyceraldehyde-3-phosphate dehydrogenase (PGAP) promoters. The maximum lipase yields in 5-l bioreactors under the control of PGCW14, P0472 and PGAP promoters were 17,353, 15,046 and 14,276U/ml, respectively. The result of this study not only identifies a few highly efficient promoters for the heterologous expression of TDL in P. pastoris, but also casts some insight into the optimization of protein production in heterologous systems.

Aspergillus oryzae S2 AmyA amylase expression in Pichia pastoris: production, purification and novel properties.

A synthetic cDNA-AmyA gene was cloned and successfully expressed in Pichia pastoris as a His-tagged enzyme under the methanol inducible AOX1 promoter. High level of extracellular amylase production of 72 U/mL was obtained after a 72h induction by methanol. As expected, the recombinant strain produced only the AmyA isoform since the host is a protease deficient strain. Besides, the purified r-AmyA showed a molecular mass of 54kDa, the same pH optimum equal to 5.6 but a higher thermoactivity of 60°C against 50°C for the native enzyme. Unlike AmyA which maintained 50% of its activity after a 10-min incubation at 60°C, r-AmyA reached 45min. The higher thermoactivity and thermostability could be related to the N-glycosylation. The r-AmyA activity was enhanced by 46% and 45% respectively in the presence of 4mM Fe2+ and Mg2+ ions. This enzyme was more efficient in bread-making since such ions were reported to have a positive impact on the nutriment quality and the rheological characteristics of the wheat flour dough. The thermoactivity/thermostability as well as the iron and magnesium activations could also be ascribed to the presence of an additional C-terminal loop containing the His tag.