Polycistronic Expression System for Pichia pastoris Composed of Chitino- and Chitosanolytic Enzymes.
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ABSTRACT:
SUBMITTER: Kaczmarek MB
PROVIDER: S-EPMC8418187 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundSustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes.Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages.ResultsIn this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes we could develop strains capable of secreting gram quantities of enzyme per liter in fed-batch cultivations. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data.ConclusionIn our experiments we could clearly show the importance of gene optimization and strain characterization for successfully improving secretion levels. We also present a basic guideline how to correctly interpret the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris.
| S-EPMC3503753 | biostudies-literature
Project description:Stylopine is a protoberberine-type alkaloid that has potential biological activities. Based on the successful microbial production of (S)-reticuline, we attempted to produce stylopine from (S)-reticuline by the reaction of berberine bridge enzyme, cheilanthifoline synthase (CYP719A5), and stylopine synthase (CYP719A2). Biosynthetic enzyme expression was examined in a methanol-utilizing yeast (Pichia pastoris), and both a "consolidated" system with all genes expressed in one cell and a "co-culture" system with three cell lines that each express a single gene were examined. Although both systems efficiently converted reticuline to stylopine, the consolidated system was more rapid and efficient than the co-culture system. However, substrate-feeding experiments revealed a decrease in the conversion efficiency in the consolidated system during successive cultures, whereas the conversion efficiency in the co-culture system remained constant. Thus, the final amount of stylopine produced from reticuline after successive feedings in the co-culture system was more than 150 nmoles from 750 nmoles of (R, S)-reticuline (375 nmoles of (S)-reticuline). The advantages and drawbacks of the "consolidated" system and the "co-culture" system are discussed.
| S-EPMC4770593 | biostudies-literature
Project description:BackgroundLipase-catalyzed biotransformation of acylglycerides or fatty acids into biodiesel via immobilized enzymes or whole cell catalysts has been considered as one of the most promising methods to produce renewable and environmentally friendly alternative liquid fuels, thus being extensively studied so far. In all previously pursued approaches, however, lipase enzymes are prepared in an independent process separated from enzymatic biodiesel production, which would unavoidably increase the cost and energy consumption during industrial manufacture of this cost-sensitive energy product. Therefore, there is an urgent need to develop novel cost-effective biocatalysts and biocatalytic processes with genuine industrial feasibility.ResultInspired by the consolidated bioprocessing of lignocellulose to generate bioethanol, an integrated process with coupled lipase production and in situ biodiesel synthesis in a recombinant P. pastoris yeast was developed in this study. The novel and efficient dual biocatalytic system based on Thermomyces lanuginosus lipase took advantage of both cell free enzymes and whole cell catalysts. The extracellular and intracellular lipases of growing yeast cells were simultaneously utilized to produce biodiesel from waste cooking oils in situ and in one pot. This integrated system effectively achieved 58% and 72% biodiesel yield via concurrent esterified-transesterified methanolysis and stepwise hydrolysis-esterification at 3:1 molar ratio between methanol and waste cooking oils, respectively. Further increasing the molar ratio of methanol to waste cooking oils to 6:1 led to an 87% biodiesel yield using the stepwise strategy. Both water tolerance and methanol tolerance of this novel system were found to be significantly improved compared to previous non-integrated biodiesel production processes using separately prepared immobilized enzymes or whole cell catalysts.ConclusionWe have proposed a new concept of integrated biodiesel production. This integrated system couples lipase production to lipase-catalyzed biodiesel synthesis in one pot. The proof-of-concept was established through construction of a recombinant P. pastoris yeast strain that was able to grow, overexpress T. lanuginosus lipase, and efficiently catalyze biodiesel production from fed waste cooking oils and methanol simultaneously. This simplified single-step process represents a significant advance toward achieving economical production of biodiesel at industrial scale via a 'green' biocatalytic route.
| S-EPMC4022340 | biostudies-literature
Project description:Margatoxin (MgTx) is a high-affinity blocker of voltage-gated potassium (Kv) channels. It inhibits Kv1.1–Kv1.3 ion channels in picomolar concentrations. This toxin is widely used to study physiological function of Kv ion channels in various cell types, including immune cells. Isolation of native MgTx in large quantities from scorpion venom is not affordable. Chemical synthesis and recombinant production in Escherichia coli need in vitro oxidative refolding for proper disulfide bond formation, resulting in a very low yield of peptide production. The Pichia pastoris expression system offers an economical approach to overcome all these limitations and gives a higher yield of correctly refolded recombinant peptides. In this study, improved heterologous expression of recombinant MgTx (rMgTx) in P. pastoris was obtained by using preferential codons, selecting the hyper-resistant clone against Zeocin, and optimizing the culturing conditions. About 36 ± 4 mg/L of >98% pure His-tagged rMgTx (TrMgTx) was produced, which is a threefold higher yield than has been previously reported. Proteolytic digestion of TrMgTx with factor Xa generated untagged rMgTx (UrMgTx). Both TrMgTx and UrMgTx blocked the Kv1.2 and Kv1.3 currents (patch-clamp) (Kd for Kv1.2 were 64 and 14 pM, and for Kv1.3, 86 and 50 pM, respectively) with comparable potency to the native MgTx. The analysis of the binding kinetics showed that TrMgTx had a lower association rate than UrMgTx for both Kv1.2 and Kv1.3. The dissociation rate of both the analogues was the same for Kv1.3. However, in the case of Kv1.2, TrMgTx showed a much higher dissociation rate with full recovery of the block than UrMgTx. Moreover, in a biological functional assay, both peptides significantly downregulated the expression of early activation markers IL2R and CD40L in activated CD4+ TEM lymphocytes whose activation was Kv1.3 dependent. In conclusion, the authors report that the Pichia expression system is a powerful method to produce disulfide-rich peptides, the overexpression of which could be enhanced noticeably through optimization strategies, making it more cost-effective. Since the presence of the His-tag on rMgTx only mildly altered the block equilibrium and binding kinetics, recombinant toxins could be used in ion channel research without removing the tag and could thus reduce the cost and time demand for toxin production.
| S-EPMC8511391 | biostudies-literature
Project description:A lysozyme gene from breast of Tibetan sheep was successfully expressed by secretion using a-factor signal sequence in the methylotrophic yeast, Pichia pastoris GS115. An expression yield and specific activity greater than 500 mg/L and 4,000 U/mg was obtained. Results at optimal pH and temperature showed recombinant lysozyme has higher lytic activity at pH 6.5 and 45°C. This study demonstrates the successful expression of recombinant lysozyme using the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, this study shows the feasibility of subsequent industrial manufacture of the enzyme with this expression system together with a high purity scheme for easy high-yield purification.
| S-EPMC4093532 | biostudies-literature
Project description:The 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.
| S-EPMC6736287 | biostudies-literature
Project description:Phospholipase B is a virulence factor for several clinically important pathogenic fungi, including Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, but its role in the thermally dimorphic fungus Talaromyces marneffei remains unclear. Here, we provide the first report of the expression of a novel phospholipase gene, designated TmPlb1, from T. marneffei in the eukaryotic expression system of Pichia pastoris GS115. Sensitive real-time quantitative reverse-transcription PCR (qRT-PCR) demonstrated that the expression of TmPlb1 increased 1.85-fold in the yeast phase compared with the mycelial phase. TmPlb1 contains an open reading frame (ORF) of 732 bp that encodes a protein of 243 amino acids. The conserved serine, aspartate and histidine catalytic triad and the G-X-S-X-G domain of TmPLB1 provide the structural basis for its molecular activity. The ORF of TmPlb1 was successfully cloned into a pPIC9K vector containing an α-mating factor secretion signal that allowed the secretory expression of TmPLB1 in P. pastoris. The heterologous protein expression began 12 h after methanol induction and peaked at 96 h. Through analysis with SDS-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting and mass spectrometry, we confirmed that TmPLB1 was successfully expressed. Through Ni-affinity chromatography, TmPLB1 was highly purified, and its concentration reached 240.4 mg/L of culture medium. With specific substrates, the phospholipase A1 and phospholipase A2 activities of TmPLB1 were calculated to be 5.96 and 1.59 U/mg, respectively. The high purity and activity of the TmPLB1 obtained here lay a solid foundation for further investigation.
| S-EPMC5148023 | biostudies-literature
Project description:BackgroundVarious protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely ?2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies.ResultsThe ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells.ConclusionCompared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in P. pastoris can be applied to the structural and biochemical studies of GPCRs.
| S-EPMC3094209 | biostudies-literature
Project description:In recent years, cell-free protein synthesis (CFPS) systems have been used to synthesize proteins, prototype genetic elements, manufacture chemicals, and diagnose diseases. These exciting, novel applications lead to a new wave of interest in the development of new CFPS systems that are derived from prokaryotic and eukaryotic organisms. The eukaryotic Pichia pastoris is emerging as a robust chassis host for recombinant protein production. To expand the current CFPS repertoire, we report here the development and optimization of a eukaryotic CFPS system, which is derived from a protease-deficient strain P. pastoris SMD1163. By developing a simple crude extract preparation protocol and optimizing CFPS reaction conditions, we were able to achieve superfolder green fluorescent protein (sfGFP) yields of 50.16 ± 7.49 ?g/ml in 5 h batch reactions. Our newly developed P. pastoris CFPS system fits to the range of the productivity achieved by other eukaryotic CFPS platforms, normally ranging from several to tens of micrograms protein per milliliter in batch mode reactions. Looking forward, we believe that our P. pastoris CFPS system will not only expand the CFPS toolbox for synthetic biology applications, but also provide a novel platform for cost-effective, high-yielding production of complex proteins that need post-translational modification and functionalization.
| S-EPMC7314905 | biostudies-literature
Project description:Comparison of transcription profile of Pichia pastoris cells grown on Glucose medium with Pichia pastoris cells grown on Methanol/Glycerol medium, the fermentations were done in a chemostat.
2015-08-31 | GSE67690 | GEO