Pichia pastoris

About: Pichia pastoris is a research topic. Over the lifetime, 7937 publications have been published within this topic receiving 162645 citations. The topic is also known as: Komagataella pastoris.

Heterologous expression of lcc1 gene from Trametes trogii in Pichia pastoris and characterization of the recombinant enzyme

Abstract: Background: Fungal laccases are useful enzymes for industrial applications; they exhibit broad substrate specificity and thus are able to oxidize a variety of xenobiotic compounds including chlorinated phenolics, synthetic dyes, pesticides and polycyclic aromatic hydrocarbons. Unfortunately, the biotechnological exploitation of laccases can be hampered by the difficulties concerning the enzyme production by the native hosts. Results: In order to obtain a simple and efficient source of laccase, the lcc1 cDNA isolated from the white-rot fungus Trametes trogii has been successfully expressed in the methylotrophic yeast Pichia pastoris under the control of the methanol induced alcohol oxidase promoter P AOX1 . The recombinant Lcc1 was produced as a secreted protein with the native N-terminal prepropeptide for signal trafficking, and thus easily recovered from the culture medium. At the 1-liter scale, as calculated on the basis of the specific activity, the recombinant protein was produced at a yield of 17 mg/l. The highest production level obtained in fed-batch culture was 2520 U/l, corresponding to a specific productivity of 31.5 U/g biomass. The purified recombinant laccase exhibited a behaviour similar to the main laccase produced by T. trogii. Lcc1 showed high activity in the presence of organic solvents and a high decolourization capacity towards azo, triarylmethane, indigo carmine and anthraquinonic dyes, that could be significantly enhanced in the presence of the redox mediators 1-hydroxybenzotriazole and violuric acid. Conclusion: Heterologous expression of T. trogii laccase lcc1 in the methylotrophic yeast P. pastoris was successfully achieved. The biochemical and kinetic characterization of the recombinant protein suggests potential technological applications for this enzyme.

Playing catch-up with Escherichia coli:using yeast to increase success rates in recombinant protein production experiments

Abstract: Several host systems are available for the production of recombinant proteins, ranging from Escherichia coli to mammalian cell-lines. This article highlights the benefits of using yeast, especially for more challenging targets such as membrane proteins. On account of the wide range of molecular, genetic, and microbiological tools available, use of the well-studied model organism, Saccharomyces cerevisiae, provides many opportunities to optimize the functional yields of a target protein. Despite this wealth of resources, it is surprisingly under-used. In contrast, Pichia pastoris, a relative new-comer as a host organism, is already becoming a popular choice, particularly because of the ease with which high biomass (and hence recombinant protein) yields can be achieved. In the last few years, advances have been made in understanding how a yeast cell responds to the stress of producing a recombinant protein and how this information can be used to identify improved host strains in order to increase functional yields. Given these advantages, and their industrial importance in the production of biopharmaceuticals, I argue that S. cerevisiae and P. pastoris should be considered at an early stage in any serious strategy to produce proteins.

Electrochemical Studies of a Truncated Laccase Produced in Pichia pastoris

Abstract: The cDNA that encodes an isoform of laccase from Trametes versicolor (LCCI), as well as a truncated version (LCCIa), was subcloned and expressed by using the yeast Pichia pastoris as the heterologous host. The amino acid sequence of LCCIa is identical to that of LCCI except that the final 11 amino acids at the C terminus of LCCI are replaced with a single cysteine residue. This modification was introduced for the purpose of improving the kinetics of electron transfer between an electrode and the copper-containing active site of laccase. The two laccases (LCCI and LCCIa) are compared in terms of their relative activity with two substrates that have different redox potentials. Results from electrochemical studies on solutions containing LCCI and LCCIa indicate that the redox potential of the active site of LCCIa is shifted to more negative values (411 mV versus normal hydrogen electrode voltage) than that found in other fungal laccases. In addition, replacing the 11 codons at the C terminus of the laccase gene with a single cysteine codon (i.e., LCCI→LCCIa) influences the rate of heterogeneous electron transfer between an electrode and the copper-containing active site (khet for LCCIa = 1.3 × 10−4 cm s−1). These results demonstrate for the first time that the rate of electron transfer between an oxidoreductase and an electrode can be enhanced by changes to the primary structure of a protein via site-directed mutagenesis.