Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker

Background: A commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele. Results: A K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth. Conclusions: In this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii

In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti

<p>Abstract</p> <p>Background</p> <p>The major Dengue virus vector <it>Aedes aegypti </it>requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of <it>Ae. aegypti </it>midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes.</p> <p>Results</p> <p>We used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (k_cat/K_M) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin.</p> <p>Conclusions</p> <p>These data show that <it>in vitro </it>activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.</p

Systematic Single-Cell Analysis of Pichia pastoris Reveals Secretory Capacity Limits Productivity

Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity

Detection and elimination of cellular bottlenecks in protein-producing yeasts

Yeasts are efficient cell factories and are commonly used for the production of recombinant proteins for biopharmaceutical and industrial purposes. For such products high levels of correctly folded proteins are needed, which sometimes requires improvement and engineering of the expression system. The article summarizes major breakthroughs that led to the efficient use of yeasts as production platforms and reviews bottlenecks occurring during protein production. Special focus is given to the metabolic impact of protein production. Furthermore, strategies that were shown to enhance secretion of recombinant proteins in different yeast species are presented

Laboratory intercomparison study for the analysis of nonylphenol and octylphenol in river water

7 pages, 4 figures, 2 tables.-- Available online Oct 30, 2007.In support of the implementation of the European Union (EU) Water Framework Directive (WFD), the European Commission (EC) Joint Research Centre (JRC) organized a laboratory and sampling intercomparison study for the chemical monitoring of polyaromatic hydrocarbons (PAHs), polybrominated diphenyl ethers (PBDEs), and nonylphenol and octylphenol (NP/OP), priority substances of the WFD, in river water. EU Member State laboratories were invited to analyze a standard solution with unknown concentration levels and a river-water extract, as well as a real water sample from the River Po (Italy). For the standard solution and the river-water extract, good agreement was achieved for five laboratories. Triple-quadrupole liquid chromatography with tandem mass spectrometry (LC-MS2) and gas chromatography with MS (GC-MS) with or without derivatization proved to be comparable methods for the analysis of NP and OP. Fluorescence detection can also be used to analyze NP and OP, but it is less specific. The results of the River Po water sample showed that some laboratories have problems in analyzing NP at concentration levels below 100 ng/L due to contamination of laboratory blanks. Plastics materials should not be used during extraction and sample preparation.Without the effort of the participating laboratories, this article would not have been possible. Susanne Boutrup, Stefano Polesello and Joan Staeb have given very helpful comments on the manuscript. Stefano Polesello is especially thanked for the help with the OP standards. Moreover, we would like to thank the “Servizio Risorse Idriche e Tutela Ambientale della Provincia di Ferrara” (Silvano Bencivelli, Paola Magri), Lars Håkanson, Graziella Leroy and Madeleine Rizzi.Peer reviewe