Industrial Production of Proteins with -.

Since the mid-1960s, methylotrophic yeast (previously described as ) has received increasing scientific attention. The interest for the industrial production of proteins for different applications (e.g.

Skin Commensal Malassezia globosa Secreted Protease Attenuates Staphylococcus aureus Biofilm Formation.

Skin provides the first defense against pathogenic micro-organisms and is also colonized by a diverse microbiota. Phylogenetic analysis of whole skin microbiome at different skin sites in health and disease has generated important insights on possible microbial involvement in modulating skin health. However, functional roles of the skin microbial community remain unclear.

Cloning and upscale production of monoamine oxidase N (MAO-N D5) by Pichia pastoris.

Objective: To clone monoamine oxidase N, that catalyses the selective oxidative deamination or deracemisation of amines into imines, in Pichia pastoris and prove the importance of choosing the proper expression system for its recombinant production.

Results: Monoamine oxidase, originating from Aspergillus niger and subjected to directed evolution (MAO-N D5), was cloned and expressed in Pichia pastoris CBS7435 Mut strain for the first time. Various transformants were screened at microscale level.

Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications.

Background: Recombinant protein production in the methylotrophic yeast Pichia pastoris largely relies on integrative vectors. Although the stability of integrated expression cassettes is well appreciated for most applications, the availability of reliable episomal vectors for this host would represent a useful tool to expedite cloning and high-throughput screening, ameliorating also the relatively high clonal variability reported in transformants from integrative vectors caused by off-target integration in the P. pastoris genome.

Pichia pastoris mutants as host strains for efficient secretion of recombinant branched chain aminotransferase (BCAT).

Branched chain aminotransferase (BCAT) is one of the enzymatic tools of choice for the production of chiral amines or amino acids; especially, non-natural amino acids are of interest as building blocks for the pharmaceutical industry. The expression and subsequent secretion of BCAT counteracts limited cell permeability of target substrates and facilitates downstream processing. Since Pichia pastoris secretes a negligible amount of native proteins and was previously shown to efficiently secrete recombinant proteins, it was chosen as the expression host.

Recombinant Expression of Trichoderma reesei Cel61A in Pichia pastoris: Optimizing Yield and N-terminal Processing.

The auxiliary activity family 9 (AA9, formerly GH61) harbors a recently discovered group of oxidative enzymes that boost cellulose degradation. Indeed, these lytic polysaccharide monooxygenases (LPMOs) are able to disrupt the crystalline structure of cellulose, thereby facilitating the work of hydrolytic enzymes involved in biomass degradation. Since these enzymes require an N-terminal histidine residue for activity, their recombinant production as secreted protein is not straightforward.

Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P.

Carbon source dependent promoters in yeasts.

Budding yeasts are important expression hosts for the production of recombinant proteins.The choice of the right promoter is a crucial point for efficient gene expression, as most regulations take place at the transcriptional level. A wide and constantly increasing range of inducible, derepressed and constitutive promoters have been applied for gene expression in yeasts in the past; their different behaviours were a reflection of the different needs of individual processes.

Screening for cytochrome P450 expression in Pichia pastoris whole cells by P450-carbon monoxide complex determination.

Cytochrome P450 (CYP) enzymes are useful biocatalysts for the pharmaceutical and biotechnological industries. A high-throughput method for quantification of CYP expression in yeast is needed in order to fully exploit the yeast expression system. Carbon monoxide (CO) difference spectra of whole cells have been successfully used for the quantification of heterologous CYP expressed in Escherichia coli in the 96-well format; however, very few researchers have shown whole-cell CO difference spectra with yeast cells using 1-cm path length.

Expression of recombinant human flavin monooxygenase and moclobemide-N-oxide synthesis on multi-mg scale.

A panel of human flavin monooxygenases were heterologously expressed in E. coli to obtain ready-to-use biocatalysts for the in vitro preparation of human drug metabolites. Moclobemide-N-oxide (65 mg) was the first high-priced metabolite prepared with recombinant hFMO3 on the multi-milligram scale.

Creation of cavitation activity in a microfluidic device through acoustically driven capillary waves.

We present a study on achieving intense acoustic cavitation generated by ultrasonic vibrations in polydimethylsiloxane (PDMS) based microfluidic devices. The substrate to which the PDMS is bonded was forced into oscillation with a simple piezoelectric transducer attached at 5 mm from the device to a microscopic glass slide. The transducer was operated at 100 kHz with driving voltages ranging between 20 V and 230 V.

Genome-scale metabolic reconstruction and in silico analysis of methylotrophic yeast Pichia pastoris for strain improvement.

Background: Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized.

Co-expression of Skp and FkpA chaperones improves cell viability and alters the global expression of stress response genes during scFvD1.3 production.

Background: The overexpression of scFv antibody fragments in the periplasmic space of Escherichia coli frequently results in extensive protein misfolding and loss of cell viability. Although protein folding factors such as Skp and FkpA are often exploited to restore the solubility and functionality of recombinant protein products, their exact impact on cellular metabolism during periplasmic antibody fragment expression is not clearly understood. In this study, we expressed the scFvD1.

An integrative expression vector for Actinosynnema pretiosum.

Background: The Actinomycete Actinosynnema pretiosum ssp. auranticum has commercial importance due to its production of ansamitocin P-3 (AP-3), a potent antitumor agent. One way to increase AP-3 production would be to constitutively express selected genes so as to relieve bottlenecks in the biosynthetic pathway; however, an integrative expression vector for A.

Induction by hypoxia of heterologous-protein production with the KlPDC1 promoter in yeasts.

The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the KlPDC1 promoter to direct heterologous gene expression in yeast.