Komagataella phaffii Erp41 is a protein disulfide isomerase with unprecedented disulfide bond catalyzing activity when coupled to glutathione.

In the methylotrophic yeast Komagataella phaffii, we identified an endoplasmic reticulum-resident protein disulfide isomerase (PDI) family member, Erp41, with a peculiar combination of active site motifs. Like fungal ERp38, it has two thioredoxin-like domains which contain active site motifs (a and a'), followed by an alpha-helical ERp29c C-terminal domain (c domain). However, while the a domain has a typical PDI-like active site motif (CGHC), the a' domain instead has CGYC, a glutaredoxin-like motif which confers to the protein an exceptional affinity for GSH/GSSG. This combination of active site motifs has so far been unreported in PDI-family members. Homology searches revealed ERp41 is present in the genome of some plants, fungal parasites, and a few nonconventional yeasts, among which are Komagataella spp. and Yarrowia lipolytica. These yeasts are both used for the production of secreted recombinant proteins. Here, we analyzed the activity of K. phaffii Erp41. We report that it is nonessential in K. phaffii, and that it can catalyze disulfide bond formation in partnership with the sulfhydryl oxidase Ero1 in vitro with higher turnover rates than the canonical PDI from K. phaffii, Pdi1, but slower activation times. We show how Erp41 has unusually fast glutathione-coupled oxidation activity and relate it to its unusual combination of active sites in its thioredoxin-like domains. We further describe how this determines its unusually efficient catalysis of dithiol oxidation in peptide and protein substrates.

Biochemical analysis of Komagataella phaffii oxidative folding proposes novel regulatory mechanisms of disulfide bond formation in yeast.

Oxidative protein folding in the endoplasmic reticulum (ER) is driven mainly by protein disulfide isomerase PDI and oxidoreductin Ero1. Their activity is tightly regulated and interconnected with the unfolded protein response (UPR). The mechanisms of disulfide bond formation have mainly been studied in human or in the yeast Saccharomyces cerevisiae. Here we analyze the kinetics of disulfide bond formation in the non-conventional yeast Komagataella phaffii, a common host for the production of recombinant secretory proteins. Surprisingly, we found significant differences with both the human and S. cerevisiae systems. Specifically, we report an inactive disulfide linked complex formed by K. phaffii Ero1 and Pdi1, similarly to the human orthologs, but not described in yeast before. Furthermore, we show how the interaction between K. phaffii Pdi1 and Ero1 is unaffected by the introduction of unfolded substrate into the system. This is drastically opposed to the previously observed behavior of the human pathway, suggesting a different regulation of the UPR and/or possibly different interaction mechanics between K. phaffii Pdi1 and Ero1.

A quantitative interpretation of oxidative protein folding activity in Escherichia coli.

BACKGROUND: Escherichia coli is of central interest to biotechnological research and a widely used organism for producing proteins at both lab and industrial scales. However, many proteins remain difficult to produce efficiently in E. coli. This is particularly true for proteins that require post translational modifications such as disulfide bonds. RESULTS: In this study we develop a novel approach for quantitatively investigating the ability of E. coli to produce disulfide bonds in its own proteome. We summarise the existing knowledge of the E. coli disulfide proteome and use this information to investigate the demand on this organism's quantitative oxidative folding apparatus under different growth conditions. Furthermore, we built an ordinary differential equation-based model describing the cells oxidative folding capabilities. We use the model to infer the kinetic parameters required by the cell to achieve the observed oxidative folding requirements. We find that the cellular requirement for disulfide bonded proteins changes significantly between growth conditions. Fast growing cells require most of their oxidative folding capabilities to keep up their proteome while cells growing in chemostats appear limited by their disulfide bond isomerisation capacities. CONCLUSION: This study establishes a novel approach for investigating the oxidative folding capacities of an organism. We show the capabilities and limitations of E. coli for producing disulfide bonds under different growth conditions and predict under what conditions excess capability is available for recombinant protein production.

In-Line Ultrasound-Enhanced Raman Spectroscopy Allows for Highly Sensitive Analysis with Improved Selectivity in Suspensions.

Raman spectroscopy is a nondestructive characterization method offering chemical-specific information. However, the cross-section of inelastically (Raman) scattered light is very low compared to elastically (Rayleigh) scattered light, resulting in weak signal intensities in Raman spectroscopy. Despite providing crucial information in off-line measurements, it usually is not sensitive enough for efficient, in-line process control in conjunction with low particle concentrations. To overcome this limitation, two custom-made 1.4404 stainless-steel prototype add-ons were developed for in-line Raman probes that enable ultrasound particle manipulation and thus concentration of particles in suspensions in the focus of the Raman excitation laser. Depending on size and density differences between particles and the carrier medium, particles are typically caught in the nodal planes of a quasi-standing wave field formed in an acoustic resonator in front of the sensor. Two arrangements were realized with regard to the propagation direction of the ultrasonic wave relative to the propagation direction of the laser. The parallel arrangement improved the limit of detection (LOD) by a factor of ≈30. In addition to increased sensitivity, the perpendicular arrangement offers increased selectivity: modifying the frequency of the ultrasonic wave field allows the liquid or solid phase to be moved into the focus of the Raman laser. The combination of in-line Raman spectroscopy with ultrasound particle manipulation holds promise to push the limits of conventional Raman spectroscopy, hence broadening its field of application to areas where previously Raman spectroscopy has not had sufficient sensitivity for accurate, in-line detection.