Reliable high-throughput screening with by limiting yeast cell death phenomena

Abstract

Comparative screening of gene expression libraries employing the potent industrial host Pichia pastoris for improving recombinant eukaryotic enzymes by protein engineering was an unsolved task. We simplified the protocol for protein expression by P. pastoris and scaled it down to 0.5-ml cultures. Optimising standard growth conditions and procedures, programmed cell death and necrosis of P. pastoris in microscale cultures were diminished. Uniform cell growth in 96-deep-well plates now allows for high-throughput protein expression and screening for improved enzyme variants. Furthermore, the change from one host for protein engineering to another host for enzyme production becomes dispensable, and this accelerates the protein breeding cycles and makes predictions for large-scale production more accurate.

Characterization of the Two Neurospora crassa Cellobiose Dehydrogenases and Their Connection to Oxidative Cellulose Degradation

“…The cultivation and expression of both CDHs were done with 96-deep-well plates according to methods described previously by Weis et al (38), with small modifications. Cells were grown in 250 l BMD1 (13.4 g liter After 70, 82, and 108 h of incubation, 50 l BMM10 (BMM2 with 5% methanol) was added to maintain inducing conditions.…”

Constitutive expression of Botrytis aclada laccase in Pichia pastoris

“…Expression strategies for high-throughput screenings based on 96-well deep well plates are established for expression in P. pastoris under control of the AOX1 promoter. 12 They rely on methanol feeding, which is labor-intensive and limits the amount of plates that can be handled by a single person. Furthermore, due to a batch phase before induction, the cultivation needs a minimum of four to six days until sufficient amounts of recombinant enzyme are secreted for a screening assay.…”

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

“…Only by knowing the number of (vital) cells that express the target molecule at the highest possible rate can the number of such cells within the population be maximized and the proportion of dead cells undergoing lysis be kept to a minimum. Furthermore, lysed cells not only are "unproductive" but also may release undesirable host cell proteins and/or proteases causing product degradation (32,33,58).Bacteria and yeasts can be readily differentiated by staining with appropriate fluorochromes and using epifluorescent microscopy. The combination of fluorescent staining and rapid counting using flow cytometry marked the advent of process monitoring, enabling the physiological state of both subpopulations and individual cells to be quantified and interpreted (22,23,37,55).…”

“…Only by knowing the number of (vital) cells that express the target molecule at the highest possible rate can the number of such cells within the population be maximized and the proportion of dead cells undergoing lysis be kept to a minimum. Furthermore, lysed cells not only are "unproductive" but also may release undesirable host cell proteins and/or proteases causing product degradation (32,33,58).…”