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

Generation of diploid <it>Pichia pastoris</it> strains by mating and their application for recombinant protein production

Abstract Background Yeast mating provides an efficient means for strain and library construction. However, biotechnological applications of mating in the methylotrophic yeast Pichia pastoris have been hampered because of concerns about strain stability of P. pastoris diploids. The aim of the study reported here is to investigate heterologous protein expression in diploid P. pastoris strains and to evaluate diploid strain stability using high cell density fermentation processes. Results By using a monoclonal antibody as a target protein, we demonstrate that recombinant protein production in both wild-type and glycoengineered P. pastoris diploids is stable and efficient during a nutrient rich shake flask cultivation. When diploid strains were cultivated under bioreactor conditions, sporulation was observed. Nevertheless, both wild-type and glycoengineered P. pastoris diploids showed robust productivity and secreted recombinant antibody of high quality. Specifically, the yeast culture maintained a diploid state for 240 h post-induction phase while protein titer and N-linked glycosylation profiles were comparable to that of a haploid strain expressing the same antibody. As an application of mating, we also constructed an antibody display library and used mating to generate novel full-length antibody sequences. Conclusions To the best of our knowledge, this study reports for the first time a comprehensive characterization of recombinant protein expression and fermentation using diploid P. pastoris strains. Data presented here support the use of mating for various applications including strain consolidation, variable-region glycosylation antibody display library, and process optimization.</p

Effects of the <i>PMT2</i> (F664S) mutation on <i>O-</i>mannosylglycan site occupancy and product titer.

<p><i>O-</i>mannosylglycan site occupancy (A and C) and product titers (B and D) were determined for mAbs purified from <i>PMT2</i> wild type or pmt2 (F664S) mutant strains. Panels A and B displayed results for mAb#1 expressed with 0 or 8 µM PMTi-4 inhibitor; and panels C and D showed resulted for mAb#2 produced in the presence of 0, 8, or 40 µM of PMTi-4. Results are shown as average ± stdev derived from at least two independent experiments.</p

Segregation patterns of the PMTi-resistant phenotype, the F664S mutation, and other genetic markers.

<p>Segregation patterns of the PMTi-resistant phenotype, the F664S mutation, and other genetic markers.</p

Identification of a point mutation within a highly conserved region of PpPmt2p.

<p>Sequence alignments of the predicted cytosolic loop #6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062229#pone.0062229-Girrbach2" target="_blank">[11]</a>, and the sequencing traces displaying the T to C mutation identified in the PMTi-resistant mutant (y17156). Amino acid residues identical in all PMT sequences are highlighted in yellow, residues identical in more than half of the sequences are highlighted in blue, and conserved residues are highlighted in green. The dashed down arrow indicates the T to C nucleotide exchange caused by the UV-mutagenesis, and the solid up arrow pointed to the position of the resulting F to S amino acid substitution in the protein sequence of the PpPmt2p enzyme.</p

Construction of PMT2 (F664S) mutant by gene targeting and replacement.

<p>Plasmid map of pGLY5931 and schematic representation of the DNA construct used to replace the endogenous <i>PMT2</i> ORF with the F664S mutant version. The “*” indicated the approximate location of the F664S mutation within the ORF.</p

Growth inhibitory effects of PMTi-3 and PMTi-4.

<p>Growth inhibitory curves (A, B) and serial dilution spot assay results (C) are shown. The percentages of growth inhibition by the PMTi-3 (A) and PMTi-4 (B) are plotted and curve-fitted using SigmaPlot. The values displayed in the figure were averages from at least 2 independent experiments. The inhibition curves for the pmt4Δ strain (y19376) and the wild type strain (y8316) are shown as dashed-lines, and the others are displayed as solid-lines. For the serial dilution assay (C), overnight-grown saturated cultures of each strain were serial diluted 1∶10, spotted onto YSD, YSD+PMTi-3, and YSD+PMTi-4 plates, and photographed after 72 hours' of growth at 24°C.</p

Characterization of relationships between intracellular and secreted proteins for single cells of <i>P. pastoris</i>.

<p>Density plots of the relative rates of eGFP secretion by single cells analyzed by microengraving with respect to the relative amount of intracellular eGFP determined by fluorescence microscopy for clones containing (A) one eGFP gene copy under pGAPDH or (B) two eGFP gene copies under pAOX1. Dashed line indicates the limit of detection for secreted eGFP in microengraving (background+2σ). The median amounts of internal eGFP for cells above and below this limit of detection are marked (X) and are significantly different (Mann-Whitney test, p≪0.001 for both pGAPDH and pAOX strains). (C) Density plot of the relative rates of secretion analyzed by microengraving for the glycosylated Fc fragment and eGFP produced simultaneously in single cells at two different loci using pGAPDH. Pearson's correlation coefficient for secretion of these two proteins as shown is 0.79.</p

Effects of altering relative rates of secretion and degradation on modeled distribution of intracellular and secreted protein.

<p>Density plot of the relative rates of protein secretion by single cells against the relative amount of intracellular protein for model data sets under three conditions: 1) where median k_ERAD≥k_sec (purple), 2) where median k_sec≪k_sec in Condition 1 (light pink), and 3) where median k_sec≪k_sec in Condition 1 and median k_ERAD>k_ERAD and/or k_expexp in Condition 1 (dark pink). The median amount of intracellular protein for populations in Conditions 1 and 3 are marked (X). The secretion-inhibited population (light pink) was generated by increasing t_sec to 400 min (standard deviation 40 min) while keeping all other parameters the same as the initial population (purple) derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037915#pone-0037915-g004" target="_blank">Figure 4C</a>. The stress-induced population (dark pink) was generated by reducing k_exp or decreasing t_ERAD to obtain a similar median level of protein in the ER as the original population.</p

Analysis of steady-state distributions of rates of secretion.

<p>(A) Distributions of rates of secretion of eGFP for (Top) a clone with a single copy of eGFP under transcriptional control of pGAPDH, and (Bottom) a clone with two copies of eGFP under transcriptional control of pAOX1. Red squares indicate binned single-cell secretion events following microengraving with each clone. Blue lines show the best fits using Eq. (1). Values for <i>a</i> and <i>b</i> are shown. (B) Relationship between <i>a</i> (burst frequency) and <i>b</i> (burst size) for proteins expressed using either pGAPDH (top) or pAOX1 (bottom) as a function of gene copy number and complexity. Clones secreting eGFP (green), clones secreting aglycosylated Fc fragment (blue) and clones secreting glycosylated Fc fragment (red) are shown for a single gene copy (squares), 2–3 gene copies (triangles) and 4 or more gene copies (circles). Error bars represent S.E.M. for each clone from at least three separate measurements.</p