Systematic Perturbation of Cultures of K. Phaffii Show Gene Signatures Associated with 1 Specific Productivity of Recombinant Proteins
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ABSTRACT:
ORGANISM(S): Komagataella phaffii
PROVIDER: GSE302183 | GEO | 2026/03/11
REPOSITORIES: GEO
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Project description:Demand for recombinant proteins is rapidly growing, driven by their use as 16 biotherapeutics, vaccine components, industrial enzymes, and food ingredients. The growing market requires novel strategies for increasing specific productivity (qp) of cellular hosts. Systems-level frameworks have been used to improve productivity, but have had difficulty relating complex cellular pathways with protein expression. Here, we demonstrate a method for mapping relationships between gene expression signatures and complex phenotypes related to recombinant protein productivity. Our approach induces systematic perturbations in cultures of K. Phaffii using varied co-feeds of carbon sources. The different carbon sources significantly impacted cell growth, specific productivity, and transcriptional states. With these data, we identified scale-independent metagenes that explained significant transcriptomic variance and strongly associated with complex cellular phenotypes, including qp and response to methanol induction for both IgG1 and VHH. We used these results to identify and knockout 31 novel gene targets whose expression inversely correlated with productivity. Ten of these genes improved productivity of IgG1 by up to 3x, and 17 genes increased productivity of VHH by up to 1.7x without impacting cell growth. This study identified ten groups of carbon co-feeds which induce similar transcriptomic states. We systematically perturbated cultures of K. phaffii using these co-feeds and identified phenotype-associated metagenes. Using the productivity-correlated metagene, we identified 10-17 genes which improved recombinant protein productivity when knocked out. This framework for relating gene signatures to complex cellular phenotypes could provide useful metrics for assessing 37 production processes and identifying new targets for cellular engineering.
2026-03-11 | GSE302181 | GEO
Project description:Demand for recombinant proteins is rapidly growing, driven by their use as 16 biotherapeutics, vaccine components, industrial enzymes, and food ingredients. The growing market requires novel strategies for increasing specific productivity (qp) of cellular hosts. Systems-level frameworks have been used to improve productivity, but have had difficulty relating complex cellular pathways with protein expression. Here, we demonstrate a method for mapping relationships between gene expression signatures and complex phenotypes related to recombinant protein productivity. Our approach induces systematic perturbations in cultures of K. Phaffii using varied co-feeds of carbon sources. The different carbon sources significantly impacted cell growth, specific productivity, and transcriptional states. With these data, we identified scale-independent metagenes that explained significant transcriptomic variance and strongly associated with complex cellular phenotypes, including qp and response to methanol induction for both IgG1 and VHH. We used these results to identify and knockout 31 novel gene targets whose expression inversely correlated with productivity. Ten of these genes improved productivity of IgG1 by up to 3x, and 17 genes increased productivity of VHH by up to 1.7x without impacting cell growth. This study identified ten groups of carbon co-feeds which induce similar transcriptomic states. We systematically perturbated cultures of K. phaffii using these co-feeds and identified phenotype-associated metagenes. Using the productivity-correlated metagene, we identified 10-17 genes which improved recombinant protein productivity when knocked out. This framework for relating gene signatures to complex cellular phenotypes could provide useful metrics for assessing 37 production processes and identifying new targets for cellular engineering.
2026-03-11 | GSE302182 | GEO
Project description:Systematic Perturbation of Cultures of K. Phaffii Show Gene Signatures Associated with 1 Specific Productivity of Recombinant Proteins
| PRJNA1289144 | ENA
Project description:Systematic Perturbation of Cultures of K. Phaffii Show Gene Signatures Associated with 1 Specific Productivity of Recombinant Proteins [K. phaffii S-1076 RNA-seq]
| PRJNA1289153 | ENA
Project description:Systematic Perturbation of Cultures of K. Phaffii Show Gene Signatures Associated with 1 Specific Productivity of Recombinant Proteins [K. phaffii S-953 RNA-seq]
| PRJNA1289154 | ENA
Project description:BackgroundKomagataella phaffii is a commonly used alternative host for manufacturing therapeutic proteins, in part because of its ability to secrete recombinant proteins into the extracellular space. Incorrect processing of secreted proteins by cells can, however, cause non-functional product-related variants, which are expensive to remove in purification and lower overall process yields. The secretion signal peptide, attached to the N-terminus of the recombinant protein, is a major determinant of the quality of the protein sequence and yield. In K. phaffii, the signal peptide from the Saccharomyces cerevisiae alpha mating factor often yields the highest secreted titer of recombinant proteins, but the quality of secreted protein can vary highly.ResultsWe determined that an aggregated product-related variant of the SARS-CoV-2 receptor binding domain is caused by N-terminal extension from incomplete cleavage of the signal peptide. We eliminated this variant and improved secreted protein titer up to 76% by extension of the N-terminus with a short, functional peptide moiety or with the EAEA residues from the native signal peptide. We then applied this strategy to three other recombinant subunit vaccine antigens and observed consistent elimination of the same aggregated product-related variant. Finally, we demonstrated that this benefit in quality and secreted titer can be achieved with addition of a single amino acid to the N-terminus of the recombinant protein.ConclusionsOur observations suggest that steric hindrance of proteases in the Golgi that cleave the signal peptide can cause unwanted N-terminal extension and related product variants. We demonstrated that this phenomenon occurs for multiple recombinant proteins, and can be addressed by minimal modification of the N-terminus to improve steric accessibility. This strategy may enable consistent secretion of a broad range of recombinant proteins with the highly productive alpha mating factor secretion signal peptide.
| S-EPMC9444097 | biostudies-literature
Project description:High-level expression and secretion of heterologous proteins in yeast cause an increased energy demand, which may result in altered metabolic flux distributions. Moreover, recombinant protein overproduction often results in endoplasmic reticulum (ER) stress and oxidative stress, causing deviations from the optimal NAD(P)H regeneration balance. In this context, overexpression of genes encoding enzymes catalyzing endogenous NADPH-producing reactions, such as the oxidative branch of the pentose phosphate pathway, has been previously shown to improve protein production in Pichia pastoris (syn. Komagataella spp.). In this study, we evaluate the overexpression of the Saccharomyces cerevisiaePOS5-encoded NADH kinase in a recombinant P. pastoris strain as an alternative approach to overcome such redox constraints. Specifically, POS5 was cooverexpressed in a strain secreting an antibody fragment, either by directing Pos5 to the cytosol or to the mitochondria. The physiology of the resulting strains was evaluated in continuous cultivations with glycerol or glucose as the sole carbon source, as well as under hypoxia (on glucose). Cytosolic targeting of Pos5 NADH kinase resulted in lower biomass-substrate yields but allowed for a 2-fold increase in product specific productivity. In contrast, Pos5 NADH kinase targeting to the mitochondria did not affect growth physiology and recombinant protein production significantly. Growth physiological parameters were in silico evaluated using the recent upgraded version (v3.0) of the P. pastoris consensus genome-scale metabolic model iMT1026, providing insights on the impact of POS5 overexpression on metabolic flux distributions.IMPORTANCE Recombinant protein overproduction often results in oxidative stress, causing deviations from the optimal redox cofactor regeneration balance. This becomes one of the limiting factors in obtaining high levels of heterologous protein production. Overexpression of redox-affecting enzymes has been explored in other organisms, such as Saccharomyces cerevisiae, as a means to fine tune the cofactor regeneration balance in order to obtain higher protein titers. In the present work, this strategy is explored in P. pastoris In particular, one NADH kinase enzyme from S. cerevisiae (Pos5) is used, either in the cytosol or in mitochondria of P. pastoris, and its impact on the production of a model protein (antibody fragment) is evaluated. A significant improvement in the production of the model protein is observed when the kinase is directed to the cytosol. These results are significant in the field of heterologous protein production in general and in particular in the development of improved metabolic engineering strategies for P. pastoris.
| S-EPMC7054088 | biostudies-literature
Project description:The yeast Komagataella phaffii (syn. Pichia pastoris) is a highly effective and well-established host for the production of recombinant proteins. The redox balance of its secretory pathway, which is multi-organelle dependent, is of high importance for producing secretory proteins. Redox imbalance and oxidative stress can significantly influence protein folding and secretion. Glutathione serves as the main redox buffer of the cell and cellular redox conditions can be assessed through the status of the glutathione redox couple (GSH-GSSG). Previous research often focused on the redox potential of the endoplasmic reticulum (ER), where oxidative protein folding and disulphide bond formation occur. In this study, in vivo measurements of the glutathione redox potential were extended to different subcellular compartments by targeting genetically encoded redox sensitive fluorescent proteins (roGFPs) to the cytosol, ER, mitochondria and peroxisomes. Using these biosensors, the impact of oxygen availability on the redox potentials of the different organelles was investigated in non-producing and producing K. phaffii strains in glucose-limited chemostat cultures. It was found that the transition from normoxic to hypoxic conditions affected the redox potential of all investigated organelles, while the exposure to hyperoxic conditions did not impact them. Also, as reported previously, hypoxic conditions led to increased recombinant protein secretion. Finally, transcriptome and proteome analyses provided novel insights into the short-term response of the cells from normoxic to hypoxic conditions.
| S-EPMC11816699 | biostudies-literature
Project description:BackgroundThe β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly.ResultsFirstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).ConclusionThis study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.
| S-EPMC11452983 | biostudies-literature
Project description:Recent advancements in functional genomics have provided an unprecedented ability to measure diverse molecular modalities, but learning causal regulatory relationships from observational data remains challenging. Here, we leverage pooled genetic screens and single cell sequencing (i.e. Perturb-seq) to systematically identify the targets of signaling regulators in diverse biological contexts. We demonstrate how Perturb-seq is compatible with recent and commercially available advances in combinatorial indexing and next-generation sequencing, and perform more than 1,500 perturbations split across six cell lines and five biological signaling contexts. We introduce an improved computational framework (Mixscale) to address cellular variation in perturbation efficiency, alongside optimized statistical methods to learn differentially expressed gene lists and conserved molecular signatures. Finally, we demonstrate how our Perturb-seq derived gene lists can be used to precisely infer changes in signaling pathway activation for in-vivo and in-situ samples. Our work enhances our understanding of signaling regulators and their targets, and lays a computational framework towards the data-driven inference of an ‘atlas’ of perturbation signatures.
2024-11-04 | GSE281048 | GEO