Enhancing co-translational folding of heterologous protein by deleting non-essential ribosomal proteins in Pichia pastoris

Liao, Xihao; Zhao, Jing; Liang, Shuli; Jin, Jingjie; Cheng, Li; Xiao, Ruiming; Li, Lu; Guo, Meijin; Zhang, Gong; Lin, Ying

doi:10.1186/s13068-019-1377-z

Cited by 10 publications

(4 citation statements)

References 64 publications

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“…Translational capacity or the availability of amino acids may represent a general limitation for yeasts during recombinant protein production, therefore. 23–25 This hypothesis is corroborated by another engineered strain with an upregulated translation factor that exhibited with improved secreted productivity by expanding the cellular capacity for translation. 26 Strategies to further redirect translational capacity towards the recombinant product of interest warrant further investigation.…”

Section: Discussionmentioning

confidence: 79%

CRISPR-Cas9 knockout screen informs efficient reduction of theKomagataella phaffiisecretome

Dalvie,

Lorgeree,

Yang

et al. 2024

Preprint

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The yeast Komagataella phaffii is widely used for manufacturing recombinant proteins, but secreted titers of recombinant proteins could be improved by genetic engineering. In this study, we hypothesized that cellular resources could be redirected from production of endogenous proteins to production of recombinant proteins be deletion of unneeded endogenous proteins. We identified a set of endogenous secreted proteins in K. phaffii and attempted to disrupt these genes, but our efforts were hindered by limited annotation of genes, especially essential ones. This is a common problem for genetic engineering of non-model organisms. To predict essential genes, therefore, we designed, transformed, and sequenced a pooled library of guide RNAs for CRISPR-Cas9-mediated knockout of all endogenous secreted proteins. We then used predicted gene essentiality to guide iterative disruptions of up to 11 non-essential genes. Engineered strains exhibited a ~20x increase in the production of human serum albumin and a twofold increase in the production of a monoclonal antibody. The pooled library of secretome-targeted guides for CRISPR-Cas9 and knowledge of gene essentiality reported here will facilitate future efforts to engineer K. phaffii for production of other recombinant proteins and enzymes.

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Section: Discussionmentioning

confidence: 79%

CRISPR-Cas9 knockout screen informs efficient reduction of theKomagataella phaffiisecretome

Dalvie,

Lorgeree,

Yang

et al. 2024

Preprint

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“… 2011 , Liao et al . 2019 ). As the biosynthesis of ribosomal proteins has a connection with heterologous protein production, this could represent another potential reason for the effect of the TOM1 deletion.…”

Section: Resultsmentioning

confidence: 99%

CRISPR/Cas9-mediated point mutations improve α-amylase secretion in Saccharomyces cerevisiae

Wang

Chen

et al. 2022

FEMS Yeast Research

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The rapid expansion of the application of pharmaceutical proteins and industrial enzymes requires robust microbial workhorses for high protein production. The budding yeast Saccharomyces cerevisiae is an attractive cell factory due to its ability to perform eukaryotic post-translational modifications and to secrete proteins. Many strategies have been used to engineer yeast platform strains for higher protein secretion capacity. Herein, we investigated a line of strains that have previously been selected after UV random mutagenesis for improved α-amylase secretion. A total of 42 amino acid altering point mutations identified in this strain line were re-introduced into the parental strain AAC to study their individual effects on protein secretion. These point mutations included missense mutations (amino acid substitution), nonsense mutations (stop codon generation) and frameshift mutations. For comparison, single gene deletions for the corresponding target genes were also performed in this study. Eleven point mutations and seven gene deletions were found to effectively improve α-amylase secretion. These targets were involved in several bioprocesses, including cellular stresses, protein degradation, transportation, mRNA processing and export, DNA replication and repair, which indicates that the improved protein secretion capacity in the evolved strains is the result of the interaction of multiple intracellular processes. Our findings will contribute to the construction of novel cell factories for recombinant protein secretion.

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“…Yeast ribosomal proteins play important roles in the biogenesis and function of the ribosome ( Aw et al, 2017 ). Deletion of a particular ribosome protein can delay or impair the subunit assembly, indicating that the decelerated elongation stage of translation might promote the co-translational folding rate of heterologous proteins ( Liao et al, 2019 ). In P. pastoris , overexpression of xylanase A (a foreign protein) can lead to a significant down-regulation of numerous ribosomal proteins, resulting in decelerated translation elongation and enhanced folding efficiency for xylanase A ( Navone et al, 2021b ).…”

Section: Discussionmentioning

confidence: 99%

“…In P. pastoris , overexpression of xylanase A (a foreign protein) can lead to a significant down-regulation of numerous ribosomal proteins, resulting in decelerated translation elongation and enhanced folding efficiency for xylanase A ( Navone et al, 2021b ). Meanwhile, studies have shown that knocking out the C4QXU7 gene in P. pastoris does not affect its growth, but can lead to a significant increase in the secretion levels of exogenous proteins (e.g., Pfu and Phytase), indicating that the decelerated elongation rate caused by the loss of C4QXU7 might promote the co-translational folding rate of heterologous proteins, increasing the expression of Pfu and Phytase ( Liao et al, 2019 ). Together with our data on protein expression in P. pastoris , these observations could collectively indicate that knocking out the C4QXU7 gene may promote the expression of foreign proteins that are not easily folded in P. pastoris .…”

Section: Discussionmentioning

confidence: 99%

Comparative transcriptomic analysis-based identification of the regulation of foreign proteins with different stabilities expressed in Pichia pastoris

Niu

Cui

et al. 2022

Front. Microbiol.

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IntroductionThe industrial yeast Pichia pastoris is widely used as a cell factory to produce proteins, chemicals and advanced biofuels. We have previously constructed P. pastoris strains that overexpress protein disulfide isomerase (PDI), which is a kind of molecular chaperone that can improve the expression of an exogenous protein when they are co-expressed. Chicken cystatin (cC) is a highly thermostable cysteine protease inhibitor and a homologous protein of human cystatin C (HCC). Wild-type cC and the two mutants, I66Q and ΔW (a truncated cC lacking the á-helix 2) represent proteins with different degrees of stability.MethodsWild-type cC, I66Q and ΔW were each overexpressed in P. pastoris without and with the coexpression of PDI and their extracellular levels were determined and compared. Transcriptomic profiling was performed to compare the changes in the main signaling pathways and cell components (other than endoplasmic reticulum quality control system represented by molecular chaperones) in P. pastoris in response to intracellular folding stress caused by the expression of exogenous proteins with different stabilities. Finally, hub genes hunting was also performed.Results and discussionThe coexpression of PDI was able to increase the extracellular levels of both wild-type cC and the two mutants, indicating that overexpression of PDI could prevent the misfolding of unstable proteins or promote the degradation of the misfolded proteins to some extent. For P. pastoris cells that expressed the I66Q or ΔW mutant, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses of the common DEGs in these cells revealed a significant upregulation of the genes involved in protein processing, but a significant downregulation of the genes enriched in the Ribosome, TCA and Glycolysis/Gluconeogenesis pathways. Hub genes hunting indicated that the most downregulated ribosome protein, C4QXU7 in this case, might be an important target protein that could be manipulated to increase the expression of foreign proteins, especially proteins with a certain degree of instability.ConclusionThese findings should shed new light on our understanding of the regulatory mechanism in yeast cells that responds to intracellular folding stress, providing valuable information for the development of a convenient platform that could improve the efficiency of heterologous protein expression in P. pastoris.

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Enhancing co-translational folding of heterologous protein by deleting non-essential ribosomal proteins in Pichia pastoris

Cited by 10 publications

References 64 publications

CRISPR-Cas9 knockout screen informs efficient reduction of theKomagataella phaffiisecretome

CRISPR-Cas9 knockout screen informs efficient reduction of theKomagataella phaffiisecretome

CRISPR/Cas9-mediated point mutations improve α-amylase secretion in Saccharomyces cerevisiae

Comparative transcriptomic analysis-based identification of the regulation of foreign proteins with different stabilities expressed in Pichia pastoris

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