Comprehensive Analysis of Signal Peptides in <i>Saccharomyces Cerevisiae</i> Reveals Features for Efficient Secretion

Xue, Songlyu; Liu, Xiufang; Pan, Yuyang; Xiao, Chufan; Feng, Yunzi; Zheng, Lin; Zhao, Ming; Huang, Mingtao

doi:10.1002/advs.202300302

Cited by 4 publications

(3 citation statements)

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“…With this in mind, we considered the sequence of yeast SP pre-sequences, which most frequently have an alanine residue at the −1 position relative to the signal peptidase cleavage site as part of an Ala-X-Ala or Val-X-Ala motif. 17 To join SP pre-sequences ending with Ala to coding sequences, without introducing any extra amino acids, we chose a TGCT overhang for DNA assembly, in which the GCT codes for Ala (Figure 1B). A TGCT overhang is predicted to have high fidelity when used as part of the YTK toolkit when analyzed using the NEBridge GetSet tool (Figure S1).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display in Saccharomyces cerevisiae

O’Riordan,

Jurić,

O’Neill

et al. 2024

ACS Synth. Biol.

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Saccharomyces cerevisiae is an attractive host for the expression of secreted proteins in a biotechnology context. Unfortunately, many heterologous proteins fail to enter, or efficiently progress through, the secretory pathway, resulting in poor yields. Similarly, yeast surface display has become a widely used technique in protein engineering but achieving sufficient levels of surface expression of recombinant proteins is often challenging. Signal peptides (SPs) and translational fusion partners (TFPs) can be used to direct heterologous proteins through the yeast secretory pathway, however, selection of the optimal secretion promoting sequence is largely a process of trial and error. The yeast modular cloning (MoClo) toolkit utilizes type IIS restriction enzymes to facilitate an efficient assembly of expression vectors from standardized parts. We have expanded this toolkit to enable the efficient incorporation of a panel of 16 well-characterized SPs and TFPs and five surface display anchor proteins into S. cerevisiae expression cassettes. The secretion promoting signals are validated by using five different proteins of interest. Comparison of intracellular and secreted protein levels reveals the optimal secretion promoting sequence for each individual protein. Large, protein of interestspecific variations in secretion efficiency are observed. SP sequences are also used with the five surface display anchors, and the combination of SP and anchor protein proves critical for efficient surface display. These observations highlight the value of the described panel of MoClo compatible parts to allow facile screening of SPs and TFPs and anchor proteins for optimal secretion and/ or surface display of a given protein of interest in S. cerevisiae.

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Section: ■ Results and Discussionmentioning

confidence: 99%

A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display in Saccharomyces cerevisiae

O’Riordan,

Jurić,

O’Neill

et al. 2024

ACS Synth. Biol.

View full text Add to dashboard Cite

show abstract

“…Maintaining the native amino- and/or carboxyl-terminal amino acid sequence can be critical to the functionality of recombinantly expressed proteins and may also be important for regulatory reasons. With this in mind we considered the sequence of yeast SP pre-sequences, which most frequently have an alanine residue at the -1 position relative to the signal peptidase cleavage site as part of an Ala-X-Ala or Val-X-Ala motif (17) . To join SP pre-sequences ending with Ala to coding sequences, without introducing any extra amino acids, we chose an TGCT overhang for DNA assembly in which the GCT codes for Ala (Fig 1B).…”

Section: Resultsmentioning

confidence: 99%

A yeast modular cloning (MoClo) toolkit expansion for optimization of heterologous protein secretion and surface display inSaccharomyces cerevisiae

O’Riordan,

Jurić,

O’Neill

et al. 2023

Preprint

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Saccharomyces cerevisiaeis an attractive host for expression of secreted proteins in a biotechnology context. Unfortunately, many heterologous proteins fail to enter, or efficiently progress through, the secretory pathway, resulting in poor yields. Similarly, yeast surface display has become a widely used technique in protein engineering but achieving sufficient levels of surface expression of recombinant proteins is often challenging. Signal peptides (SPs) and translational fusion partners (TFPs) can be used to direct heterologous proteins through the yeast secretory pathway, however, selection of the optimal secretion promoting sequence is largely a process of trial and error. The yeast modular cloning (MoClo) toolkit utilises Type IIS restriction enzymes to facilitate efficient assembly of expression vectors from standardized parts. We have expanded this toolkit to enable the efficient incorporation of a panel of sixteen well-characterized SPs and TFPs and five surface display anchor proteins intoS. cerevisiaeexpression cassettes. The secretion promoting signals were validated using five different proteins of interest. Comparison of intracellular and secreted protein levels revealed the optimal secretion promoting sequence for each individual protein. Large, protein of interest-specific variations in secretion efficiency were observed. SP sequences were also used with the five surface display anchors and the combination of SP and anchor protein proved critical for efficient surface display. These observations highlight the value of the described panel of MoClo compatible parts to allow facile screening of SPs, TFPs and anchor proteins for optimal secretion and/or surface display of a given protein of interest inS. cerevisiae.GRAPHICAL ABSTRACT

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“…[15] Gene deletion and gene integration were performed in strain AMP003 using the CRISPR/Cas9 system. [60,61] The gRNA sequence was selected from the CRISPOR website (http://crispor.tefor.net/). The DNA fragments were amplified using PCR with appropriate primers.…”

Section: Strains and Plasmids Constructionmentioning

confidence: 99%

ER stress‐induced transcriptional response reveals tolerance genes in yeast

Xie,

Xiao,

Pan

et al. 2024

Biotechnology Journal

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Saccharomyces cerevisiae is important for protein secretion studies, yet the complexities of protein synthesis and secretion under endoplasmic reticulum (ER) stress conditions remain not fully understood. ER stress, triggered by alterations in the ER protein folding environment, poses substantial challenges to cells, especially during heterologous protein production. In this study, we used RNA‐seq to analyze the transcriptional responses of yeast strains to ER stress induced by reagents such as tunicamycin (Tm) or dithiothreitol (DTT). Our gene expression analysis revealed several crucial genes, such as HMO1 and BIO5, that are involved in ER‐stress tolerance. Through metabolic engineering, the best engineered strain R23 with HMO1 overexpression and BIO5 deletion, showed enhanced ER stress tolerance and improved protein folding efficiency, leading to a 2.14‐fold increase in α‐amylase production under Tm treatment and a 2.04‐fold increase in cell density under DTT treatment. Our findings contribute to the understanding of cellular responses to ER stress and provide a basis for further investigations into the mechanisms of ER stress at the cellular level.

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Comprehensive Analysis of Signal Peptides in Saccharomyces Cerevisiae Reveals Features for Efficient Secretion

Cited by 4 publications

References 0 publications

A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display in Saccharomyces cerevisiae

A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display in Saccharomyces cerevisiae

A yeast modular cloning (MoClo) toolkit expansion for optimization of heterologous protein secretion and surface display inSaccharomyces cerevisiae

ER stress‐induced transcriptional response reveals tolerance genes in yeast

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