Secretome-based screening of fusion partners and their application in recombinant protein secretion in Saccharomyces cerevisiae

Bae, Jung-Hoon; Yun, Sung-Ho; Kim, Mi Jin; Kim, Hyun‐Jin; Sung, Bong Hyun; Kim, Kyung Sik; Sohn, Jung‐Hoon

doi:10.1007/s00253-021-11750-9

Cited by 10 publications

(11 citation statements)

References 29 publications

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“…We denote these parts as 3a′ and the coding sequence parts as 3b′ to highlight this distinction. The choice of TGCT overhang necessitated changing glycine to alanine at the −1 position for two SP pre-sequences and an amino acid change at the −2 position for one of these (Table 1 18 ). For the MFαpp8 sequence, a Glu-Ala to Asp-Ala change was made for similar (A) The indicated types of amino-terminal secretion promoting sequences were combined with a protein of interest and a carboxylterminal 3xFLAG-6xHistidine tag to assess their secretion efficiency or an HA epitope-tagged anchor protein for surface display experiments.…”

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.

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“…While S. cerevisiae does not secrete heterologous proteins as efficiently as other yeast species (e.g., Komagataella phaffii ), it has been reported to secrete several industrially significant proteins at relatively high levels 22 . Various strategies, such as testing diverse secretion signals and rational engineering of the secretion pathway, have led to strains secreting as much as 5 g/L heterologous protein in recombinant S. cerevisiae 40 . The P.tr.xln43 was also fused with S. cerevisiae SED1 , which encodes a stress‐induced GPI‐cell wall glycoprotein, and they were incorporated into the xylose utilizing strain to confer cell‐associated xylosidase activity.…”

Section: Resultsmentioning

confidence: 99%

“…22 Various strategies, such as testing diverse secretion signals and rational engineering of the secretion pathway, have led to strains secreting as much as 5 g/L heterologous protein in recombinant S. cerevisiae. 40 The P.tr.xln43 was also fused with S. cerevisiae SED1, which encodes a stress-induced GPI-cell wall glycoprotein, and they were incorporated into the xylose utilizing strain to confer cell-associated xylosidase activity. These strains were both subsequently engineered using CRISPR-Cas9 to integrate the xylanase-encoding gene T.r.xyn2 into an intergenic region on chromosome 11.…”

Section: Strain Constructionmentioning

confidence: 99%

Surface tethered xylosidase activity improved xylan conversion in engineered strains of Saccharomyces cerevisiae

Kruger

Haan

2022

J of Chemical Tech & Biotech

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BACKGROUND Second‐generation biofuel production strategies require utilization of the largest possible fraction of the available sugars in renewable biomass. Xylose can make up as much as 35% of plant dry cell weight (DCW), primarily in the form of the hemicellulose polymer xylan. Xylo‐oligosaccharides (XOS) are breakdown products of xylan released during pre‐treatment and hydrolysis that may inhibit the fermentation process. To enable growth on xylan and the removal of inhibitory XOS, CRISPR‐Cas9 was used to confer xylanase and GH43 xylosidase activity to xylose‐assimilating Saccharomyces cerevisiae strains. Xylosidase activity was engineered to be cell free or tethered to the yeast cell surface. RESULTS Gene‐integration and expression of both xylanolytic enzyme‐encoding genes was successful. Secretion yielded higher overall xylosidase activity when strains were cultivated on glucose, but cell‐associated activity was higher when strains were cultivated in the presence of xylose. Growth trials on both xylan and XOS showed that a combination of both enzymes, along with cell‐associated xylosidase production, resulted in the best growth on both substrates. The increased growth on xylan also translated to substantial improvements in ethanol production from polymeric xylan as sole carbon source. CONCLUSION Increased enzyme production, growth capabilities, and hemicellulosic substrate conversion due to cell‐tethered activity over secreted activity were shown for the first time. The use of a GH43 xylosidase to help avoid problematic transglycosylation for this purpose is also novel. The development of S. cerevisiae strains capable of xylan utilization and fermentation brings the industry a step closer to the ideal of utilizing the full range of sugars in biomass feedstocks for large scale ethanol production. © 2022 Society of Chemical Industry (SCI).

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“…Currently, more than 50 heterologous proteins have been successfully expressed in K. marxianus . The highest yield of heterologous proteins in K. marxianus was 12.2 g/l 6 , which is higher than that of S. cerevisiae (5 g/l) 30 , but is significantly lagging behind that of Pichia pastoris (22 g/l) 31 . This indicates that there is still significant room to improve the yield of heterologous proteins in K. marxianus .…”

Section: Introductionmentioning

confidence: 92%

Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus

Wu,

Mo,

Tian

et al. 2024

mLife

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Kluyveromyces marxianus is a food‐safe yeast with great potential for producing heterologous proteins. Improving the yield in K. marxianus remains a challenge and incorporating large‐scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K. marxianus. These modules contained up to seven genes with a maximum size of 15 kb. KmYACs carried telomeres either from K. marxianus or Tetrahymena. KmYACs were transferred successfully into K. marxianus and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high‐density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l, the highest reported level to date in K. marxianus. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis, enhanced flux entering the tricarboxylic acid cycle, and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories.

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Secretome-based screening of fusion partners and their application in recombinant protein secretion in Saccharomyces cerevisiae

Cited by 10 publications

References 29 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

Surface tethered xylosidase activity improved xylan conversion in engineered strains of Saccharomyces cerevisiae

Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus

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