Improved protein production in yeast using cell engineering with genes related to a key factor in the unfolded protein response

Lin, Yeping; Feng, Yunzi; Zheng, Lin; Zhao, Ming; Huang, Mingtao

doi:10.1016/j.ymben.2023.04.004

Cited by 19 publications

(6 citation statements)

References 70 publications

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“…Pry1 reached an export index of (99%) when expressed from a low-copy plasmid, which is comparable with that of the two human proteins, indicating that they were indeed secreted efficiently ( Figure 2C ). Except for Pry1 ss -Fabp4, expression of all three proteins from a high-copy number plasmid resulted in reduced secretion efficiency, possibly due to saturation of the translocation and/or ER folding machinery ( Besada-Lombana and Da Silva, 2019 ; Lin et al, 2023 ).…”

Section: Resultsmentioning

confidence: 99%

Human lipocalins bind and export fatty acids through the secretory pathway of yeast cells

Ekim Kocabey,

Schneiter

2024

Front. Microbiol.

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The activation of fatty acids to their acyl-CoA derivatives is a crucial step for their integration into more complex lipids or their degradation via beta-oxidation. Yeast cells employ five distinct acyl-CoA synthases to facilitate this ATP-dependent activation of acyl chains. Notably, mutant cells that are deficient in two of these fatty acid-activating (FAA) enzymes, namely, Faa1 and Faa4, do not take up free fatty acids but rather export them out of the cell. This unique fatty acid export pathway depends on small, secreted pathogenesis-related yeast proteins (Pry). In this study, we investigate whether the expression of human fatty acid-binding proteins, including Albumin, fatty acid-binding protein 4 (Fabp4), and three distinct lipocalins (ApoD, Lcn1, and Obp2a), could promote fatty acid secretion in yeast. To optimize the expression and secretion of these proteins, we systematically examined various signal sequences in both low-copy and high-copy number plasmids. Our findings reveal that directing these fatty-acid binding proteins into the secretory pathway effectively promotes fatty acid secretion from a sensitized quadruple mutant model strain (faa1∆ faa4∆ pry1∆ pry3∆). Furthermore, the level of fatty acid secretion exhibited a positive correlation with the efficiency of protein secretion. Importantly, the expression of all human lipid-binding proteins rescued Pry-dependent fatty acid secretion, resulting in the secretion of both long-chain saturated and unsaturated fatty acids. These results not only affirm the in vitro binding capabilities of lipocalins to fatty acids but also present a novel avenue for enhancing the secretion of valuable lipidic compounds. Given the growing interest in utilizing yeast as a cellular factory for producing poorly soluble compounds and the potential of lipocalins as platforms for engineering substrate-binding specificity, our model is considered as a powerful tool for promoting the secretion of high-value lipid-based molecules.

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Section: Resultsmentioning

confidence: 99%

Human lipocalins bind and export fatty acids through the secretory pathway of yeast cells

Ekim Kocabey,

Schneiter

2024

Front. Microbiol.

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“…cerevisiae are largely produced during the ethanol consumption phase. Meanwhile, glycerol and acetate levels offer insights into the metabolic activity and energy status of the yeast cells. ,,, To investigate the metabolic characteristics and production processes of the amylase-HSA fusion protein, samples were taken from cultures of strains C0 (harboring the empty plasmid CPOTud), AH0, and AH28 at different time points (Figure ). Compared with strain C0, strain AH0 containing plasmid pCP-AGSH exhibited slower growth and lower final biomass (Figure B).…”

Section: Resultsmentioning

confidence: 99%

“…To better manage the ER stress caused by expressing complex recombinant proteins in S. cerevisiae strains, the pCP-AGSH plasmid was transformed to a high HSA production strain that had been genetically modified in genes related to the key node Hac1 in the UPR pathway. 50 Strain AH27, with the pCP-AGSH plasmid, exhibited a 4-fold increase in amylase-HSA fusion protein production compared with that of strain AH0 (Figure 5B). Next, S. cerevisiae PDI1 + ERO1 and B. taurus PDIA1 + ERO1β were introduced into strain AH27, resulting in strains AH28 and AH29, respectively.…”

Section: Overexpression Of B Taurus Pdia1 B Subtilis Bdbd and Pmentioning

confidence: 99%

“…To enhance HSA fusion protein production, future studies can explore the use of nonfermentable carbon sources like ethanol. 50 A comparison between strain AH0 and strain AH28 revealed that engineered strain AH28 exhibited faster growth than original strain AH0 approximately from 24 to 54 h, after which the growth rate gradually declined. However, the final biomass of strain AH28 was 15.8% lower than that of strain AH0 (Figure 6B).…”

Section: Overexpression Of B Taurus Pdia1 B Subtilis Bdbd and Pmentioning

confidence: 99%

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Optimization of Protein Folding for Improved Secretion of Human Serum Albumin Fusion Proteins in Saccharomyces cerevisiae

Li,

Xiao,

Pan

et al. 2023

J. Agric. Food Chem.

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The successful expression and secretion of recombinant proteins in cell factories significantly depend on the correct folding of nascent peptides, primarily achieved through disulfide bond formation. Thus, optimizing cellular protein folding is crucial, especially for proteins with complex spatial structures. In this study, protein disulfide isomerases (PDIs) from various species were introduced into Saccharomyces cerevisiae to facilitate proper disulfide bond formation and enhance recombinant protein secretion. The impacts of these PDIs on recombinant protein production and yeast growth metabolism were evaluated by substituting the endogenous PDI1. Heterologous PDIs cannot fully compensate the endogenous PDI. Furthermore, protein folding mediators, PDI and ER oxidoreductase 1 (Ero1), from different species were used to increase the production of complex human serum albumin (HSA) fusion proteins. The validated folding mediators were then introduced into unfolded protein response (UPR)-optimized strains, resulting in a 7.8-fold increase in amylase-HSA and an 18.2-fold increase in albiglutide compared with the control strain. These findings provide valuable insights for optimizing protein folding and expressing HSA-based drugs.

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“…Therefore, protein yield and quality can be significantly improved by designing and engineering protein secretion pathways. Secretion signal engineering, ER folding engineering [ 86 ], and vesicle trafficking engineering are the major strategies used to modify S. cerevisiae protein secretion pathway system [ 87 ] (Fig. 5 ).…”

Section: Protein Secretion Engineeringmentioning

confidence: 99%

Engineering strategies for enhanced heterologous protein production by Saccharomyces cerevisiae

Zhao,

Ma,

Zhang

et al. 2024

Microb Cell Fact

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Microbial proteins are promising substitutes for animal- and plant-based proteins. S. cerevisiae, a generally recognized as safe (GRAS) microorganism, has been frequently employed to generate heterologous proteins. However, constructing a universal yeast chassis for efficient protein production is still a challenge due to the varying properties of different proteins. With progress in synthetic biology, a multitude of molecular biology tools and metabolic engineering strategies have been employed to alleviate these issues. This review first analyses the advantages of protein production by S. cerevisiae. The most recent advances in improving heterologous protein yield are summarized and discussed in terms of protein hyperexpression systems, protein secretion engineering, glycosylation pathway engineering and systems metabolic engineering. Furthermore, the prospects for efficient and sustainable heterologous protein production by S. cerevisiae are also provided.

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Improved protein production in yeast using cell engineering with genes related to a key factor in the unfolded protein response

Cited by 19 publications

References 70 publications

Human lipocalins bind and export fatty acids through the secretory pathway of yeast cells

Human lipocalins bind and export fatty acids through the secretory pathway of yeast cells

Optimization of Protein Folding for Improved Secretion of Human Serum Albumin Fusion Proteins in Saccharomyces cerevisiae

Engineering strategies for enhanced heterologous protein production by Saccharomyces cerevisiae

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