Overexpression of genes by stress-responsive promoters increases protein secretion in Saccharomyces cerevisiae

Xiao, Chufan; Xue, Songlyu; Pan, Yong; Liu, Xiufang; Huang, Mingtao

doi:10.1007/s11274-023-03646-9

Cited by 4 publications

(2 citation statements)

References 62 publications

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“…Another constitutive promoter (pTEF1) controls the expression of the translation elongation factor EF1 alpha, showing higher expression intensity than promoter alcohol dehydrogenase 1 and being unrepressed by ethanol ( Partow et al, 2010 ). pTEF1 has been used to enhance the production of r-proteins and homologous enzymes by S. cerevisiae ( Steinborn et al, 2007 ; Zhang et al, 2017 ; Xiao et al, 2023 ). Other constitutive promoters have also been applied to the construction of expression vectors ( Partow et al, 2010 ; Lee et al, 2013 ).…”

Section: Well-developed Technologies Used In Saccharomyces ...mentioning

confidence: 99%

“…Synthetic promoters can be defined by inserting functional cis-elements into the distal promoter region to either positively or negatively recruit the corresponding transcription factors, specific regulation of transcription efficiency and strength can be achieved without being constrained by endogenous factors ( Machens et al, 2017 ; Yasmeen et al, 2023 ). For example, the α -amylase titer increased by 95% in S. cerevisiae by using an unfolded protein response synthetic responsive promoter, which is constructed by inserting the unfolded protein response element in front of the upstream enhancer region of yeast native promoters ( Xiao et al, 2023 ). Artificially synthesized promoters commonly feature shorter sequences, which are more accurately regulated and more stably condition-induced for heterologous metabolic transcription in dynamic metabolic networks ( Wichmann et al, 2023 ).…”

Section: Well-developed Technologies Used In Saccharomyces ...mentioning

confidence: 99%

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An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system

Wu,

Feng,

Sun

et al. 2023

Front. Bioeng. Biotechnol.

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Saccharomyces cerevisiae is one of the most extensively used biosynthetic systems for the production of diverse bioproducts, especially biotherapeutics and recombinant proteins. Because the expression and insertion of foreign genes are always impaired by the endogenous factors of Saccharomyces cerevisiae and nonproductive procedures, various technologies have been developed to enhance the strength and efficiency of transcription and facilitate gene editing procedures. Thus, the limitations that block heterologous protein secretion have been overcome. Highly efficient promoters responsible for the initiation of transcription and the accurate regulation of expression have been developed that can be precisely regulated with synthetic promoters and double promoter expression systems. Appropriate codon optimization and harmonization for adaption to the genomic codon abundance of S. cerevisiae are expected to further improve the transcription and translation efficiency. Efficient and accurate translocation can be achieved by fusing a specifically designed signal peptide to an upstream foreign gene to facilitate the secretion of newly synthesized proteins. In addition to the widely applied promoter engineering technology and the clear mechanism of the endoplasmic reticulum secretory pathway, the innovative genome editing technique CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated system) and its derivative tools allow for more precise and efficient gene disruption, site-directed mutation, and foreign gene insertion. This review focuses on sophisticated engineering techniques and emerging genetic technologies developed for the accurate metabolic regulation of the S. cerevisiae expression system.

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Section: Well-developed Technologies Used In Saccharomyces ...mentioning

confidence: 99%

Section: Well-developed Technologies Used In Saccharomyces ...mentioning

confidence: 99%

An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system

Wu,

Feng,

Sun

et al. 2023

Front. Bioeng. Biotechnol.

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Synthetic Promoter Design and Functional Evaluation in Saccharomyces cerevisiae

Xiao,

Liu,

Huang

2024

Methods in Molecular Biology

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Tailored UPRE2 variants for dynamic gene regulation in yeast

Xiao,

Liu,

Pan

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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Genetic elements are foundational in synthetic biology serving as vital building blocks. They enable programming host cells for efficient production of valuable chemicals and recombinant proteins. The unfolded protein response (UPR) is a stress pathway in which the transcription factor Hac1 interacts with the upstream unfolded protein response element (UPRE) of the promoter to restore endoplasmic reticulum (ER) homeostasis. Here, we created a UPRE2 mutant (UPRE2m) library. Several rounds of screening identified many elements with enhanced responsiveness and a wider dynamic range. The most active element m84 displayed a response activity 3.72 times higher than the native UPRE2. These potent elements are versatile and compatible with various promoters. Overexpression of HAC1 enhanced stress signal transduction, expanding the signal output range of UPRE2m. Through molecular modeling and site-directed mutagenesis, we pinpointed the DNA-binding residue Lys60 in Hac1(Hac1-K60). We also confirmed that UPRE2m exhibited a higher binding affinity to Hac1. This shed light on the mechanism underlying the Hac1-UPRE2m interaction. Importantly, applying UPRE2m for target gene regulation effectively increased both recombinant protein production and natural product synthesis. These genetic elements provide valuable tools for dynamically regulating gene expression in yeast cell factories.

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Overexpression of genes by stress-responsive promoters increases protein secretion in Saccharomyces cerevisiae

Cited by 4 publications

References 62 publications

An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system

An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system

Synthetic Promoter Design and Functional Evaluation in Saccharomyces cerevisiae

Tailored UPRE2 variants for dynamic gene regulation in yeast

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