Identification of a novel promoter for driving antibiotic-resistant genes to reduce the metabolic burden during protein expression and effectively select ... View Full Text


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Article Info

DATE

2021-04-05

AUTHORS

Qi Shen, Zhuang Yu, Xiao-Ting Zhou, Shi-Jia Zhang, Shu-Ping Zou, Neng Xiong, Ya-Ping Xue, Zhi-Qiang Liu, Yu-Guo Zheng

ABSTRACT

Routine approaches for the efficient expression of heterogenous proteins in Pichia pastoris include using the strong methanol-regulated alcohol oxidase (AOX1) promoter and multiple inserts of expression cassettes. To screen the transformants harboring multiple integrations, antibiotic-resistant genes such as the Streptoalloteichus hindustanus bleomycin gene are constructed into expression vectors, given that higher numbers of insertions of antibiotic-resistant genes on the expression vector confer resistance to higher concentrations of the antibiotic for transformants. The antibiotic-resistant genes are normally driven by the strong constitutive translational elongation factor 1a promoter (PTEF1). However, antibiotic-resistant proteins are necessary only for the selection process. Their production during the heterogenous protein expression process may increase the burden in cells, especially for the high-copy strains which harbor multiple copies of the expression cassette of antibiotic-resistant genes. Besides, a high concentration of the expensive antibiotic is required for the selection of multiple inserts because of the effective expression of the antibiotic-resistant gene by the TEF1 promoter. To address these limitations, we replaced the TEF1 promoter with a weaker promoter (PDog2p300) derived from the potential promoter region of 2-deoxyglucose-6-phosphate phosphatase gene for driving the antibiotic-resistant gene expression. Importantly, the PDog2p300 has even lower activity under carbon sources (glycerol and methanol) used for the AOX1 promoter–based production of recombinant proteins compared with glucose that is usually used for the selection process. This strategy has proven to be successful in screening of transformants harboring more than 3 copies of the gene of interest by using plates containing 100 μg/ml of Zeocin. Meanwhile, levels of Zeocin resistance protein were undetectable by immunoblotting in these multiple-copy strains during expression of heterogenous proteins.Key points• PDog2p300was identified as a novel glucose-regulated promoter.• The expression of antibiotic-resistant gene driven by PDog2p300was suppressed during the recombinant protein expression, resulting in reducing the metabolic burden.• The transformants harboring multiple integrations were cost-effectively selected by using the PDog2p300for driving antibiotic-resistant genes. More... »

PAGES

3211-3223

References to SciGraph publications

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  • 2013-06-26. Identification and characterization of PGCW14: a novel, strong constitutive promoter of Pichia pastoris in BIOTECHNOLOGY LETTERS
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  • 2014-01-04. Engineering of a Pichia pastoris Expression System for High-Level Secretion of HSA/GH Fusion Protein in APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
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  • 2013-12-20. Can too many copies spoil the broth? in MICROBIAL CELL FACTORIES
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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1007/s00253-021-11195-0

    DOI

    http://dx.doi.org/10.1007/s00253-021-11195-0

    DIMENSIONS

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    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/33818673


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    23 schema:description Routine approaches for the efficient expression of heterogenous proteins in Pichia pastoris include using the strong methanol-regulated alcohol oxidase (AOX1) promoter and multiple inserts of expression cassettes. To screen the transformants harboring multiple integrations, antibiotic-resistant genes such as the Streptoalloteichus hindustanus bleomycin gene are constructed into expression vectors, given that higher numbers of insertions of antibiotic-resistant genes on the expression vector confer resistance to higher concentrations of the antibiotic for transformants. The antibiotic-resistant genes are normally driven by the strong constitutive translational elongation factor 1a promoter (PTEF1). However, antibiotic-resistant proteins are necessary only for the selection process. Their production during the heterogenous protein expression process may increase the burden in cells, especially for the high-copy strains which harbor multiple copies of the expression cassette of antibiotic-resistant genes. Besides, a high concentration of the expensive antibiotic is required for the selection of multiple inserts because of the effective expression of the antibiotic-resistant gene by the TEF1 promoter. To address these limitations, we replaced the TEF1 promoter with a weaker promoter (PDog2p300) derived from the potential promoter region of 2-deoxyglucose-6-phosphate phosphatase gene for driving the antibiotic-resistant gene expression. Importantly, the PDog2p300 has even lower activity under carbon sources (glycerol and methanol) used for the AOX1 promoter–based production of recombinant proteins compared with glucose that is usually used for the selection process. This strategy has proven to be successful in screening of transformants harboring more than 3 copies of the gene of interest by using plates containing 100 μg/ml of Zeocin. Meanwhile, levels of Zeocin resistance protein were undetectable by immunoblotting in these multiple-copy strains during expression of heterogenous proteins.Key points• PDog2p300was identified as a novel glucose-regulated promoter.• The expression of antibiotic-resistant gene driven by PDog2p300was suppressed during the recombinant protein expression, resulting in reducing the metabolic burden.• The transformants harboring multiple integrations were cost-effectively selected by using the PDog2p300for driving antibiotic-resistant genes.
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    33 Pichia pastoris
    34 Streptoalloteichus hindustanus bleomycin gene
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