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Enhancing metabolic efficiency via novel constitutive promoters to produce protocatechuic acid in Escherichia coli

Englund Örn, Oliver LU ; Hagman, Arne LU ; Ismail, Mohamed LU orcid ; Leiva Eriksson, Nélida LU orcid and Hatti-Kaul, Rajni LU (2024) In Applied Microbiology and Biotechnology 108.
Abstract

Abstract: The antioxidant molecule protocatechuic acid (PCA) can also serve as a precursor for polymer building blocks. PCA can be produced in Escherichia coli overexpressing 3-dehydroshikimate dehydratase (DSD), an enzyme that catalyses the transformation of 3-dehydroshikimate to PCA. Nevertheless, optimizing the expression rate of recombinant enzymes is a key factor in metabolic engineering when producing biobased chemicals. In this study, a degenerate synthetic promoter approach was investigated to improve further the production of PCA. By limited screening of a randomized promoter library made using pSEVA221 plasmid in E. coli, three novel synthetic constitutive promoters were selected that increased the PCA yield from glucose by... (More)

Abstract: The antioxidant molecule protocatechuic acid (PCA) can also serve as a precursor for polymer building blocks. PCA can be produced in Escherichia coli overexpressing 3-dehydroshikimate dehydratase (DSD), an enzyme that catalyses the transformation of 3-dehydroshikimate to PCA. Nevertheless, optimizing the expression rate of recombinant enzymes is a key factor in metabolic engineering when producing biobased chemicals. In this study, a degenerate synthetic promoter approach was investigated to improve further the production of PCA. By limited screening of a randomized promoter library made using pSEVA221 plasmid in E. coli, three novel synthetic constitutive promoters were selected that increased the PCA yield from glucose by 10–21% compared to the inducible T7-promoter. RT-qPCR analysis showed that the DSD gene, regulated by the synthetic promoters, had high expression during the exponential phase, albeit the gene expression level dropped 250-fold during stationary phase. Besides the increased product yield, the synthetic promoters avoided the need for a costly inducer for gene expression. Screening of the entire promoter library is likely to provide more positive hits. The study also shows that E. coli transformed with the DSD gene on either pSEVA221 or pCDFDuet plasmids exhibit background PCA levels (~ 0.04 g/L) in the absence of a transcriptional regulatory element.

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author
; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Inducer free expression system, Promoter library, Protocatechuic acid production, RT-qPCR, Synthetic constitutive promoter
in
Applied Microbiology and Biotechnology
volume
108
article number
442
pages
13 pages
publisher
Springer
external identifiers
  • pmid:39153079
  • scopus:85201433321
ISSN
0175-7598
DOI
10.1007/s00253-024-13256-6
language
English
LU publication?
yes
additional info
Publisher Copyright: © The Author(s) 2024.
id
444361cf-3923-4d7e-9e8c-6c3a3b44b6a8
date added to LUP
2024-08-24 20:26:10
date last changed
2025-06-30 01:21:58
@article{444361cf-3923-4d7e-9e8c-6c3a3b44b6a8,
  abstract     = {{<p>Abstract: The antioxidant molecule protocatechuic acid (PCA) can also serve as a precursor for polymer building blocks. PCA can be produced in Escherichia coli overexpressing 3-dehydroshikimate dehydratase (DSD), an enzyme that catalyses the transformation of 3-dehydroshikimate to PCA. Nevertheless, optimizing the expression rate of recombinant enzymes is a key factor in metabolic engineering when producing biobased chemicals. In this study, a degenerate synthetic promoter approach was investigated to improve further the production of PCA. By limited screening of a randomized promoter library made using pSEVA221 plasmid in E. coli, three novel synthetic constitutive promoters were selected that increased the PCA yield from glucose by 10–21% compared to the inducible T7-promoter. RT-qPCR analysis showed that the DSD gene, regulated by the synthetic promoters, had high expression during the exponential phase, albeit the gene expression level dropped 250-fold during stationary phase. Besides the increased product yield, the synthetic promoters avoided the need for a costly inducer for gene expression. Screening of the entire promoter library is likely to provide more positive hits. The study also shows that E. coli transformed with the DSD gene on either pSEVA221 or pCDFDuet plasmids exhibit background PCA levels (~ 0.04 g/L) in the absence of a transcriptional regulatory element.<br/></p>}},
  author       = {{Englund Örn, Oliver and Hagman, Arne and Ismail, Mohamed and Leiva Eriksson, Nélida and Hatti-Kaul, Rajni}},
  issn         = {{0175-7598}},
  keywords     = {{Inducer free expression system; Promoter library; Protocatechuic acid production; RT-qPCR; Synthetic constitutive promoter}},
  language     = {{eng}},
  publisher    = {{Springer}},
  series       = {{Applied Microbiology and Biotechnology}},
  title        = {{Enhancing metabolic efficiency via novel constitutive promoters to produce protocatechuic acid in<i> Escherichia coli</i>}},
  url          = {{http://dx.doi.org/10.1007/s00253-024-13256-6}},
  doi          = {{10.1007/s00253-024-13256-6}},
  volume       = {{108}},
  year         = {{2024}},
}