Enhancing metabolic efficiency via novel constitutive promoters to produce protocatechuic acid in Escherichia coli
Englund Örn, Oliver LU ; Hagman, Arne LU ; Ismail, Mohamed LU
; Leiva Eriksson, Nélida
LU
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.
(Less)
- author
- Englund Örn, Oliver
LU
; Hagman, Arne
LU
; Ismail, Mohamed
LU
; Leiva Eriksson, Nélida
LU
and Hatti-Kaul, Rajni
LU
- organization
- publishing date
- 2024-12
- 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
- 2024-08-27 02:47:50
@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}},
}