Engineering and application of synthetic nar promoter for fine-tuning the expression of metabolic pathway genes in Escherichia coli

Hwang, Hee Jin; Lee, Sang Yup; Lee, Pyung Cheon

doi:10.1186/s13068-018-1104-1

Cited by 48 publications

(42 citation statements)

References 31 publications

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“…They affect system behaviour by switching on and off gene transcription in response to various stimuli and by mediating the rate of transcription. Exploiting of these features has enabled the construction of genetic circuits (Nielsen et al, 2016) and environmental sensors (Dietrich et al, 2013), as well as the overexpression of biosynthetic pathway genes for the production of value-added chemicals (Hwang et al, 2018). Some natural promoters have been well characterized and employed to drive gene expression (Dahl et al, 2013;Huo et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Context‐dependency of synthetic minimal promoters in driving gene expression: a case study

Jin

Nawab

Xia

et al. 2019

Microbial Biotechnology

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Summary Synthetic promoters are considered ideal candidates in driving robust gene expression. Most of the available synthetic promoters are minimal promoters, for which the upstream sequence of the 5′ end of the core region is usually excluded. Although the upstream sequence has been shown to mediate transcription of natural promoters, its impact on synthetic promoters has not been widely studied. Here, a library of chromosomal DNA fragments is randomly fused with the 5′ end of the J23119 synthetic promoter, and the transcriptional performance of the promoter is evaluated through β‐galactosidase assay, fluorescence intensity and chemical biosynthesis. Results show that changes in the upstream sequence can induce significant variation in the promoter strength of up to 5.8‐fold. The effect is independent of the length of the insertions and the number of potential transcription factor binding sites. Several DNA fragments that are able to enhance the transcription of both the natural and the synthetic promoters are identified. This study indicates that the synthetic minimal promoters are susceptible to the surrounding sequence context. Therefore, the upstream sequence should be treated as an indispensable component in the design and application of synthetic promoters, or as an independent genetic part for the fine‐tuning of gene expression.

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Section: Introductionmentioning

confidence: 99%

Context‐dependency of synthetic minimal promoters in driving gene expression: a case study

Jin

Nawab

Xia

et al. 2019

Microbial Biotechnology

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“…lpdE354 K Δmdh ΔarcA gltAR164L ilvBN ( E. coli ), aldB ( L. lactis subsp . lactis ), bdh1 ( S. cerevisiae ) Glucose + YE + tryptone Fed-batch 88 0.35 1.87 [ 33 ] Escherichia coli JM109 ΔldhA Δpta ΔadhE ΔpoxB alsS ( B. subtilis , alsD ( B. subtilis ), budC ( K. pneumoniae ) Glucose + YE Shake flask 14.5 0.30 0.30 [ 34 ] Escherichia coli W budA , budB , budC ( Enterobacter cloacae subsp . dissolvens ) Glucose Fed-batch High oxygen 52.1 a 0.27 a 4.53 a This study Escherichia coli W ΔldhA ΔadhE Δpta ΔfrdA budA , budB , budC ( Enterobacter cloacae subsp .…”

Section: Introductionmentioning

confidence: 99%

“…Using this promoter library, fine-tuning of expression levels was used to optimize expression of the three pathway genes for 2,3-butanediol production. Fed-batch cultivations with the optimized construct under microaerobic conditions using glucose supplemented with yeast extract and peptone showed a titer of 88.0 g l −1 2,3-butanediol [ 33 ].…”

Section: Introductionmentioning

confidence: 99%

Engineered E. coli W enables efficient 2,3-butanediol production from glucose and sugar beet molasses using defined minimal medium as economic basis

2018

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BackgroundEfficient microbial production of chemicals is often hindered by the cytotoxicity of the products or by the pathogenicity of the host strains. Hence 2,3-butanediol, an important drop-in chemical, is an interesting alternative target molecule for microbial synthesis since it is non-cytotoxic. Metabolic engineering of non-pathogenic and industrially relevant microorganisms, such as Escherichia coli, have already yielded in promising 2,3-butanediol titers showing the potential of microbial synthesis of 2,3-butanediol. However, current microbial 2,3-butanediol production processes often rely on yeast extract as expensive additive, rendering these processes infeasible for industrial production.ResultsThe aim of this study was to develop an efficient 2,3-butanediol production process with E. coli operating on the premise of using cost-effective medium without complex supplements, considering second generation feedstocks. Different gene donors and promoter fine-tuning allowed for construction of a potent E. coli strain for the production of 2,3-butanediol as important drop-in chemical. Pulsed fed-batch cultivations of E. coli W using microaerobic conditions showed high diol productivity of 4.5 g l−1 h−1. Optimizing oxygen supply and elimination of acetoin and by-product formation improved the 2,3-butanediol titer to 68 g l−1, 76% of the theoretical maximum yield, however, at the expense of productivity. Sugar beet molasses was tested as a potential substrate for industrial production of chemicals. Pulsed fed-batch cultivations produced 56 g l−1 2,3-butanediol, underlining the great potential of E. coli W as production organism for high value-added chemicals.ConclusionA potent 2,3-butanediol producing E. coli strain was generated by considering promoter fine-tuning to balance cell fitness and production capacity. For the first time, 2,3-butanediol production was achieved with promising titer, rate and yield and no acetoin formation from glucose in pulsed fed-batch cultivations using chemically defined medium without complex hydrolysates. Furthermore, versatility of E. coli W as production host was demonstrated by efficiently converting sucrose from sugar beet molasses into 2,3-butanediol.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1038-0) contains supplementary material, which is available to authorized users.

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“…Klebsiella oxytoca or Enterobacter cloacae, some hosts are pathogenic and require complex and expensive media additives [6]. Therefore, 2,3-butanediol production has recently been developed in Escherichia coli [6][7][8]. Since E. coli is not a natural producer, a heterologous pathway consisting of three genes has to be overexpressed: acetolactate synthase, acetolactate decarboxylase and butanediol dehydrogenase ( Fig.…”

Section: Introductionmentioning

confidence: 99%

“…1) [9]. So far, 2,3-butanediol has been produced from glucose [7,8] and alternative sugar sources such as sugar beet molasse [6].…”

Section: Introductionmentioning

confidence: 99%

Microbial Upgrading of Acetate into 2,3-Butanediol and Acetoin by E. coli W

Novak

Kutscha

Pflügl

2020

Preprint

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BackgroundAcetate is an abundant carbon source and its use as an alternative feedstock has great potential for the production of fuel and platform chemicals. Acetoin and 2,3-butanediol represent two of these potential platform chemicals. ResultsThe aim of this study was to produce 2,3-butanediol and acetoin from acetate in Escherichia coli W. The key strategies to achieve this goal were: strain engineering, in detail the deletion of mixed acid fermentation pathways E. coli W ΔldhA ΔadhE Δpta ΔfrdA 445_Ediss and the development of a new defined medium containing five amino acids and seven vitamins. Stepwise reduction of the media additives further revealed that diol production from acetate is mediated by the availability of aspartate. Other amino acids or TCA cycle intermediates did not enable growth on acetate. Cultivation under controlled conditions in batch and fed-batch experiments showed that aspartate was consumed before acetate, indicating that co-utilization is not a prerequisite for diol production. The addition of aspartate gave cultures a start-kick and was not required for feeding. Pulsed fed-batches resulted in the production of 1.43 g l-1 from aspartate and acetate and 1.16 g l-1 diols (2,3-butanediol and acetoin) from acetate alone. The yield reached 0.09 g diols per g acetate, which accounts for 26 % of the theoretical maximum.ConclusionThis study for the first time showed acetoin and 2,3-butanediol production from acetate as well as the use of chemically defined medium for product formation from acetate in E. coli. Hereby, we provide a solid base for process intensification and the investigation of other potential products.

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Engineering and application of synthetic nar promoter for fine-tuning the expression of metabolic pathway genes in Escherichia coli

Cited by 48 publications

References 31 publications

Context‐dependency of synthetic minimal promoters in driving gene expression: a case study

Context‐dependency of synthetic minimal promoters in driving gene expression: a case study

Engineered E. coli W enables efficient 2,3-butanediol production from glucose and sugar beet molasses using defined minimal medium as economic basis

Microbial Upgrading of Acetate into 2,3-Butanediol and Acetoin by E. coli W

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