Site-directed mutagenesis and over expression of aroG gene of Escherichia coli K-12

Lin, Songyi; Meng, Xianyu; Jiang, Jie; Pang, Daxing; Jones, Gregory S.; Ouyang, Hongsheng; Ren, Linzhu

doi:10.1016/j.ijbiomac.2012.07.013

Cited by 12 publications

(6 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For instance, regulation mechanisms of enzyme such as feed-back or feed-forward inhibitions (Chen et al., 2018; Zurawski et al., 1981) restricting the catalytic efficiency. In this regard, the construction of an efficient pathway inevitably requires dedicated protein engineering (Chen et al., 2011b, 2013; Ger et al., 1994; Lin et al., 2012).…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli

Chen

Zeng

2019

Metabolic Engineering Communications

View full text Add to dashboard Cite

Protein engineering plays an increasingly important role in developing new and optimizing existing metabolic pathways for biosynthesis. Conventional screening approach of libraries of gene and enzyme variants is often done using a host strain under conditions not relevant to the cultivation or intracellular conditions of the later production strain. This does not necessarily result in the identification of the best enzyme variant for in vivo use in the production strain. In this work, we propose a method which integrates CRISPR/Cas9-facilitated engineering of the target gene(s) with growth-coupled and sensor-guided in vivo screening (CGSS) for protein engineering and pathway optimization. The efficiency of the method is demonstrated for engineering 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase AroG, a key enzyme in the chorismate pathway for the synthesis of aromatic amino acids (AAAs), to obtain variants of AroG (AroG fbr ) with increased resistance to feedback inhibition of Phe. Starting from a tryptophan (Trp)-producing E. coli strain (harboring a reported Phe-resistant AroG variant AroG S180F ), the removal of all the endogenous DAHP synthases makes the growth of this strain dependent on the activity of an introduced AroG variant. The different catalytic efficiencies of AroG variants lead to different intracellular concentration of Trp which is sensed by a Trp biosensor (TnaC-eGFP). Using the growth rate and the signal strength of the biosensor as criteria, we successfully identified several novel Phe-resistant AroG variants (including the best one AroG D6G−D7A ) which exhibited higher specific enzyme activity than that of the reference variant AroG S180F at the presence of 40 mM Phe. The replacement of AroG S180F with the newly identified AroG D6G−D7A in the Trp-producing strain significantly improved the Trp production by 38.5% (24.03 ± 1.02 g/L at 36 h) in a simple fed-batch fermentation.

show abstract

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli

Chen

Zeng

2019

Metabolic Engineering Communications

View full text Add to dashboard Cite

show abstract

“…The dSA05-3 showed less decreased biomass, 25.2% lower than that of the control, which was suitable for further modifications. These results suggested that it was crucial to select an appropriate aroG mutant, as different mutations of this gene could affect the activity of DAHP synthase to different extents [ 34 ]. In the PTS - strains, pyruvate, one of the most important central metabolites, is generated from PEP only by pyruvate kinases.…”

Section: Resultsmentioning

confidence: 99%

Multiple Metabolic Engineering Strategies to Improve Shikimate Titer in Escherichia coli

Wang

et al. 2023

Metabolites

View full text Add to dashboard Cite

Shikimate is a valuable chiral precursor for synthesizing oseltamivir (Tamiflu®) and other chemicals. High production of shikimate via microbial fermentation has attracted increasing attention to overcome the unstable and expensive supply of shikimate extracted from plant resources. The current cost of microbial production of shikimate via engineered strains is still unsatisfactory, and thus more metabolic strategies need to be investigated to further increase the production efficiency. In this study, we first constructed a shikimate E. coli producer through the application of the non-phosphoenolpyruvate: carbohydrate phosphotransferase system (non-PTS) glucose uptake pathway, the attenuation of the shikimate degradation metabolism, and the introduction of a mutant of feedback-resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase. Inspired by the natural presence of bifunctional 3-dehydroquinate dehydratase (DHD)-shikimate dehydrogenase (SDH) enzyme in plants, we then designed an artificial fusion protein of DHD-SDH to decrease the accumulation of the byproduct 3-dehydroshikimate (DHS). Subsequently, a repressed shikimate kinase (SK) mutant was selected to promote shikimate accumulation without the supplementation of expensive aromatic substances. Furthermore, EsaR-based quorum sensing (QS) circuits were employed to regulate the metabolic flux distribution between cell growth and product synthesis. The final engineered strain dSA10 produced 60.31 g/L shikimate with a yield of 0.30 g/g glucose in a 5 L bioreactor.

show abstract

“…We obtained regulatory information from an earlier kinetic modeling study 56 . Considering the focus of our work on anthranilate production through the shikimate pathway, we also added the inhibition of aroG by phenylalanine 57 and the inhibition of ANS by tryptophan 58 . Overall, we incorporated regulatory information for 31 reactions, including interactions for 5 reactions in the Shikimate pathway (Supplementary Note 11 , Supplementary Data 2 ).…”

Section: Methodsmentioning

confidence: 99%

Rational strain design with minimal phenotype perturbation

Narayanan,

Weilandt,

Masid

et al. 2024

Nat Commun

View full text Add to dashboard Cite

Devising genetic interventions for desired cellular phenotypes remains challenging regarding time and resources. Kinetic models can accelerate this task by simulating metabolic responses to genetic perturbations. However, exhaustive design evaluations with kinetic models are computationally impractical, especially when targeting multiple enzymes. Here, we introduce a framework for efficiently scouting the design space while respecting cellular physiological requirements. The framework employs mixed-integer linear programming and nonlinear simulations with large-scale nonlinear kinetic models to devise genetic interventions while accounting for the network effects of these perturbations. Importantly, it ensures the engineered strain’s robustness by maintaining its phenotype close to that of the reference strain. The framework, applied to improve the anthranilate production in E. coli, devises designs for experimental implementation, including eight previously experimentally validated targets. We expect this framework to play a crucial role in future design-build-test-learn cycles, significantly expediting the strain design compared to exhaustive design enumeration.

show abstract

Site-directed mutagenesis and over expression of aroG gene of Escherichia coli K-12

Cited by 12 publications

References 21 publications

CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli

CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli

Multiple Metabolic Engineering Strategies to Improve Shikimate Titer in Escherichia coli

Rational strain design with minimal phenotype perturbation

Contact Info

Product

Resources

About