A high-throughput screening assay for the directed evolution-guided discovery of halohydrin dehalogenase mutants for epoxide ring-opening reaction

Gul, Ijaz; Bogale, Tadesse Fantaye; Deng, Jiao; Wang, Le; Feng, Jia‐Xun; Tang, Lixia

doi:10.1016/j.jbiotec.2020.02.007

Cited by 11 publications

(11 citation statements)

References 22 publications

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“…5,6 However, its use is limited to easy-to-screen phenotypes such as growth or color. [7][8][9] Targeted genome editing usually involves multiple rounds of genomic modification with one gene at a time, which is laborious and time consuming. The parallel disruption and/or integration of multicopy genes in the genome can not only speed up construction of strains, but also generate gene dosage diversity of multiple genes or pathways.…”

Section: Introductionmentioning

confidence: 99%

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Zhang

Yang

et al. 2021

The CRISPR Journal

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Directed evolution and targeted genome editing have been deployed to create genetic variants with usefully altered phenotypes. However, these methods are limited to high-throughput screening methods or serial manipulation of single genes. In this study, we implemented multicopy chromosomal integration using CRISPRassociated transposases (MUCICAT) to simultaneously target up to 11 sites on the Escherichia coli chromosome for multiplex gene interruption and/or insertion, generating combinatorial genomic diversity. The MUCICAT system was improved by replacing the isopropyl-beta-D-thiogalactoside (IPTG)-dependent promoter to decouple gene editing and product synthesis and truncating the right end to reduce the leakage expression of cargo. We applied MUCICAT to engineer and optimize the N-acetylglucosamine (GlcNAc) biosynthesis pathway in E. coli to overproduce the industrially important GlcNAc in only 8 days. Two rounds of transformation, the first round for disruption of two degradation pathways related gene clusters and the second round for multiplex integration of the GlcNAc gene cassette, would generate a library with 1-11 copies of the GlcNAc cassette. We isolated a best variant with five copies of GlcNAc cassettes, producing 11.59 g/L GlcNAc, which was more than sixfold than that of the strain containing the pET-GNAc plasmid. Our multiplex approach MUCICAT has potential to become a powerful tool of cell programing and can be widely applied in many fields such as synthetic biology.

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Section: Introductionmentioning

confidence: 99%

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Zhang

Yang

et al. 2021

The CRISPR Journal

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“…Directed evolution is a widely used technique in biology and interdisciplinary fields, particularly important for improving enzyme expression. 14 This method is effective for detecting and achieving protein modifications and is often considered the first choice for such alterations. The directed evolution process can result in genetic differences between the evolved strain or enzyme and the original one, which may lead to variations in the expression of related proteins or metabolites.…”

Section: ■ Introductionmentioning

confidence: 99%

“…In recent years, the low expression of heterologous LAI limits its industrial application, and the optimization of expression is gaining researchers’ attention. Directed evolution is a widely used technique in biology and interdisciplinary fields, particularly important for improving enzyme expression . This method is effective for detecting and achieving protein modifications and is often considered the first choice for such alterations.…”

Section: Introductionmentioning

confidence: 99%

Proteomics and Metabolomics Analysis Reveal the Regulation Mechanism of Linoleate Isomerase Activity and Function in Propionibacterium acnes

Liu,

Chen,

Yue

et al. 2023

ACS Omega

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Conjugated linoleic acid (CLA) holds significant application prospects due to its anticancer, anti-atherosclerosis, lipid-lowering, weight-loss, and growth-promoting functions. The key to its efficient production lies in optimizing the biocatalytic performance of linoleic acid isomerase (LAI). Here, we constructed a Propionibacterium acnes mutant library and screened positive mutants with high linoleate isomerase activity. The proteomics and metabolomics were used to explore the mechanism in the regulation of linoleic acid isomerase activity. High-throughput proteomics revealed 104 differentially expressed proteins unique to positive mutant strains of linoleic acid isomerase of which 57 were upregulated and 47 were downregulated. These differentially expressed proteins were primarily involved in galactose metabolism, the phosphotransferase system, starch metabolism, and sucrose metabolism. Differential metabolic pathways were mainly enriched in amino acid biosynthesis, including glutamate metabolism, the Aminoacyl-tRNA biosynthesis pathway, and the ABC transporter pathway. The upregulated metabolites include dl-valine and Acetyl coA, while the downregulated metabolites include Glutamic acid and Phosphoenolpyruvate. Overall, the activity of linoleic acid isomerase in the mutant strain was increased by the regulation of key proteins involved in galactose metabolism, sucrose metabolism, and the phosphotransferase system. This study provides a theoretical basis for the development of high-yield CLA food.

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“…Additionally, specific activities based on initial reaction velocities of HheG and HheG-682 in the azidolysis of the different epoxide substrates were determined using an adapted, pH-based spectrophotometric assay. [10] Reactions were performed at 30°C using 10 mM epoxide, 20 mM azide and 100 µg mL -1 purified enzyme. As expected, both HheG and HheG-682 displayed low specific activity with epoxide 1 (Figure 2), while for all other tested epoxides the specific activities of HheG-682 were significantly higher compared to HheG.…”

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confidence: 99%

“…7), (+)-cis/trans-limonene oxide (9) and trans-1-phenylpropylene oxide (11) determined with a spectrophotometric assay. [10] Reactions were carried out in duplicate in a total volume of 1 mL with 10 mM epoxide and 20 mM azide in 2 mM buffer at 30 °C using 100 µg mL -1 purified enzyme. Samples were taken after 30, 60, 180, 270 and 360 s and quenched in equal volumes of MeOH.…”

mentioning

confidence: 99%

Characterization of a new G-type halohydrin dehalogenase with enhanced catalytic activity

Günther

Schallmey

2022

Preprint

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A high-throughput screening assay for the directed evolution-guided discovery of halohydrin dehalogenase mutants for epoxide ring-opening reaction

Cited by 11 publications

References 22 publications

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Proteomics and Metabolomics Analysis Reveal the Regulation Mechanism of Linoleate Isomerase Activity and Function in Propionibacterium acnes

Characterization of a new G-type halohydrin dehalogenase with enhanced catalytic activity

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