Development of a new Geobacillus lipase variant GDlip43 via directed evolution leading to identification of new activity-regulating amino acids

Druteika, Gytis; Sadauskas, Mikas; Malunavicius, Vilius; Lastauskienė, Eglė; Taujenis, Lukas; Gegeckas, Audrius; Gudiukaitė, Renata

doi:10.1016/j.ijbiomac.2019.10.163

Cited by 8 publications

(3 citation statements)

References 72 publications

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“…This mutant strain showed a 240-fold increase in demethylation activity of ethoxycoumarin and a 10-fold increase in demethylation activity of 7-methoxycoumarin . Directionally evolved lipase activity was found to be 2-fold higher than the parent, while directionally evolved metalloproteinases showed enhanced tolerance to organic solvents …”

Section: Introductionmentioning

confidence: 93%

“…This mutant strain showed a 240-fold increase in demethylation activity of ethoxycoumarin and a 10fold increase in demethylation activity of 7-methoxycoumarin. 15 Directionally evolved lipase activity was found to be 2fold higher than the parent, 16 while directionally evolved metalloproteinases showed enhanced tolerance to organic solvents. 17 In recent years, the rapid development of genomics technology has enabled the comparison and analysis of different aspects of proteins, including their fine structure, translation processes, and modification processes.…”

Section: ■ Introductionmentioning

confidence: 99%

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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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Section: Introductionmentioning

confidence: 93%

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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“…Because of the rapid expansion of enzyme application scenarios, natural enzymes often need to be improved to optimize their properties, such as catalytic activity, thermostability, and tolerance to organic solvents, in non-native environments. Directed evolution has been established to be a powerful technique to enhance catalytic activities of enzymes or develop novel enzymes for catalyzing unnatural reactions. , More importantly, directed evolution offers the possibility to enhance the catalytic activity of enzymes through “undesignable routes,” such as reshaping global dynamical networks and energy landscapes. , In directed evolution, the target mutants are always sorted out from libraries with different scales generated by error-prone PCR (EP-PCR), DNA shuffling, or site saturation mutagenesis. − For instance, it is conceivable that the combined saturation mutagenesis in two residues would theoretically contain 4 × 10 2 mutations, while merely five residues would amount to 3.2 × 10 6 . Therefore, it would be an extremely time- and labor-consuming process without a convenient high-throughput screening technique.…”

Section: Introductionmentioning

confidence: 99%

Construction and Application of a High-Throughput In Vivo Screening Platform for the Evolution of Nitrile Metabolism-Related Enzymes Based on a Desensitized Repressive Biosensor

et al. 2022

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Transcription factor (TF)-based biosensors are expected to serve as powerful tools for the high-throughput screening of biocatalytic systems; however, most of them respond to ligands in a narrow concentration range, which limits their application. In this study, we constructed a heterogenous niacin biosensor using the repressive TF BsNadR and its target promoters from Bacillus subtilis. The fine-tunable output of the niacin biosensor was expanded to a wide range of niacin concentrations (0−50 mM) through desensitization engineering, which was suitable for the accurate identification of differences in enzyme activity. Structural mechanism analysis indicated that weakening the affinity of BsNadR with the ligand niacin and with DNA alters its regulatory properties. Based on the desensitized niacin biosensor, a high-throughput in vivo screening platform was developed for evolving nitrile metabolismrelated enzymes. The evolved nitrilase, amidase, and nitrile hydratase with 6.6-, 2.1-, and 21.3-fold improvements in activity were achieved, respectively. In addition, these mutants also exhibited elevated activity toward other cognate substrates, indicating the broad applicability of the screening platform. This study not only provided a universal high-throughput screening platform for different nitrile metabolism-related enzymes but also demonstrated the advantages of repressive biosensors and the vital role of desensitization engineering of the TF in the development of high-throughput screening platforms for enzymes.

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New engineered Geobacillus lipase GD-95RM for industry focusing on the cleaner production of fatty esters and household washing product formulations

Druteika

Sadauskas

Malunavicius

et al. 2020

World J Microbiol Biotechnol

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Development of a new Geobacillus lipase variant GDlip43 via directed evolution leading to identification of new activity-regulating amino acids

Cited by 8 publications

References 72 publications

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

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

Construction and Application of a High-Throughput In Vivo Screening Platform for the Evolution of Nitrile Metabolism-Related Enzymes Based on a Desensitized Repressive Biosensor

New engineered Geobacillus lipase GD-95RM for industry focusing on the cleaner production of fatty esters and household washing product formulations

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