Directed Evolution Methods for Enzyme Engineering

Nirantar, Saurabh

doi:10.3390/molecules26185599

Cited by 22 publications

(9 citation statements)

References 113 publications

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“…Within any family of enzymes that catalyze a specific transformation, the physiological substrate specificity is achieved by evolutionary pressures that lead to multiple specific mutations that adapt the enzyme for a particular substrate, without compromising catalytic efficiency. In the last several decades, these lessons have been implemented in the laboratory settings by directed evolution experiments that have been able to greatly expand the substrate and reaction scope of many enzymes, though often the expanded substrate scope is accompanied by altered catalytic efficiency. − In the context of catalysts, enzymes are unique in their ability to recognize specific structures, often through multiple interactions that are generally remote from the site of chemistry. By contrast, small molecular catalysts often function by specifically recognizing the reacting groups and effecting their transformations.…”

Section: Discussionmentioning

confidence: 99%

A Promiscuous rSAM Enzyme Enables Diverse Peptide Cross-linking

Eastman

Mifflin

Oblad

et al. 2023

ACS Bio Med Chem Au

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Ribosomally produced and post-translationally modified polypeptides (RiPPs) are a diverse group of natural products that are processed by a variety of enzymes to their biologically relevant forms. PapB is a member of the radical S-adenosyl-l-methionine (rSAM) superfamily that introduces thioether cross-links between Cys and Asp residues in the PapA RiPP. We report that PapB has high tolerance for variations in the peptide substrate. Our results demonstrate that branched side chains in the thiol- and carboxylate-containing residues are processed and that lengthening of these groups to homocysteine and homoglutamate does not impair the ability of PapB to form thioether cross-links. Remarkably, the enzyme can even cross-link a peptide substrate where the native Asp carboxylate moiety is replaced with a tetrazole. We show that variations to residues embedded between the thiol- and carboxylate-containing residues are tolerated by PapB, as peptides containing both bulky (e.g., Phe) and charged (e.g., Lys) side chains in both natural L- and unnatural D-forms are efficiently cross-linked. Diastereomeric peptides bearing (2S,3R)- and (2S,3S)-methylaspartate are processed by PapB to form cyclic thioethers with markedly different rates, suggesting the enzymatic hydrogen atom abstraction event for the native Asp-containing substrate is diastereospecific. Finally, we synthesized two diastereomeric peptide substrates bearing E- and Z-configured γ,δ-dehydrohomoglutamate and show that PapB promotes addition of the deoxyadenosyl radical (dAdo•) instead of hydrogen atom abstraction. In the Z-configured γ,δ-dehydrohomoglutamate substrate, a fraction of the dAdo-adduct peptide is thioether cross-linked. In both cases, there is evidence for product inhibition of PapB, as the dAdo-adducts likely mimic the native transition state where dAdo• is poised to abstract a substrate hydrogen atom. Collectively, these findings provide critical insights into the arrangement of reacting species in the active site of the PapB, reveal unusual promiscuity, and highlight the potential of PapB as a tool in the development peptide therapeutics.

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Section: Discussionmentioning

confidence: 99%

A Promiscuous rSAM Enzyme Enables Diverse Peptide Cross-linking

Eastman

Mifflin

Oblad

et al. 2023

ACS Bio Med Chem Au

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“…To increase the speed of screening, automated equipment such as robotic liquid handling units and colony picking systems have been developed (Nirantar, 2021). In highthroughput screening methods, colorimetric or fluorogenic substrates are often used to measure enzymatic activity (Giger et al, 2013).…”

Section: High-throughput Screeningmentioning

confidence: 99%

Enabling technology and core theory of synthetic biology

Zhang

Liu

Dai

et al. 2023

Sci. China Life Sci.

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Synthetic biology provides a new paradigm for life science research (“build to learn”) and opens the future journey of biotechnology (“build to use”). Here, we discuss advances of various principles and technologies in the mainstream of the enabling technology of synthetic biology, including synthesis and assembly of a genome, DNA storage, gene editing, molecular evolution and de novo design of function proteins, cell and gene circuit engineering, cell-free synthetic biology, artificial intelligence (AI)-aided synthetic biology, as well as biofoundries. We also introduce the concept of quantitative synthetic biology, which is guiding synthetic biology towards increased accuracy and predictability or the real rational design. We conclude that synthetic biology will establish its disciplinary system with the iterative development of enabling technologies and the maturity of the core theory.

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“…It largely depends on prior knowledge regarding catalytic mechanisms and physicochemical properties of the protein. Second, through directed evolution of protein stability and activity, improved enzymes can be obtained directly . This method usually involves induction of random mutations and high-throughput screening to drive the process of protein evolution in a certain direction without depending on the structure and catalytic mechanism of proteins.…”

Section: Prospectivesmentioning

confidence: 99%

“…Second, through directed evolution of protein stability and activity, improved enzymes can be obtained directly. 116 This method usually involves induction of random mutations and high-throughput screening to drive the process of protein evolution in a certain direction without depending on the structure and catalytic mechanism of proteins. Third, the semirational design strategy combines rational design and directed evolution to improve efficiency by constructing more reasonable mutant libraries.…”

Section: Prospectivesmentioning

confidence: 99%

Progress and Prospects of Natural Glycoside Sweetener Biosynthesis: A Review

Qu,

Liu,

et al. 2023

J. Agric. Food Chem.

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To achieve an adequate sense of sweetness with a healthy low-sugar diet, it is necessary to explore and produce sugar alternatives. Recently, glycoside sweeteners and their biosynthetic approaches have attracted the attention of researchers. In this review, we first outlined the synthetic pathways of glycoside sweeteners, including the key enzymes and rate-limiting steps. Next, we reviewed the progress in engineered microorganisms producing glycoside sweeteners, including de novo synthesis, whole-cell catalysis synthesis, and in vitro synthesis. The applications of metabolic engineering strategies, such as cofactor engineering and enzyme modification, in the optimization of glycoside sweetener biosynthesis were summarized. Finally, the prospects of combining enzyme engineering and machine learning strategies to enhance the production of glycoside sweeteners were discussed. This review provides a perspective on synthesizing glycoside sweeteners in microbial cells, theoretically guiding the bioproduction of glycoside sweeteners.

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Directed Evolution Methods for Enzyme Engineering

Cited by 22 publications

References 113 publications

A Promiscuous rSAM Enzyme Enables Diverse Peptide Cross-linking

A Promiscuous rSAM Enzyme Enables Diverse Peptide Cross-linking

Enabling technology and core theory of synthetic biology

Progress and Prospects of Natural Glycoside Sweetener Biosynthesis: A Review

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