Engineered enzymes for the synthesis of pharmaceuticals and other high-value products

Reetz, Manfred T.; Qu, Ge; Sun, Zhoutong

doi:10.1038/s44160-023-00417-0

Cited by 53 publications

(18 citation statements)

References 135 publications

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“…Directed evolution has become a powerful tool for positive engineering of almost any enzyme. 45,46 Enzymatic activity undoubtedly plays a pivotal part in enzymatic engineering and is a key focus in the industrialization of enzymes, whether through mutagenesis of the protein itself or methods such as enzyme immobilization. Synergistic enzymatic properties, including cofactor affinity, thermal stability, and substrate tolerance must be taken into consideration in enzymatic engineering, especially for an enzyme with already promising activity such as 7α-HSDH.…”

Section: Discussionmentioning

confidence: 99%

Not exclusively the activity, but the sweet spot: a dehydrogenase point mutation synergistically boosts activity, substrate tolerance, thermal stability and yield

Cen,

Zhang,

Jiang

et al. 2024

Org. Biomol. Chem.

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Section: Discussionmentioning

confidence: 99%

Not exclusively the activity, but the sweet spot: a dehydrogenase point mutation synergistically boosts activity, substrate tolerance, thermal stability and yield

Cen,

Zhang,

Jiang

et al. 2024

Org. Biomol. Chem.

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“…With the rapid growth in recombinase technology and protein engineering, tremendous efforts in site-directed mutagenesis and rational design have made it possible to artificially modify enzymes with higher thermal tolerance. 13,14 In reality, owing to the well-known stability−activity trade-off, especially when the mutation site is located near the active site, it is extremely challenging to balance enzyme activity and thermal tolerance. 15 Rational design requires many iterative experiments, which is extremely difficult for enzymes that lack high-throughput screening methods.…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, proteins obtained from thermophilic bacteria typically exhibit optimal catalytic efficiency at high temperatures, which is expensive for biotransformation processes. With the rapid growth in recombinase technology and protein engineering, tremendous efforts in site-directed mutagenesis and rational design have made it possible to artificially modify enzymes with higher thermal tolerance. , In reality, owing to the well-known stability–activity trade-off, especially when the mutation site is located near the active site, it is extremely challenging to balance enzyme activity and thermal tolerance . Rational design requires many iterative experiments, which is extremely difficult for enzymes that lack high-throughput screening methods. , Owing to these factors, a universal method without extensive structural knowledge or complex experimental screening is undoubtedly more promising, as it can provide faster and more widely applicable strategies to improve enzyme thermal tolerance.…”

Section: Introductionmentioning

confidence: 99%

Rational Design of Spontaneous Self-Cyclization Enzymes In Vivo and In Vitro with Improved Thermal Tolerance and Activity

Wei,

Kang,

Zhao

et al. 2024

ACS Catal.

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Enzymes have selectivity, require mild catalytic conditions, and are important cornerstones in many industrial catalytic processes. Protein self-cyclization has opened up the possibility of preserving fragile enzymes during long-term high-temperature catalysis. However, the mechanism for self-cyclization and improvement of thermal tolerance have not been elucidated, severely limiting their industrial applications. Herein, we provide a strategy for the rational design of fusion proteins based on structural analysis to obtain cyclized enzymes with improved properties. First, we constructed fusion proteins that preferentially translated SpyCatcher (CBT) or SpyTag (TBC), both of which could form stable single selfcyclization with significantly improved thermal tolerance. Especially, the thermal half-life of TBC obtained by modifying the N-terminal SpyTag at 40 °C was 10.83 times that of wild enzymes. Structural analysis revealed that the terminus of the protein, which preferentially translated to SpyCatcher, folded into a conformation that adversely affected stability. In addition, the structure of the catalytic pocket and the orientation of the catalytic residues of CBT were significantly different from those of the wild-type enzymes, which led to a decrease in the catalytic activity. These conclusions were confirmed when another industrial enzyme, sucrose phosphorylase, was cyclized. Finally, the cyclized glucosidase was also superior to the wild-type strain for the preparation of ginsenoside F1 at high titers and as a whole-cell catalyst for extended use. In conclusion, we demonstrated for the first time that conjugated long oligopeptide SpyCatcher significantly affected the catalytic activity and stability of cyclized enzymes. It was necessary to preferentially translate units with less steric hindrance to reduce their impact on the protein structure. The rational design of cyclized enzymes based on structural analysis provides a simple and effective strategy for the modification of industrial enzymes with poor thermal tolerance, providing considerable prospects for biosynthesis in vivo and in vitro.

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“…The utilization of enzymes in stereoselective synthesis has experienced a resurgence in both academic and industrial domains over the past few decades . Notably, the gradual adoption of enzyme catalysts in main stream organic chemistry changed with the advent of directed enzyme evolution, first with the aim to enhance resistance to hostile organic solvents and subsequently with the purpose to control enantioselectivity .…”

Section: Introductionmentioning

confidence: 99%

Enzymatic Stereodivergent Access to Fluorinated β-Lactam Pharmacophores via Triple-Parameter Engineered Ketoreductases

Mei,

Li,

Han

et al. 2024

ACS Catal.

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Enzyme-catalyzed stereodivergent synthesis to access all possible stereoisomers of organofluorine compounds bearing multiple stereogenic centers remains an important and challenging subject. By integrative data-driven mining and mechanism-guided engineering of ketoreductases, we identified a stereodivergent biocatalytic platform to produce four stereoisomeric fluoroalkyl amino acid esters bearing two vicinal stereocenters. Fast triple-parameter coevolution via a semirational CAST/ISM strategy provided the quadruple mutant M5 (A140K/ L203T/G92A/V84I) of ketoreductase BgADH not only displayed high stereoselectivity toward the target stereoisomers (99:1 dr, 99% ee) but also observed with enhanced activity (k cat /K m , 6.3 folds) and improved thermostability (T 50 15 , 4 °C). Crystal structural analysis and molecular dynamics (MD) simulation studies unveil two residues (A140 and F148) of BgADH to be the key sites that are responsible for the control of the stereoselectivity. The L203T/G92A mutation enhanced activity by affecting the conformational distribution of the α-helix within the active-site region, and V84I improved thermal stability by strengthening the hydrogen bonding network with neighboring residues. The synthetic utility was further demonstrated by fluoroalkyl substrate scope expansion, gramscale reactions (648 g L −1 day −1 ), and synthetic transformations to chiral fluorinated β-lactams that are the antibiotic carbapenem cores.

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Engineered enzymes for the synthesis of pharmaceuticals and other high-value products

Cited by 53 publications

References 135 publications

Not exclusively the activity, but the sweet spot: a dehydrogenase point mutation synergistically boosts activity, substrate tolerance, thermal stability and yield

Not exclusively the activity, but the sweet spot: a dehydrogenase point mutation synergistically boosts activity, substrate tolerance, thermal stability and yield

Rational Design of Spontaneous Self-Cyclization Enzymes In Vivo and In Vitro with Improved Thermal Tolerance and Activity

Enzymatic Stereodivergent Access to Fluorinated β-Lactam Pharmacophores via Triple-Parameter Engineered Ketoreductases

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