The identification and application of a robust ω-transaminase with high tolerance towards substrates and isopropylamine from a directed soil metagenome

Xie, Youyu; Wang, Jiguo; Yang, Lin; Wang, Wei; Liu, Qinghai; Wang, Hualei; Wei, Dongzhi

doi:10.1039/d1cy02032c

Cited by 10 publications

(9 citation statements)

References 67 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…CC1012 is a robust TA that was characterized by our group [18] in a previous study. It showed not only excellent thermostability but also high tolerance towards IPA (up to 2 M).…”

Section: Resultsmentioning

confidence: 96%

“…It showed not only excellent thermostability but also high tolerance towards IPA (up to 2 M). As an industry‐favored amine donor owing to its low cost and achirality, [19] IPA is not widely accepted by most natural ω‐TAs [18] …”

Section: Resultsmentioning

confidence: 99%

“…As an industry-favored amine donor owing to its low cost and achirality, [19] IPA is not widely accepted by most natural ω-TAs. [18] Although a few ω-TAs can accept IPA, they usually require 10-200 equivalents to drive unfavorable equilibrium. [20] CC1012 could use only two equivalents of IPA to completely aminate 1-Boc-3-piperidone in a previous report.…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Mechanism‐Guided Computational Design of ω‐Transaminase by Reprograming of High‐Energy‐Barrier Steps

Yang

Zhang

et al. 2022

Angew Chem Int Ed

Self Cite

View full text Add to dashboard Cite

ω-Transaminases (ω-TAs) show considerable potential for the synthesis of chiral amines. However, their low catalytic efficiency towards bulky substrates limits their application, and complicated catalytic mechanisms prevent precise enzyme design. Herein, we address this challenge using a mechanism-guided computational enzyme design strategy by reprograming the transition and ground states in key reaction steps. The common features among the three high-energy-barrier steps responsible for the low catalytic efficiency were revealed using quantum mechanics (QM). Five key residues were simultaneously tailored to stabilize the rate-limiting transition state with the aid of the Rosetta design. The 14 top-ranked variants showed 16.9-143fold improved catalytic activity. The catalytic efficiency of the best variant, M9 (Q25F/M60W/W64F/I266A), was significantly increased, with a 1660-fold increase in k cat / K m and a 1.5-26.8-fold increase in turnover number (TON) towards various indanone derivatives.

show abstract

“…CC1012 is a robust TA that was characterized by our group [18] in a previous study. It showed not only excellent thermostability but also high tolerance towards IPA (up to 2 M).…”

Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Mechanism‐Guided Computational Design of ω‐Transaminase by Reprograming of High‐Energy‐Barrier Steps

Yang

Zhang

et al. 2022

Angew Chem Int Ed

Self Cite

View full text Add to dashboard Cite

show abstract

“…[18][19][20] Metagenomic approaches provide a reasonable and promising method for obtaining enzymes with target qualities. A robust ω-transaminase with high tolerance towards substrates (0.75 M) and isopropylamine (2 M) was identified from amidogen-enriched soil metagenome by Xie et al [21] Similarly, a robust transaminase that exhibits significant tolerance to IPA and DMSO was identified by screening the metagenome from the environment with surfactants and cleaning chemicals. [22] These discoveries of novel enzymes exhibited potential for industrial applications using the metagenomic approach, demonstrating the unrivalled ability of metagenomic approaches to identify robust enzymes.…”

mentioning

confidence: 99%

The identification of a robust leucine dehydrogenase from a directed soil metagenome for efficient synthesis of L‐2‐aminobutyric acid

Liu

Zhong

Luo

et al. 2023

Biotechnology Journal

Self Cite

View full text Add to dashboard Cite

L‐2‐aminobutyric acid (L‐2‐ABA) is a chiral precursor for the synthesis of anti‐epileptic drug levetiracetam and anti‐tuberculosis drug ethambutol. Asymmetric synthesis of L‐2‐ABA by leucine dehydrogenases has been widely developed. However, the limitations of natural enzymes, such as poor stability, low catalytic efficiency, and inhibition of high‐concentration substrates, limit large‐scale applications. Herein, by directed screening of a metagenomic library from unnatural amino acid‐enriched environments, a robust leucine dehydrogenase, TvLeuDH, was identified, which exhibited high substrate tolerance and excellent enzymatic activity towards 2‐oxobutyric acid. In addition, TvLeuDH has strong affinity for NADH. Subsequently, a three‐enzyme co‐expression system containing L‐threonine deaminase, TvLeuDH, and glucose dehydrogenase was established. By optimizing reaction conditions, 1.5 M L‐threonine could be converted to L‐2‐ABA with a 99% molar conversion rate and a space‐time yield of 51.5 g·L−1·h−1. In this process, no external coenzyme was added. The robustness of TvLeuDH allowed the reaction to be performed without the addition of extra salt as the buffer, demonstrating the simplest reaction system currently reported. These unique properties for the efficient and environmentally friendly production of chiral amino acids make TvLeuDH a particularly promising candidate for industrial applications, which reveals the great potential of directed metagenomics for industrial biotechnology.

show abstract

“…[20] CC1012 could use only two equivalents of IPA to completely aminate 1-Boc-3-piperidone in a previous report. [18] Thus, we chose CC1012 for further studies and used IPA as the amine donor.…”

Section: Resultsmentioning

confidence: 99%

Mechanism‐Guided Computational Design of ω‐Transaminase by Reprograming of High‐Energy‐Barrier Steps

Yang

Zhang

et al. 2022

Angewandte Chemie

Self Cite

View full text Add to dashboard Cite

ω‐Transaminases (ω‐TAs) show considerable potential for the synthesis of chiral amines. However, their low catalytic efficiency towards bulky substrates limits their application, and complicated catalytic mechanisms prevent precise enzyme design. Herein, we address this challenge using a mechanism‐guided computational enzyme design strategy by reprograming the transition and ground states in key reaction steps. The common features among the three high‐energy‐barrier steps responsible for the low catalytic efficiency were revealed using quantum mechanics (QM). Five key residues were simultaneously tailored to stabilize the rate‐limiting transition state with the aid of the Rosetta design. The 14 top‐ranked variants showed 16.9–143‐fold improved catalytic activity. The catalytic efficiency of the best variant, M9 (Q25F/M60W/W64F/I266A), was significantly increased, with a 1660‐fold increase in kcat/Km and a 1.5–26.8‐fold increase in turnover number (TON) towards various indanone derivatives.

show abstract

The identification and application of a robust ω-transaminase with high tolerance towards substrates and isopropylamine from a directed soil metagenome

Abstract: The ω-transaminase-mediated asymmetric amination of a ketone substrate has gained significant attention for its immense potential to synthesize chiral amine pharmaceuticals and precursors. However, few of these have been authentically...

Cited by 10 publications

References 67 publications

Mechanism‐Guided Computational Design of ω‐Transaminase by Reprograming of High‐Energy‐Barrier Steps

Mechanism‐Guided Computational Design of ω‐Transaminase by Reprograming of High‐Energy‐Barrier Steps

The identification of a robust leucine dehydrogenase from a directed soil metagenome for efficient synthesis of L‐2‐aminobutyric acid

Mechanism‐Guided Computational Design of ω‐Transaminase by Reprograming of High‐Energy‐Barrier Steps

Contact Info

Product

Resources

About