Biocatalytic asymmetric synthesis of l-phosphinothricin using a one-pot three enzyme system and a continuous substrate fed-batch strategy

Zhou, Haisheng; Meng, Lijun; Yin, Xinjian; Liu, Yayun; Wu, Jianping; Xu, Gang; Wu, Mianbin; Yang, Lirong

doi:10.1016/j.apcata.2019.117239

Cited by 16 publications

(16 citation statements)

References 30 publications

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“…Another example of a reaction run as fed-batch was reported by Zhou and co-workers (2020) in the production of Lphosphinothricin, an active ingredient in common commercial herbicides. [115] Here substrate inhibition was found at concentrations above 35 mM substrate, with decreasing initial reaction rate after this point. For this reason, they chose to run the reaction in a 100 L stainless-steel bioreactor operated as fedbatch, with a start-and end working volume of 40 L and 90 L, respectively.…”

Section: Substrate Solubility and Inhibitionmentioning

confidence: 82%

“…Another example is the glutamate dehydrogenase/alcohol dehydrogenase (GLDH/ADH) system as reported by Zhou and co-workers and seen in Scheme 8. [115] Unlike the LDH/GDH co-factor regeneration system, the GLDH/ADH system regenerates the amine donor, here Lglutamate, using ammonia, NADH and isopropanol as amine supplement, co-factor and reducing agent, respectively. By utilizing an amine donor recycling system instead of reduction, only stoichiometric amounts of amine donor would be required in theory, ensuring a cheaper overall process.…”

Section: Amine Donor Regeneration or Reductionmentioning

confidence: 99%

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Considerations for the Scale‐up of in vitro Transaminase‐Catalyzed Asymmetric Synthesis of Chiral Amines

Madsen

Woodley

2023

ChemCatChem

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In vitro transaminase catalyzed asymmetric synthesis is not a new phenomenon in the biocatalytic toolbox. Our group published a review on process consideration for these reactions in 2011, in which process metrics and the unfavorable reaction equilibrium was deduced to be the main bottlenecks at the time. Now, a decade later, this has been solved by different process methods presented in this review, with the development of enzymatic cascades one of the main solutions. Today, the new bottleneck is the downstream processing, due to the complexity of transaminase reactions, in which a ketone and an amine as substrates and a new ketone and a new amine as products. How to recover the produced amine from the resulting product mixture is considered and will be the key challenge for scale‐up of such systems in the near future.

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Section: Substrate Solubility and Inhibitionmentioning

confidence: 82%

Section: Amine Donor Regeneration or Reductionmentioning

confidence: 99%

Considerations for the Scale‐up of in vitro Transaminase‐Catalyzed Asymmetric Synthesis of Chiral Amines

Madsen

Woodley

2023

ChemCatChem

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“…[8] In recent years, a variety of synthetic methods of non-natural amino acids, such as reductive amination of α-keto acids, transaminase-mediated amino transfer, asymmetric addition of ammonia, and aldol condensation, have been developed. [9][10][11][12] Among these methods, the reductive amination catalysed by amino acid dehydrogenases (AADHs) has been widely studied in the synthesis of l-PPT, due to its high stereoselectivity of product, no by-product, and high equilibrium constant. [13,14] Rencently, nicotinamide adenine dinucleotide phosphate (NADPH)-dependent glutamate dehydrogenase (GluDH) has been developed, and its catalytic activity was substantially increased toward l-PPT proketone 2-oxo-4-[(hydroxy) (methyl) phosphino] buty-ric acid (PPO).…”

Section: Introductionmentioning

confidence: 99%

Development of an NAD(H)‐Driven Biocatalytic System for Asymmetric Synthesis of Chiral Amino Acids

et al. 2022

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Chiral amino acids are extensively applied in the pharmaceutical, food, cosmetic, and agricultural industries. As a representative example, l‐phosphinothricin (l‐PPT, a chiral non‐natural amino acid) is a broad‐spectrum herbicide. An NAD(H)‐driven biocatalytic system for the asymmetric synthesis of chiral amino acids (focused on l‐PPT) with high efficiency and low cost is highly desired. The key for the development of such biocatalytic system is to obtain an NADH‐dependent biocatalyst with high catalytic performance toward l‐PPT pro‐ketone PPO. Herein, an engineered glutamate dehydrogenase from Lysinibacillus composti (LcGluDH) with desired activity was obtained by gene mining and protein engineering. In silico analyses suggested that the volume of substrate‐binding pocket was substantially enlarged from 330.5 Å3 to 409.6 Å3. The stability of LcGluDH was increased (Tm value increased from 47.3 °C to 65.3 °C) by introducing positively charged amino acids or aromatic amino acids at position 375. The desired biocatalytic system was constructed by coupling the engineered LcGluDH and an NAD+‐dependent FDH. Through this biocatalytic system, the batch production of l‐PPT exhibited high space‐time yield (207.3 g ⋅ L−1 ⋅ day−1) with strict stereoselectivity (ee of l‐PPT>99%). Furthermore, eight other chiral amino acids were synthesised by the developed NAD(H)‐driven biocatalytic system with high ee values.

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“…Noncanonical amino acids, which are not naturally encoded into proteins, have unusual biological activities in their optically pure form and are important building blocks of agrochemicals and pharmaceuticals. − For example, l -phosphinothricin is a promising herbicide; l - tert -leucine, l -homoalanine, and l -phenylglycine are used to synthesize the anti-AIDS drug atazanavir, the anti-epileptic levetiracetam, and the antibiotic clopidogrel, respectively; − l -norvaline and l -homophenylalanine are the building blocks for angiotensin converting enzyme (ACE) inhibitors, such as perindopril, benazepril, captopril, enalapril, lisinopril, and quinapril. − Biocatalytic synthesis of these chiral amino acids with high optical purity is a reliable, scalable, and preferred route owing to its remarkable chemo-, regio-, and stereoselectivity features, coupled with safety and environmental benefits. , Among them, the asymmetric reductive amination of prochiral keto acids catalyzed by amino acid dehydrogenase (AADH) is a very efficient strategy for the production of noncanonical chiral amino acids, − with advantages like direct use of cheap ammonia as the amino donor, high atom utilization, and high optical purity of the products (often exceeding 99%). However, though numerous AADHs have been identified and classified according to their substrate specificities, such as glutamate dehydrogenase (GluDH), leucine dehydrogenase (LeuDH), and phenylalanine dehydrogenase (PheDH), only a few AADHs have considerable potential as biocatalysts for the reductive amination processes .…”

Section: Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Computational Redesign of the Substrate Binding Pocket of Glutamate Dehydrogenase for Efficient Synthesis of Noncanonical l-Amino Acids

Wang

Zhou²,

et al. 2022

ACS Catal.

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Noncanonical amino acids are attractive molecules in synthetic chemistry as important building blocks for pharmaceuticals and agrochemicals. Asymmetric reductive amination of prochiral keto acids catalyzed by glutamate dehydrogenase (GluDH) is an efficient process for the production of these molecules. However, the stringent substrate specificity and the lack of an engineering strategy hampered its wide application in substrate-oriented chiral carbon−nitrogen bond formation. To address this issue, first, the substrate recognition mechanism of GluDH was investigated using CdGluDH (an NADHdependent GluDH from Clostridium difficile) and four molecules including the natural and three non-natural substrates as models through computational simulation and experimental verification. Second, a computational engineering strategy based on reducing the pocket steric hindrance and tuning enzyme−substrate electrostatic interactions and/or X−H•••π interactions was developed to systematically redesign the binding pocket with mutations of 10 sites to accept distinct substrates. As a result, tailor-made variants were created to synthesize the corresponding high-value products, L-norvaline, L-phosphinothricin, and L-homophenylalanine, with specific activities improved 2.3-fold, 916.2-fold, and from no activity to 66.64 U/mg, respectively. Finally, reductive amination efficiency of the variants was tested by a simulated industrial catalytic reaction, demonstrating that the computational enzyme redesign strategy is promising in enhancing the production of noncanonical amino acids using GluDHs.

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Biocatalytic asymmetric synthesis of l-phosphinothricin using a one-pot three enzyme system and a continuous substrate fed-batch strategy

Cited by 16 publications

References 30 publications

Considerations for the Scale‐up of in vitro Transaminase‐Catalyzed Asymmetric Synthesis of Chiral Amines

Considerations for the Scale‐up of in vitro Transaminase‐Catalyzed Asymmetric Synthesis of Chiral Amines

Development of an NAD(H)‐Driven Biocatalytic System for Asymmetric Synthesis of Chiral Amino Acids

Computational Redesign of the Substrate Binding Pocket of Glutamate Dehydrogenase for Efficient Synthesis of Noncanonical l-Amino Acids

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