Development of an aminotransferase‐driven biocatalytic cascade for deracemization of d,l‐phosphinothricin

Abstract

Abstract2‐oxo‐4‐[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) is the essential precursor keto acid for the asymmetric biosynthesis of herbicide l‐phosphinothricin (l‐PPT). Developing a biocatalytic cascade for PPO production with high efficiency and low cost is highly desired. Herein, a d‐amino acid aminotransferase from Bacillus sp. YM‐1 (Ym DAAT) with high activity (48.95 U/mg) and affinity (Km = 27.49 mM) toward d‐PPT was evaluated. To circumvent the inhibition of by‐product d‐glutamate (d‐Glu), an amino acceptor (α‐ketoglutarate) regeneration cascade was constructed as a recombinant Escherichia coli (E. coli D), by coupling Ym d‐AAT, d‐aspartate oxidase from Thermomyces dupontii (TdDDO) and catalase from Geobacillus sp. CHB1. Moreover, the regulation of the ribosome binding site was employed to overcome the limiting step of expression toxic protein TdDDO in E. coli BL21(DE3). The aminotransferase‐driven whole‐cell biocatalytic cascade (E. coli D) showed superior catalytic efficiency for the synthesis of PPO from d,l‐phosphinothricin (d,l‐PPT). It revealed the production of PPO exhibited high space–time yield (2.59 g L−1 h−1) with complete conversion of d‐PPT to PPO at high substrate concentration (600 mM d,l‐PPT) in 1.5 L reaction system. This study first provides the synthesis of PPO from d,l‐PPT employing an aminotransferase‐driven biocatalytic cascade.

One-Pot Two-Stage Biocatalytic Cascade to Produce l-Phosphinothricin by Two Enantioselective Complementary Aminotransferases at High Substrate Loading via a Deracemization Process

“…As summarized in Table , the K m value of ( R )- Ach AT was 14.20 mM, indicating its high affinity to the D-PPT. The k cat /K m value of ( R )- Ach AT was 2.01 × 10 3 s –1 M –1 , which was comparable to that of reported Ym AT (2.04 × 10 3 s –1 M –1 ) and 2.09 fold higher than that of reported Rg DAAO (0.96 × 10 3 s –1 M –1 ), indicating the significant advantage of catalytic efficiency of ( R )- Ach AT toward D-PPT. For ( S )- Ab AT, the K m and k cat values of PPO were 22.8 mM and 15.81 s –1 , respectively.…”

Identification of a novel thermostable transaminase and its application in L-phosphinothricin biosynthesis

“…1 b). Based on our previous report (Liu et al 2023 ), the Ym DAAT exhibited high activity (48.95 U/mg) and affinity ( K m = 27.49 mM) towards D-PPT. Thus, Ym DAAT was employed to catalyze the amino transformation between D-PPT and PPO, followed by L-PPT synthesis catalyzed by Pt TA.…”

“…The asymmetric synthesis of L-PPT from 4-(hydroxy(methyl)phosphoryl)-2-oxobutanoic acid (PPO) catalyzed by fold-type I TAs with 100% theoretical yield and strict ( S )-enantioselectivity has continuously attracted the interest of researchers (Cheng et al 2021 ; Horsman and Zechel 2017 ). However, several TAs developed for the asymmetric synthesis of L-PPT suffered low equilibrium constant, resulting in an unfavorable thermodynamic equilibrium, which further led to the inability to achieve the theoretical yield (Liu et al 2023 ; Zhu and Hua 2009 ), for example, the TA from Citrobacter koseri ( Ck TA), which showed a maximum conversion of 93.3% at 100 mM substrate concentration (Jia et al 2019 ), and the TA from Pseudomonas fluorescens ( Pf TA), which we previously reported to yield 79% L-PPT after 24 h reaction at 500 mM substrate (Jin et al 2019 ). Removal of by-product can effectively shift the equilibrium in the transamination reaction, one of the most attractive strategies was to construct an enzymatic cascade by coupling amino acid dehydrogenases (AADHs, EC 1.4.1.X) (Hepworth et al 2017 ; Mathew et al 2023 ), such as alanine dehydrogenase (AlaDH, EC 1.4.1.1) or glutamate dehydrogenase (GluDH, EC 1.4.1.2) (Wu et al 2023 ).…”