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

Transaminase (TA) is a crucial biocatalyst for enantioselective production of the herbicide L-phosphinothricin (L-PPT). The use of enzymatic cascades has been shown to effectively overcome the unfavorable thermodynamic equilibrium of TA-catalyzed transamination reaction, also increasing demand for TA stability. In this work, a novel thermostable transaminase (PtTA) from Pseudomonas thermotolerans was mined and characterized. The PtTA showed a high specific activity (28.63 U/mg) towards 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO), with excellent thermostability and substrate tolerance. Two cascade systems driven by PtTA were developed for L-PPT biosynthesis, including asymmetric synthesis of L-PPT from PPO and deracemization of D, L-PPT. For the asymmetric synthesis of L-PPT from PPO, a three-enzyme cascade was constructed as a recombinant Escherichia coli (E. coli G), by co-expressing PtTA, glutamate dehydrogenase (GluDH) and D-glucose dehydrogenase (GDH). Complete conversion of 400 mM PPO was achieved using only 40 mM amino donor L-glutamate. Furthermore, by coupling D-amino acid aminotransferase (Ym DAAT) from Bacillus sp. YM-1 and PtTA, a two-transaminase cascade was developed for the one-pot deracemization of D, L-PPT. Under the highest reported substrate concentration (800 mM D, L-PPT), a 90.43% L-PPT yield was realized. The superior catalytic performance of the PtTA-driven cascade demonstrated that the thermodynamic limitation was overcome, highlighting its application prospect for L-PPT biosynthesis. KEY POINTS: • A novel thermostable transaminase was mined for L-phosphinothricin biosynthesis. • The asymmetric synthesis of L-phosphinothricin was achieved via a three-enzyme cascade. • Development of a two-transaminase cascade for D, L-phosphinothricin deracemization.

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

2-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.

Bioconversion of food waste to crayfish feed using solid-state fermentation with yeast.

In order to realize the value-added utilization of food waste (FW), the preparation of crayfish (Procambarus clarkii) feed by yeast fermentation was investigated. Firstly, the suitable fermentation condition was obtained through a single factor experiment as follows: the initial moisture of the FW was adjusted to 60% with bran and inoculated with a 2% yeast mixture (Saccharomyces cerevisiae, Candida utilis, and Yarrowia lipolytica, 3:2:1) followed by aerobic solid-state fermentation for 7 days. The crude protein and acid-soluble protein contents in the fermented feed were 25.14% and 5.16%, which were increased by 8% and 140.67%, respectively. The crude fat content was 0.74%, decreased by 68.29%. The content of antioxidant glutathione (571.78 µg/g) increased 63.33%, and the activities of protease and amylase increased nearly 9 and 3 times, respectively. The maximum degradation rates of aflatoxin B1, zearalenone, and deoxynivalenol were 63.83%, 77.52%, and 80.16%, respectively. The fermented feeds were evaluated by substituting (0%, 10%, 30%, 50%, and 100%) commercial diet for crayfish (30-day culture period). When the replacement proportion was 30%, the weight gain of crayfish reached 44.87% (initial body weight 13.98 ± 0.41 g), which was significantly increased by 10.25% compared with the control (p = 0.0005). In addition, the lysozyme and SOD enzyme activities in crayfish hepatopancreas were also increased significantly. Our findings suggest that yeast-fermented feed from FW can replace 30% of crayfish's conventional diet, which may improve crayfish's antioxidant capacity and enhance non-specific immunity by providing molecules such as glutathione.