L-Phosphinothricin (L-PPT) is the active compound of a broad-spectrum herbicide. In this study, we biosynthesized L-PPT via the asymmetric reduction of 2-carboxy-4-(hydroxymethylphosphoryl) butyric acid (PPO) using a whole-cell biocatalyst. Glutamate dehydrogenase from Pseudomonas moorei ( Pm GluDH), Glucose dehydrogenase from Exiguobacterium sibiricum ( Es GDH) and NAD kinase from Corynebacterium glutamicum ( Cg NADK) were coexpressed in E. coli , significantly increasing the catalytic efficiency by regenerating more NADPH to promote PPO reduction. Following the optimization of balancing the co-expression of the three enzymes in pETDuet-1 and pCDFDuet-1, the coexpressing strain increases in the intracellular NADP(H) concentration to 54.9 μmol/g DCW, which was 3.8-fold higher than the control. The co-expression system exhibited optimal activity at a temperature of 35°C, pH 7.5, and the glucose to PPO molar ratio of 1.4:1. In a 30 L bioconversion system the final concentration and productivity of L-PPT reached 79.8 g/L (>99% ee ) and 1440 g/(L·d), respectively. These results will contribute to the industrial biological production of L-PPT.
Hydroxysteroid dehydrogenases (HSDHs) catalyze the oxidation/reduction of hydroxyl/keto groups of steroids with high regio- or stereoselectivity, playing an essential role in producing optically pure chemicals. In this work, a novel approach was developed to simultaneously improve the stability and activity of 7β-hydroxysteroid dehydrogenase (7β-HSDH) by combining B-factor analysis and computer-aided prediction. Several advantageous mutants were identified, and the most promising variant, S51Y/P202Y, exhibited 2.3-fold improvements in catalytic activity, 3.3-fold in half-life at 40°C, and 4.7-fold in catalytic efficiency (kcat/Km), respectively. Structural modeling analysis showed that the shortened reversible oxidation reaction catalytic distance and the strengthened residue interactions compared to the wild type were attributed to the improved stability and activity of the obtained mutants. To synthesize ursodeoxycholic acid cost-effectively by mutant S51Y/P202Y, a NAD-kinase was employed to facilitate the substitution of nicotinamide adenine dinucleotide phosphate (NADP+) with nicotinamide adenine dinucleotide (NAD+) in the whole-cell catalysis system. The substrate 7-ketolithocholic acid (100 mM) was converted completely in 0.5 h, achieving a space-time yield of 1,887.3 g L−1 d−1. This work provided a general target-oriented strategy for obtaining stable and highly active dehydrogenase for efficient biosynthesis.
NAD(P)H-dependent oxidoreductases are crucial biocatalysts for synthesizing chiral compounds. Yet, the industrial implementation of enzymatic redox reactions is often hampered by an insufficient supply of expensive nicotinamide cofactors. Here, a cofactor self-sufficient whole-cell biocatalyst was developed for the enzymatic asymmetric reduction of 2-oxo-4-[(hydroxy)(-methyl)phosphinyl] butyric acid (PPO) to L-phosphinothricin (L-PPT). The endogenous NADP + pool was significantly enhanced by regulating Preiss-Handler pathway toward NAD(H) synthesis and, in the meantime, introducing NAD kinase to phosphorylate NAD(H) toward NADP + . The intracellular NADP(H) concentration displayed a 2.97-fold increase with the strategy compared with the wild-type strain. Furthermore, a recombinant multi-enzyme cascade biocatalytic system was constructed based on the Escherichia coli chassis. In order to balance multi-enzyme co-expression levels, the strategy of modulating rate-limiting enzyme Pm GluDH by RBS strengths regulation successfully increased the catalytic efficiency of PPO conversion. Finally, the cofactor self-sufficient whole-cell biocatalyst effectively converted 300 mM PPO to L-PPT in 2 h without the need to add exogenous cofactors, resulting in a 2.3-fold increase in PPO conversion (%) from 43% to 100%, with a high space-time yield of 706.2 g L −1 d −1 and 99.9% ee . Overall, this work demonstrates a technological example for constructing a cofactor self-sufficient system for NADPH-dependent redox biocatalysis.
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