A High‐Throughput Visual Screen for the Directed Evolution of Cβ‐stereoselectivity of L‐threonine Aldolase

• Published in: Chembiochem : a European journal of chemical biology Vol. 25; no. 24; pp. e202400637 - n/a
• Main Authors: Zhao, You‐Xue, Li, Hai‐Peng, Cheng, Li‐Hang, Li, Chun‐Xiu, Pan, Jiang, Xu, Jian‐He
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 16.12.2024
• Subjects: Aldehydes; Aldolase; Amino acids; Diastereoselectivity; Directed evolution; Directed Molecular Evolution; Glycine; Glycine Hydroxymethyltransferase - chemistry; Glycine Hydroxymethyltransferase - genetics; Glycine Hydroxymethyltransferase - metabolism; High-throughput screening; High-Throughput Screening Assays; L-threonine aldolase; NADP - chemistry; NADP - metabolism; NADP+ reductases; Oxidation; Screening; Stereoisomerism; Stereoselectivity; Threonine aldolase; High-throughput screening; NADP+ reductases; Directed evolution; L-threonine aldolase; Diastereoselectivity
• ISSN: 1439-4227; 1439-7633; 1439-7633
• DOI: 10.1002/cbic.202400637

L‐Threonine aldolase (L‐TA) is a pyridoxal phosphate‐dependent enzyme that catalyzes the reversible condensation of glycine and aldehydes to form β‐hydroxy‐α‐amino acids. The combination of directed evolution and efficient high‐throughput screening methods is an effective strategy for enhancing the enzyme's catalytic performance. However, few feasible high‐throughput methods exist for engineering the Cβ‐stereoselectivity of L‐TAs. Here, we present a novel method of screening for variants with improved Cβ‐stereoselectivity; this method couples an L‐threo‐phenylserine dehydrogenase, which catalyzes the specific oxidation of L‐threo‐4‐methylsulfonylphenylserine (L‐threo‐MTPS), with the concurrent synthesis of NADPH, which is easily detectable via 340‐nm UV absorption. This enables the visual detection of L‐threo‐MTPS produced by L‐TA through the measurement of generated NADPH. Using this method, we discover an L‐TA variant with significantly higher diastereoselectivity, increasing from 0.98 % de (for the wild‐type) to 71.9 % de. L‐threonine aldolase (L‐TA) catalyzes the reversible condensation of glycine and aldehydes to form β‐hydroxy‐α‐amino acids. Currently, few high‐throughput methods are available for engineering L‐TAs with enhanced Cβ‐stereoselectivity. This study introduces a novel screening method that couples ketone reductase, which specifically catalyzes the oxidation of L‐threo‐4‐methylsulfonylphenylserine (L‐threo‐MTPS), with the simultaneous synthesis of NADPH.