Directed Evolution Of L-Threonine Aldolase For The Diastereoselective Synthesis Of Beta-Hydroxy-Alpha-Amino Acids

L-Threonine aldolase (LTA) is an attractive tool in organic chemistry for catalyzing the formation of beta-hydroxy-alpha-amino acids with two chiral centers. The enzyme has a strict selectivity for C-alpha of beta-hydroxy-alpha-amino acids but a moderate selectivity for C-beta, limiting its wide applications in stereospecific carbon-carbon bond synthesis. Here, a combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was applied to accelerate directed evolution of LTA in diastereoselectivity. A total of 27 amino acid residues lining the substrate pocket were selected and divided into two groups based on their functional region. In silico screening and site-directed saturation mutagenesis identified six (3 + 3) amino acid residues of them with a significant effect on diastereoselectivity. The ISM strategy was then performed in and between each group to obtain the best combinatorial mutation. As a result, a variant RS1 (Y8H/Y31H/I143R/N305R) was obtained with a dramatically improved preference for the synthesis of L-syn-3-[4-(methylsulfonyl)phenylserine]. The product with a de value of 99.5% (73.2% (conv)) was produced in a 20 L reactor, which is promising in the industrial synthesis of aromatic L-syn-beta-hydroxy-a-amino acids with LTA. The variant also represented a significant selective improvement to other L-phenylserine derivatives. The de values of 2-NO2-, 4-NO2-, H-, and 4-CH4-substituted L-phenylserine derivatives were more than 99%(syn) by dynamic control. The insight of the mutant model suggests that the binding pocket of the active center was reshaped.