Stereodivergent Protein Engineering of a Lipase To Access All Possible Stereoisomers of Chiral Esters with Two Stereocenters

Abstract

Enzymatic stereodivergent synthesis to access all possible product stereoisomers bearing multiple stereocenters is relatively undeveloped, although enzymes are being increasingly used in both academic and industrial areas. When two stereocenters and thus four stereoisomeric products are involved, obtaining stereodivergent enzyme mutants for individually accessing all four stereoisomers would be ideal. Although significant success has been achieved in directed evolution of enzymes in general, stereodivergent engineering of one enzyme into four highly stereocomplementary variants for obtaining the full complement of stereoisomers bearing multiple stereocenters remains a challenge. Using Candida antarctica lipase B (CALB) as a model, we report the protein engineering of this enzyme into four highly stereocomplementary variants needed for obtaining all four stereoisomers in transesterification reactions between racemic acids and racemic alcohols in organic solvents. By generating and screening less than 25 variants each isomer, we achieved >90% selectivity for all of the four possible stereoisomers in the model reaction. This difficult feat was accomplished by developing a strategy dubbed "focused rational iterative site-specific mutagenesis" (FRISM) at sites lining the enzyme's binding pocket. The accumulation of single mutations by iterative site-specific mutagenesis using a restricted set of rationally chosen amino acids allows the formation of ultra-small mutant libraries requiring minimal screening for stereoselectivity. The crystal structure of all stereodivergent CALB variants, flanked by MD simulations, uncovered the source of selectivity.

The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes

“…Most recently, we proposed and implemented experimentally a different concept in rational design of stereoselective enzymes, dubbed Focused Rational Iterative Site‐specific Mutagenesis (FRISM) . As illustrated in Figure , it means that at a given CAST residue a limited number of predictive suggestions for single mutations are made first, requiring the site‐specific generation of typically only 3–4 mutants.…”

Die zentrale Rolle der Methodenentwicklung in der gerichteten Evolution selektiver Enzyme

“…In einer jüngsten Studie entwickelten wir ein andersartiges Konzept für das rationale Design stereoselektiver Enzyme, das wir FRISM (Focused Rational Iterative Site‐specific Mutagenesis) nennen . Wie in Abbildung illustriert, werden für eine gegebene CAST‐Position zunächst eine begrenzte Anzahl von prädiktiven Vorschlägen von Einzelmutationen gemacht, was die ortsspezifische Erzeugung von typischerweise nur 3–4 Mutanten erforderlich ist.…”

Engineering Improves Enzymatic Synthesis of L-Tryptophan by Tryptophan Synthase from Escherichia coli

“…Reetz et al successfully improved the stability of Bacillus subtilis lipase based on B-FITTER [18]. In this study, B-FITTER software was used to analyze the temperature factors of 397 amino acid residues of tryptophan synthase trpB (PDB: 2dh6), and the temperature factors were obtained.…”

“…An efficient protein engineering strategy based on the fusion of directed evolution and rational design was used to obtain lipase mutant strains with high stereoselectivity, and the strategy required minimal screening. Crystal structure analysis and molecular dynamics simulation of the nonaqueous phase were used to explain the specific stereoselectivity and the molecular mechanism of the four mutant strains, respectively [18]. Directed evolution was used to modify threonine aldolase to improve the synergistic effect of its amino acid residues [19].…”