A Novel Genetic Selection System for Improved Enantioselectivity of Bacillus subtilis Lipase A

Abstract

In directed evolution experiments, success often depends on the efficacy of screening or selection methods. Genetic selections have proven to be extremely valuable for evolving enzymes with improved catalytic activity, improved stability, or with altered substrate specificity. In contrast, enantioselectivity is a difficult parameter to select for. In this study, we present a successful strategy that not only selects for catalytic activity, but for the first time also for enantioselectivity, as demonstrated by the selection of Bacillus subtilis lipase A variants with inverted and improved enantioselectivity. A lipase mutant library in an aspartate auxotroph Escherichia coli was plated on minimal medium that was supplemented with the aspartate ester of the desired enantiomer (S)-(+)-1,2-O-isopropylidene-sn-glycerol. To inhibit growth of less enantioselective variants, a covalently binding phosphonate ester of the opposite (R)-(-)-1,2-O-isopropylidene-sn-glycerol enantiomer was added as well. After three selection rounds in which the selection pressure was increased by raising the phosphonate ester concentration, a mutant was selected with an improved enantioselectivity increased from an ee of -29.6 % (conversion 23.4 %) to an ee of +73.1 % (conversion 28.9 %) towards the (S)-(+)-enantiomer. Interestingly, its amino acid sequence showed that the acid of the catalytic triad had migrated to a position further along the loop that connects beta7 and alphaE; this shows that the position of the catalytic acid is not necessarily conserved in this lipase.

An Esterase with Superior Activity and Enantioselectivity towards 1,2‐O‐Isopropylideneglycerol Esters Obtained by Protein Design

“…The major advantage of this structure-based approach is that it allows for the generation of smaller libraries, reducing the time of screening or selection, with higher functional diversity. [8][9][10][11][12] Herein, we describe molecular docking experiments of (R)-1 into the YbfF active site in order to identify active site residues that are in close proximity to the substrate. These docking experiments helped to identify four positions (25, 124, 185 and 235) in the YbfF active site that might be good targets for engineering the enantioselectivity of YbfF towards 1 and 3.…”

“…In the last two decades novel laboratory evolution methods have been devised in order to improve enzyme properties (such as activity, stability and selectivity) towards non-natural substrates. [8][9][10] Random evolution approaches have been the preferred method of choice, since they do not require detailed knowledge of the enzyme structure or reaction mechanism. However, with the increased number of 3D structures solved and the growth of computational methods, structure-based enzyme engineering strategies are becoming more in reach for improving enzyme properties.…”

A roadmap to directed enzyme evolution and screening systems for biotechnological applications

“…Selection assays are therefore mostly limited for detoxifying enzymes or enzymes synthetizing compounds necessary for cell growth and survival. Selection assays can be designed to screen for enantio-selective enzymes (Boersma et al, 2008, where the desired enantiopure substrate releases essential nutrients upon reaction, while the undesired enantiomer was supplied as an inhibitor or poison releasing substrate, allowing the growth only of the variants having the desired activity.…”

A Combination of In Vivo Selection and Cell Sorting for the Identification of Enantioselective Biocatalysts

“…[10] In a follow-up study, they could further refine the principle and identified variants of lipase CAL-B with slightly inverted but not high enantioselectivity towards 1,2-O-isopropylidene glycerol (IPG, WT (wild type): E = 1.9, favoring the (R)-enantiomer to mutants with E = 3-8 favoring the (S)-enantiomer). [11] Similarly, Quax and co-workers [12] investigated a lipase from Bacillus subtilis using IPG, but linked either to aspartate or a phosphonate. Release of the amino acid supported growth of an aspartate auxotroph E. coli strain whereas the phosphonate lead to inhibition of the lipase.…”