Towards Understanding Directed Evolution: More than Half of All Amino Acid Positions Contribute to Ionic Liquid Resistance of Bacillus subtilis Lipase A

Abstract

Ionic liquids (ILs) are attractive (co-)solvents for biocatalysis. However, in high concentration (>10 % IL), enzymes usually show decreased activity. No general principles have been discovered to improve IL resistance of enzymes by protein engineering. We present a systematic study to elucidate general engineering principles by site saturation mutagenesis on the complete gene bsla. Screening in presence of four [BMIM]-based ILs revealed two unexpected lessons on directed evolution: 1) resistance improvement was obtainable at 50-69 % of all amino acid positions, thus explaining the success of small sized random mutant libraries; 2) 6-13 % of substitutions led to improved resistance. Among these, 66-95 % were substitutions by chemically different amino acids (e.g., aromatic to polar/aliphatic/charged amino acids), thus indicating that mutagenesis methods introducing such changes should, at least for lipases like BSLA, be favored to improve IL resistance.

Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents?

“…Organic cosolvent concentrations for BSLA SSM library screening were chosen to ensure a residual BSLA WT activity of~30%. Similar values have proven to be a good compromise between the enzyme's resistance on the one hand and reliably determining the enzyme's activity compared to BSLA WT in a previous study [28]. The pH of the incubation mixture (BSLA lipase, TEA buffer, organic cosolvent) and the pH of the reaction mixture were measured and pH shifts of less than 0.22 pH units were observed.…”

“…A detailed description of the BSLA SSM library generation was reported in our previous studies [28,29]. Briefly, a library of 3620 unique variants (20 amino acids × 181 positions) was generated by site-saturation mutagenesis (SSM) and site-directed mutagenesis (SDM).…”

“…Cultivation of BSLA SSM library variants was performed as previously described [28]. Briefly, three wild type (WT) clones and one empty vector clone (controls) alongside 92 clones of the BSLA SSM libraries were incubated in 150 mL of a modified auto-induction medium [41] (see Supplementary Materials) supplemented with ampicillin in a 96-well plate (V-bottomed, polystyrene MTP, Greiner Bio-One, Frickenhausen, Germany) for 16 h (37 • C, 70% humidity, 900 rpm).…”

“…BSLA activity measurements were performed as described previously [28]. BSLA converts pNPB to p-nitrophenolate upon hydrolysis, which was detected photometrically at 410 nm.…”

Directed evolution 2.0: improving and deciphering enzyme properties

“…A detailed analysis of 3000 mutations in the lipase gene bsla which were generated with three random mutagenesis methods (1000 mutations each: epPCR-low, 1000 PCR-high, and SeSaM-Tv P/P) and their comparison to a site saturation mutagenesis (SSM) library of bsla which covers the natural diversity at each amino acid position of BSLA (in total 181 positions), yielded first experimental and quantitative data on the obtainable diversity. 22,23 The SSM-BSLA library was generated in 181 site saturation mutagenesis experiments, subsequent sequencing of 18547 clones and subsequent site-directed mutagenesis experiments to obtain missing substitutions and to ensure that the full natural diversity is available. 23 BSLA resistances towards an ionic liquid [C 4 mim][TfO] were studied by screening the SSM-BSLA library and in total 292 beneficial substitutions at 104 different amino acid positions were identified.…”