Folding Assessment of Incorporation of Noncanonical Amino Acids Facilitates Expansion of Functional‐Group Diversity for Enzyme Engineering

Abstract Protein design is limited by the diversity of functional groups provided by the canonical protein „building blocks“. Incorporating noncanonical amino acids (ncAAs) into enzymes enables a dramatic expansion of their catalytic features. For this, quick identification of fully translated and correctly folded variants is decisive. Herein, we report the engineering of the enantioselectivity of an esterase utilizing several ncAAs. Key for the identification of active and soluble protein variants was the use of the split‐GFP method, which is crucial as it allows simple determination of the expression levels of enzyme variants with ncAA incorporations by fluorescence. Several identified variants led to improved enantioselectivity or even inverted enantiopreference in the kinetic resolution of ethyl 3‐phenylbutyrate.

gel after the electrophoresis, (the gel extraction kit, Thermo Fisher, Germany). After obtaining the insert, both insert and vector pBAD_GFP11_T7LysH17A were digested with restriction enzymes Xho I and Spe I (Thermo Fisher, Germany). 40 μL mixture contained: 25 μL of a vector/insert, 1 μL of each restriction enzyme, 4 μL of FastDigest Green Buffer and 9 μL of double-distilled water. The reaction took place at 37 °C overnight. Fragments of interest were then separated by electrophoresis on a 1 % agarose gel. Cut DNA fragment was isolated from the gel band using the gel extraction kit (Thermo Fisher, Germany). 20 μL ligation reaction mixture contained: 7 μL of cut insert DNA solution, 2 μL of cut vector DNA solution, 10 μL of T4 DNA reaction buffer (2X) 1 μL T4 DNA ligase (Thermo Fisher, Germany). Such reaction mixture was incubated overnight at 16 °C. After incubation, 5 μL from the mixture was used to transform E. coli TOP10 cells.
coli XL-1-Blue cells and successful mutation was confirmed by sequencing. The list of primers used is given in Supplementary Table 2 (3-21).

Small-scale expression in 96-well format
In each well of the autoclaved 96-deep well plates (DWPs) 950 μL of LB medium (Carl Roth, Germany) with appropriate antibiotics (Supplementary Table 1) were added. 50 μL of overnight cultures were added as the inoculum. Cultures were grown at 400 rpm at 37 °C. After 4.5 h growth, when OD reached value between 0.6-1, inducers and respective non-canonical amino acid were added (Supplementary Table 1). Then, DWPs were placed to the shaker at 28 °C, 400 rpm for the overnight expression. After the expression, cultures in DWPs were pelleted in the centrifuge (Eppendorf, Germany) (3220 g, 30 min, 4 °C). Pellets were resuspended in 200 μL of BugBuster® Master Mix (Merck Millipore, Germany), then incubated at the 28 °C on the shaker for 2 hours and freeze -20 °C until further used. For the pAF lysates, 20μl of Tris(2-carboxyethyl)phosphine stock solution (100 mM) was added and the reduction was performed for an additional 2 hours at 30 °C. The resuspended cells were frozen -20 °C till further used Cell-free extracts were obtained as supernatants after centrifugation (3220 g, 60 min, 4 °C) and transferred to fresh microtiter plates (MTPs).

p-nitrophenylacetate assay
In a MTP, 10 μL of cleared lysates were mixed with 180 μL of Tris-HCl buffer (100 mM Tris-HCl, 150 mM NaCl, pH 7.5). Absorbance over time (λ = 410 nm) was measured in the S5 spectrophotometer (BioTek, USA) immediately after the addition of 10 μL of 20 mM pnitrophenyl acetate in DMSO. The PFE activity in a CFE was calculated based on the calibration as the initial reaction velocity (μM -1 s -1 ).

Split-GFP Method
PFE variants were expressed with the GFP11 fragment as a fusion tag. Content (expression level) of such fusion proteins was determined using fluorescence (Santos-Aberturas et al., 2018) [1] . In brief, 180 μL of reporter solution (see Preparation of GFP 1-10 Reporter Solution) were mixed in a MTP with 20 μL of CFE. The MTP was incubated for 18 hours at 4 °C then fluorescence (λex = 488 nm/ λem = 530 nm) was measured in the spectrophotometer (BioTek, USA).

Large Scale Protein Expression and Protein Purification
In an autoclaved Erlenmeyer flask with 0.5 L of LB medium (10 g/l peptone, 5 g/l yeast extract, 10 g/l NaCl) with respective antibiotics (Supplementary Table 1 inoculum from an overnight culture was added. The culture was grown under agitation at 37 °C. After ~4 h growth, when OD reached value between 0.4 -0.6, inducers (Supplementary Table 1 conversion reached between 10-80%. [2] Enantiomeric excess and conversion were calculated as functions of the peak areas, measured by chiral GC-FID. The results shown in Table 1 are average values obtained by at least two independent measurements (in case of PFE_F158NapA only a single measurement). Standard deviations are below 5%. GC-FID parameters used for the analytics S7

Preparation of GFP 1-10 Reporter Solution
The reporter solution for fluorescence determination of expression level was prepared following the protocol by Santos-Aberturas et al. [1] with minor changes. 500 mL LB medium was inoculated by adding 5 mL of an overnight culture of E. coli BL21(DE3) containing plasmid pET41b_GFP (1-10) and placed to the shaker at 37 °C. After OD ≈ 0.6 had been reached, the protein expression was induced by adding IPTG to final concertation 0.1 mM.
Then the culture was placed to the shaker at 28 °C for overnight expression of GFP1-10 inclusion bodies. After expression, the culture was centrifuged (4000 g, 20 min, 4 °C). Pelleted cells were resuspended in 20 mL cold TNG buffer (100 mM Tris/HCl, 150 mM NaCl, 10% (v/v) Glycerol) and transferred to fresh Falcon-type tube. Then the cells were disrupted by sonication (10 min, 50% work/rest, 50% power input). The produced suspension, which included GFP1-10 inclusion bodies was centrifuged at 30 000 g (30 min, 4 °C). The supernatant was discarded and the pellet that contained inclusion bodies was resuspended in 10 mL of BugBuster® Master Mix with the help of sonication (2 min, 50% work/rest, 50% power input).
The generated suspension was centrifuged (30 000 g, 30 min, 4 °C), the supernatant discarded, and the pellet resuspended again in 10 mL of BugBuster® Master Mix (sonication: 2 min, 50% work/rest, 50% power input). This step (resuspension in BugBuster® Master Mix followed by centrifugation) was repeated two more times. To remove the detergent, the pellet was resuspended with the help of sonication in 10 mL of TNG buffer. Inclusion bodies were pelleted by centrifugation (30 000 g, 30 min, 4 °C). This step was repeated one more time. The obtained inclusion bodies were weighted and resuspended in TNG buffer to reach a concentration of 75 mg/mL. Sonication was implied again to stimulate the resuspension. This suspension was split to fresh tubes in 1 mL aliquots, centrifuged (16 000 g, 10 min, 4 °C), the supernatant was discarded, and pelleted inclusion bodies stored at -20 °C. Each aliquot was resuspended in 1 mL of urea solution (urea 9 mol/L and DTT 5 mmol/L). The insoluble fraction was removed by centrifugation (16 000 g, 1 min) and supernatant, which contained solubilized GFP1-10 fragments was added into 50 mL of buffer solution (100 mM Tris-HCl, 150 mM NaCl).

30000
32000 34000 36000 38000 40000 m/z S14 Split-GFP assay Figure S4. Calibration curve of the split-GFP assay (measured at each split-GFP solution preparation) S15 Figure S5. Bar graph comparison of expression levels of PFE variants measured in cell-free extracts (in μM) using the split-GFP assay and corresponding reaction rates measured for pNpA assay of the chosen PFE variants and wt. S16 GC chromatograms Figure S6. Chiral GC-FID analysis of results of the PFE-catalyzed of rac-ethyl 3phenylbutyrate kinetic resolution.