Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis

Abstract

Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded substrate scope is a contemporary challenge. One approach to address this challenge is Substrate Multiplexed Screening (SUMS), where enzyme activity is measured on competing substrates. SUMS has long been used to rigorously quantitate native enzyme specificity, primarily for in vivo settings. SUMS has more recently found sporadic use as a protein engineering approach but has not been widely adopted by the field, despite its potential utility. Here, we develop principles of how to design and interpret SUMS assays to guide protein engineering. This rich information enables improving activity with multiple substrates simultaneously, identifies enzyme variants with altered scope, and indicates potential mutational hot-spots as sites for further engineering. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering.

Fig. 1. Overview of substrate multiplexed screening (SUMS).

a In SUMS, activity is measured for multiple competing substrates simultaneously. b SUMS can be leveraged to address a range of goals when engineering biocatalysts. Here, we apply SUMS to a model biosynthetic cascade, forming tryptamines from indoles using Ruminococcus gnavus tryptophan decarboxylase (RgnTDC) and Pyrococcus furiosus tryptophan synthase β-subunit (PfTrpB).

Fig. 2. Substrate multiplexed screening (SUMS)-based engineering of Ruminoccus gnavus tryptophan decarboxylase (RgnTDC).

a i. Substrate competition model with equation describing relative rates of product formation. ii. Timecourse of a substrate multiplexed reaction of RgnTDC with 2-Me-, 4-Br-, 6-Cl-, and Trp. Full reaction conditions found in Supplementary Fig. 42. b General reaction of RgnTDC, with the labile bond highlighted. R = halo, alkyl, nitro, ether, etc. See ref. for detailed scope of the wild-type enzyme. c SUMS results from a W349X library using a mixture of Trp substrates where R = 5-OEt-, 5-acetyl-, 5-CONH₂-, 5-OMe-, and 5-OMe-2-Me-. Colored bars indicate relative abundances of each product, and black diamonds indicate total intensity of single ion retention (SIR) for each product’s unique m/z. No product was observed from 5-OMe-2-Me-Trp. d Fold-activity relative to wild-type from a single-substrate screen of the W349X library with 5-OMe-Trp corresponding to classical protein engineering techniques. Retention of function curves with full sequence analyses are shown in Supplementary Figs. 3–4.

Fig. 3. SUMS identifies RgnTDC active site mutations that improve activity for a range of substrates.

a Active site model of RgnTDC (built from PDB ID: 4OBV) with residues highlighted at which mutations were found that significantly altered promiscuity or improved activity. b Select improved variants from active site libraries. Substrate screening conditions: 0.2 mM Trp and 7-I-Trp, and 2 mM 2-Me-Trp, 4-Br-Trp, 5-OMe-Trp, and 6-Cl-Trp, 4 h, 37 °C. Colored bars indicate the relative abundances of each product, and black diamonds indicate the total product formed. Full screening results found in Supplementary Figs. 6–S14. c Turnover numbers of wild-type RgnTDC and the top improved variant for each substrate. Different variants are depicted by different colored bars. Turnover numbers are presented as the averages of technical triplicate measurements with standard deviation shown as a bar. d Michaelis-Menten parameters for wild-type RgnTDC and activated variants for Trp and Trp analogs. Kinetic and turnover data were conducted in triplicate and complete data including error analysis are shown in Supplementary Tables 1 and 2 and Supplementary Figs. 17–21.

Fig. 4. SUMS-based engineering of the β-subunit of Pyrococcus furiosus tryptophan synthase (PfTrpB).

a General reaction scheme for PfTrpB. Indole analogs were used to screen PfTrpB libraries. The nucleophilic atom is shown with a circle. R = halo, alkyl, nitro, ether, etc. See ref. for a detailed scope of engineered TrpB enzymes. b SUMS results for generally activated variants detected during globally random mutagenesis library screening. Complete library results and experimental conditions are shown in Supplementary Figs. 23–25. c Comparison of Trp and DIT production under competition and single substrate reaction conditions, using purified 2B9 and H275R enzymes. Indoline was present at 10-fold excess (15 mM indoline, 1.5 mM indole) for both competition and single substrate reactions. Complete duplicate data and conditions are shown in Supplementary Fig. 28. d H275 is a second-sphere residue that forms hydrogen bonds with neighboring residues, N166, and Y301 (PDB ID: 6AM8). e SUMS results for 2B9 and two variants from the H275X SSM library. 4-CN-indole was also included in reactions, but no product was observed. Complete library results are shown in Supplementary Fig. 27. f Product formation in single substrate reactions. Activity of H275R and H275E is shown relative to activity of 2B9 (black dashed line). g Michaelis–Menten parameters for 2B9, H275R, and H275E with either indole or indoline as the nucleophilic substrate. Kinetic data were measured in triplicate, and complete data including error analysis are shown in Supplementary Fig. 29.

Fig. 5. Crystallographic and spectroscopic characterization of H275E.

a Internal aldimine structure of H275E (light blue, PDB: 7RNQ) is superimposed with the corresponding structure of 2B9 (gray, PDB: 6AM7). b Addition of 20 mM l-serine (Ser) to 2B9 (gray), results in two peaks corresponding to external aldimine E(Aex₁) and amino acrylate, E(A-A), intermediates. Addition of 20 mM Ser to H275E (pink) shows a dominant peak corresponding to E(A-A). A representative enzyme-only trace is shown in black. Spectra were collected at 37 °C. c X-ray structures of H275E. i. Trp binding is shown in magenta from PDB: 7ROF. ii. 4-Cl-Trp binding is shown in cyan from PDB: 7RNP. Hydrogen and halogen bonds are shown in orange and purple dashes, respectively.

Fig. 6. Engineered biocatalytic cascade for synthesis of tryptamine analogs.

a Utilized biocatalytic cascade for the telescoped biosynthesis of tryptamine analogs. R = 4-Br-, 5-OMe-, 5-OEt-, 2-Me-, 6-Cl-, and 6-NO₂-. b Synthesized tryptamines, with the RgnTDC variants used for different syntheses highlighted. Isolated yields of tryptamine products after both steps are listed. *1.4 mmol substrate used for 6-chlorotryptamine synthesis. PfTrpB^H275E loading was 0.05 mol% and RgnTDC variant loading was 0.02 mol% for each reaction except for 2-methyltryptamine, which had 0.2 mol% catalyst.