Close this search box.

Self-driving laboratories to autonomously navigate the protein fitness landscape – Nature Chemical Engineering

Benchmarking BO methods on P450 data

We compiled a cytochrome P450 dataset to benchmark the modeling and BO methods. The dataset consists of 518 data points with binary active/inactive data from ref. 22 and thermostability measurements from ref. 21. We tested the multi-output GP model by performing tenfold cross-validation, where a GP classifier was trained on binary active/inactive data and a GP regression model was trained on thermostability data. The models used a linear Hamming kernel (sklearn36 DotProduct with sigma_0 = 1) with an additive noise term (sklearn WhiteKernel noise_level = 1). For the test-set predictions, we categorized sequences as either true negative (TN), false negative (FN), false positive (FP) or true positive (TP), and for true positives we calculated the Pearson correlation between predicted thermostability and true thermostability values.

We used the cytochrome P450 data to benchmark the BO methods. The random method randomly selects a sequence from the pool of untested sequences. The UCB method chooses the sequence with the largest upper confidence bound (GP thermostability model mean + 95% prediction interval) from the pool of untested sequences. The UCB method does not have an active/inactive classifier and, if it observes an inactive sequence, it does not update the GP regression model. The UCB positive method incorporates the active/inactive classifier and only considers the subset of sequences that are predicted to be active by the GP classifier (Pactive > 0.5). From this subset of sequences it selects the sequence with the top UCB (GP thermostability model mean + 95% prediction interval) value. The expected UCB method takes the expected value of the UCB score by (1) subtracting the minimum value from all thermostability predictions to set the baseline to zero, (2) adding the 95% prediction interval and (3) multiplying by the active/inactive classifier Pactive. The sequence with the top expected UCB value is chosen from the pool of untested sequences.

We tested the performance of these four methods by running 10,000 simulated protein engineering trials using the cytochrome P450 data. For each simulated protein engineering trial, the first sequence was chosen randomly, and subsequent experiments were chosen according to the different BO criteria. A trial’s performance at a given round is the maximum observed thermostability from that round and all prior rounds. We averaged each performance profile over the 10,000 simulated trials.

We also developed and tested batch methods that select multiple sequences each round. For the batch methods we use the same UCB variants described above to choose the first sequence in the batch, then we update the GP model assuming the chosen sequence is equal to its predicted mean, and then we select the second sequence according to the specified UCB criteria. We continue to select sequences and update the GP model until the target batch size is met. We assessed how the batch size affects performance by running 10,000 simulated protein engineering trials at different batch sizes and evaluating how many learning cycles were needed to reach 90% of the maximum thermostability.

Glycoside hydrolase combinatorial sequence space design

We designed a combinatorial glycoside hydrolase family 1 (GH1) sequence space composed of sequence elements from natural GH1 family members, elements designed using Rosetta26, and elements designed using evolutionary information27. The combinatorial sequence space mixes and matches these sequence elements to create new sequences. The sequences are assembled using Golden Gate cloning and thus require common four-base-pair overhangs to facilitate assembly between adjacent elements.

We chose six natural sequences by running a BLAST search on Bgl337 and selecting five additional sequences that fell within the 70–80% sequence identity range (Supplementary Fig. 3). We aligned these six natural sequences and chose breakpoints using SCHEMA recombination38,39 with the wild-type Bgl3 crystal structure (PDB 1GNX). The breakpoints for the Rosetta and evolution-designed sequence fragments were chosen to interface with the natural fragments and also introduce new breakpoints to promote further sequence diversity. For the Rosetta fragments, we started with the crystal structure of wild-type Bgl3 (PDB 1GNX), relaxed the structure using FastRelax, and used RosettaDesign to design a sequence segment for a given fragment while leaving the remainder of the sequence and structure as wild-type Bgl3. At each position, we only allowed residues that were observed within the six aligned natural sequences. For the evolution-designed fragments, we used Jackhmmer40 to build a large family of multiple sequence alignment and designed sequence segments containing the most frequent amino acid from residues that were observed within the six natural sequences. The GH1 family’s active site involves a glutamic acid catalytic nucleophile around position 360 and a glutamic acid general acid/base catalyst around position 180. As all fragments were designed based on aligned sequences, these conserved active-site residues will all fall within the same fragment position. The Glu nucleophile is present in blocks P1F3, P2F3, P3F3, P4F3, P5F3, P6F3, PrF6 and PcF6. The Glu general acid/base is present in blocks P1F1, P2F1, P3F1, P4F1, P5F1, P6F1, PrF4, PcF4, PrF5 and PcF5.

We designed DNA constructs to assemble sequences from the combinatorial sequence space using Golden Gate cloning. The designed amino-acid sequence elements were reverse-translated using the Twist codon optimization tool, and the endpoints were fixed to preserve the correct Golden Gate overhangs. We added BsaI sites to both ends to allow restriction digestion and ordered the 34 gene fragments cloned into the pTwist Amp High Copy vector. Each sequence element’s amino acid and gene sequence are given in Supplementary Data 5.

Automated gene assembly, expression and characterization

We implemented our fully automated protein testing pipeline on an in-house Tecan liquid-handling system and the Strateos Cloud Lab. The system was initialized with a plate of the 34 gene fragments (5 ng μl−1), an NEB Golden Gate Assembly Kit (E1601L) diluted to a 2× stock solution, a 2 μM solution of forward and reverse PCR primers, Phusion 2X Master Mix (ThermoFisher F531L), 2× EvaGreen stock solution, Bioneer AccuRapid Cell Free Protein Expression Kit (Bioneer K-7260) Master Mix diluted in water to 0.66×, AccuRapid E. coli extract with added 40 μM fluorescein, a fluorogenic substrate master mix (139 μM 4-methylumbelliferyl-α-d-glucopyranoside, 0.278% vol/vol dimethylsulfoxide (DMSO), 11 mM phosphate and 56 mM NaCl, pH 7.0) and water.

Golden Gate assembly of DNA fragments

For a given assembly, 5 μl of each DNA fragment were mixed and 10 μl of the resultant mixture was then combined with 10 μl of 2× Golden Gate Assembly Kit. This reaction mix was heated at 37 °C for 1 h, followed by a 5-min inactivation at 55 °C.

PCR amplification of assembled genes

A 10-μl volume of the Golden Gate assembly product was combined with 90 μl of the PCR primers stock, and 10 μl of this mixture was then added to 10 μl Phusion 2X Master Mix. PCR was carried out with a 5-min melt at 98 °C, followed by 35 cycles of 56 °C anneal for 30 s, 72 °C extension for 60 s, and 95 °C melt for 30 s. This was followed by one final extension for 5 min at 72 °C.

Verification of PCR amplification

A 10-μl volume of the PCR product was combined with 90 μl of water, and 50 μl of this mixture was then combined with 50 μl 2× EvaGreen. The fluorescence of the sample was read on a microplate reader (excitation, 485 nm; emission, 535 nm) and the signal was compared to previous positive/negative control PCRs to determine whether PCR amplification was successful.

Cell-free protein expression

A 30-μl volume of the 10× PCR dilution from the previous step was added to 40 μl of AccuRapid E. coli extract and mixed with 80 μl of AccuRapid Master Mix. The protein expression reaction was incubated at 30 °C for 3 h.

Thermostability assay

We used T50 measurements to assess GH1 thermostability. T50 is defined as the temperature where 50% of the enzyme is irreversibly inactivated in 10 min and is measured by heating enzyme samples across a range of temperatures, evaluating residual enzyme activity, and fitting a sigmoid function to the temperature profile to obtain the curve midpoint. T50 represents the fractional activity lost as a function of temperature and is therefore independent of absolute enzyme concentration and expression level.

A 70-μl volume of the expressed protein was diluted with 600 μl of water, and 70-μl aliquots of this diluted protein were added to a column of a 96-well PCR plate for temperature gradient heating. The plate was heated for 10 min on a gradient thermocycler such that each protein sample experienced a different incubation temperature. After incubation, 10 μl of the heated sample was added to 90 μl of the fluorogenic substrate master mix and mixed by pipetting. The fluorescein internal standard was analyzed on a microplate reader (excitation, 494 nm; emission, 512 nm) for sample normalization, and the enzyme reaction progress was monitored by analyzing the sample fluorescence (excitation, 372 nm; emission, 445 nm) every 2 min for an hour. Any wells with fluorescein fluorescence less than 20% of the average for a given run were assumed to reflect pipetting failure and were not considered when fitting a thermostability curve.

Human characterization of top designed enzymes

Bacterial protein expression and purification

The designs were built using Golden Gate cloning to assemble the constituent gene fragments, and the full gene was cloned into the pET-22b expression plasmid. The assemblies were transformed into E. coli DH5α cells and the gene sequences were verified using Sanger sequencing. The plasmids were then transformed into E. coli BL21(DE3) and preserved as glycerol stocks at −80 °C. The glycerol stocks were used to inoculate an overnight Luria broth (LB) starter culture and the next day this culture was diluted 100× into a 50-ml LB expression culture with 50 μg ml−1 carbenicillin. The culture was incubated while shaking at 37 °C until the optical density at 600 nm reached 0.5–0.6 and then induced with 1 mM isopropyl β-d-1-thiogalactopyranoside. The expression cultures were incubated while shaking overnight at 16 °C, and the next day the cultures were collected by centrifugation at 3,600g for 10 min, discarding the supernatant. The cell pellets were resuspended in 5 ml of phosphate-buffered saline and lysed by sonication at 22 W for 20 cycles of 5 s on and 15 s off. The lysates were clarified by centrifugation at 10,000g for 15 min.

The enzymes were purified by loading the clarified lysates onto a Ni-NTA agarose column (Cytiva 17531801), washing with 20 ml of wash buffer (25 mM Tris, 400 mM NaCl, 20 mM imidazole, 10% vol/vol glycerol, pH 7.5) and eluting with 5 ml of elution buffer (25 mM Tris, 400 mM NaCl, 250 mM imidazole, 10% vol/vol glycerol, pH 7.5). The eluted samples were concentrated using an Amicon filter concentrator and concurrently transitioned to storage buffer (25 mM Tris, 100 mM NaCl, 10% vol/vol glycerol, pH 7.5). The final protein concentration was determined using the Bio-Rad protein assay, the samples were diluted to 2 mg ml−1 in storage buffer, and frozen at −80 °C.

Thermostability assay

The clarified cell lysate from the protein expression was diluted 100× in phosphate-buffered saline, then 100 μl of the diluted lysate was arrayed into a 96-well PCR plate and heated for 10 min on a gradient thermocycler from 40 °C to 75 °C. The heated samples were assayed for enzyme activity in quadruplicate with final reaction conditions of 10% heated lysate, 125 μM 4-methylumbelliferyl-β-d-glucopyranoside, 0.125% vol/vol DMSO, 10 mM phosphate buffer pH 7 and 50 mM NaCl. The reaction progress was monitored using a microplate reader analyzing sample fluorescence (excitation, 372 nm; emission, 445 nm) every 2 min for 30 min. The reaction progress curves were fit using linear regression to obtain the reaction rate, and a shifted sigmoid function was fit to the rate as a function of temperature incubation to obtain the T50 value.

Michaelis–Menten kinetic assay

The purified enzymes were assayed in quadruplicate along an eight-point twofold dilution series of 4-methylumbelliferyl-β-d-glucopyranoside starting from 500 μM. The assays were performed with 10 nM enzyme, 0.5% vol/vol DMSO, 10 mM phosphate buffer pH 7 and 50 mM NaCl. The reaction progress was monitored using a microplate reader analyzing the sample fluorescence (excitation, 372 nm; emission, 445 nm) every 2 min for 30 min. A standard curve of 4-methylumbelliferone (4MU) ranging from 3.91 to 62.5 µM was used to determine the assay’s linear range. The initial rate for each reaction was determined by fitting a linear function to 4MU fluoresence (excitation, 372 nm; emission, 445 nm) at 0-, 2- and 4-min reaction times. The initial rate data were fit to the Michaelis–Menten equation using the scikit-learn36 curve_fit function to determine the enzyme kcat and KM.

SAMPLE code execution

A detailed description of the software loop driving SAMPLE is provided in the Supplementary Information under the heading Detailed description of SAMPLE code functionality.