Most chemicals used in this work were bought from either Bioshop Canada, Gold Biotechnology, Sigma Aldrich, Thermo Fisher Scientific or p212121. Else, the Pfu polymerase was purchased from Bio Basic, DNA primers were obtained from IDT technologies, p-nitrophenoxydecanoic acid  was purchased from TCI Chemicals and N-benzyl-1,4-dihydronicotinamide was produced by Kemitek as reported in . The B. megaterium strain used in this work, B. megaterium de Bary (ATCC® 14581™), was obtained from ATCC as was the pBR322 vector. The Escherichia coli dh5α and BL21 (DE3) pLysS strains were both acquired from Thermo Fischer Scientific. The restriction enzymes SpeI, BamHI, and DpnI were acquired from New England Biolabs. The pET-15B vector was acquired from Novagen.
Bacillus megaterium transformation
Prior to the transformation procedure, B. megaterium cells were grown at 30°C in 25 mL lysogeny broth (LB) medium (5 g/L yeast extract, 10 g/L tryptone, 5 g/L NaCl, adjusted to pH 7 with HCl) in a 250 mL shake flask at 210 rpm until cells reached an optical density at 600 nm of 1. The cells were then centrifuged at 3000 g for 10 min, resuspended into 1.5 mL fresh LB medium and then kept on ice. Following this, 475 µL of B. megaterium cell suspension was mixed with 15 µL of pBR322 vector DNA in 0.1 cm gapped electroporation cuvette (Bio-Rad) and incubated on ice for 30 min. The resuspendend cells were then subjected to a single electric pulse (1000 V, 50 µF, 200 Ω) using the Bio-Rad Gene Pulser Xcelltm. The electroporated cells were then transferred to a 1.5 mL microtube and incubated 1 hour at 37°C on a heating block, plated on LB agar plates containing 15 mg/mL tetracyline and incubated overnight at 30°C.
Experimental evolution cultures of Bacillus megaterium
Every culture following the initial B. megaterium transformation contained 15 mg/mL tetracyline, were grown at 30°C and stirred at 210 rpm with the exception of the 20 L bioreactor culture were cultures were stirred at 150 rpm. Plated cells were grown at 30°C. Unless stated otherwise, all centrifugation steps were performed at 3000 g for 10 min at room temperature. Oleic acid was dissolved in dimethyl sulfoxide (DMSO) to generate a stock solution of 158.5 µM which was used to prepare cultures containing up to 5 µM of oleic acid. A stock solution of oleic acid in DMSO of 20 mM was used to prepare cultures whose oleic acid concentration was set to be between 10 µM and 300 µM. These stock solutions were selected to ensure that DMSO concentration in cultures remained below 5% (v/v) so as to not interfere with bacterial growth. To establish the maximum oleic acid concentration B. megaterium could handle, plated transformants were first grown in a 50 mL preculture of LB medium without oleic acid in a 250 mL shake flask for 12 hours. Subsequently, cells were centrifuged, resuspended in 25 mL of fresh LB medium and 500 µl of these resuspended cells were added to 50 mL of LB medium in 250 mL shake flask containing concentrations of Oleic acid ranging from 0 µM to 100 µM. These cultures were then incubated for 6 hours and their optical density at 600 nm measured every hour. The standard deviation and mean for each data point were calculated from three experimental replicates.
For the experimental evolution of B. megaterium Supplementary Info 1 was added for clarity to better describe the procedure utilised for this crucial experiment. The experiment was initiated by incubating a single colony of B. megaterium harbouring the pBR322 vector in a 50 mL preculture of LB medium without oleic acid in a 250 mL shake flask for overnight. Cultured cells were then centrifuged, resuspended in fresh LB medium and poured inside a 20 L bioreactor containing 19.5 L of LB medium and 2.5 µM of Oleic acid. Cells were grown for 12 hours at 150 rpm with pH maintained at 7 with ammonium hydroxide. These above listed tasks encompass step 1 described within Supplementary Info 1. At the end of the incubation, 25 mL of cultured cells were harvested and used to streak LB agar plates containing 0, 2.5 and 5 µM oleic acid for overnight incubation. Afterwards the same 25 mL was mixed with 25 mL of glycerol and stored at -80°C in a 50 mL falcon tube. An isolated colony grown on the LB agar plates containing 5 µM of oleic acid was then grown further for 10 hours in a 100 mL preculture of LB medium with 5 µM oleic acid in a 500 mL shake flask. Thereafter, 8.5 mL of the preculture was added to each of the four 2 L shake flasks containing 850 mL of LB medium with a concentration of oleic acid of either 5, 10, 20 or 30 µM. Cells were incubated for overnight. This would conclude step 2 and begin step 3 in Supplementary Info 1. By the end of the incubation, 25 mL of cultured cells from the shake flask with 30 µM of oleic acid were harvested and used to streak LB agar plates containing 30, 40 and 50 µM of oleic acid for overnight incubation. Afterwards the same 25 mL was mixed with 25 mL of glycerol and stored at -80°C in a 50 mL falcon tube. Following this, an isolated colony grown on LB agar plates with 50 µM of oleic acid was then further grown for 8 hours in a 100 mL preculture of LB medium with 50 µM oleic acid in a 500 mL shake flask. Again, 8.5 mL of preculture was added to four 2 L shake flasks containing 850 mL of LB medium with a concentration of oleic acid of either 50, 100, 200 or 300 µM. Cells were incubated overnight. Finally, 25 mL of the 300 µM oleic acid culture was mixed with 25 mL of glycerol and stored at -80°C in a conical 50 mL tube. Another 10 mL fraction was centrifuged in a conical 15 mL tube at 8000 g for 10 min at 4°C and stored at -20°C for later genomic DNA extraction.
Genomic DNA extraction
Following experimental evolution procedures, cell pellet obtained from the centrifuged 10 mL fraction was thawed on ice while CTAB lysis buffer solution for genomic DNA extraction (2% w/v CTAB, 20 mM EDTA, 100 mM Tris-HCl, 1.4 M NaCl, pH 8) was incubated in a 65°C bath to dissolve all CTAB crystals. Once ready, 400 µl of CTAB solution was used to resuspend the pellet. The mixture was then transferred to a 2 mL microtube and incubated at 65°C for 30 min. Following this, 200 µl of phenol was added to the CTAB cell pellet mixture after which 200 µl of a 96/4 solution of chloroform and isoamyl alcohol was added to the mixture and gently shaked. The preparation was then centrifuged at 10000 g for 2 min. Then, 375 µl of aqueous phase at the top was removed and transferred to a 2 mL microtube. Thereafter, 375 µl of a 96/4 solution of chloroform and isoamyl alcohol was added to the sample, gently shaked and centrifuged at 10000 g for 2 min. Then, 350 µl of aqueous phase at the top was removed and transferred to a new 2 mL microtube to which 350 µl of isopropanol was added. The sample was then gently inverted and incubated 10 min at room temperature. Following this, the sample was centrifuged at 10000 g for 10 min after which the supernatant was removed. In place, 750 µl of ice cold 70% ethanol was added and allowed to incubate for 2 minutes at room temperature. The sample was then centrifuged at 10000 g for 5 min. Ethanol was then removed and the microtube was opened and allowed to dry. Once dry, 100 µL of milli-Q water was added to the microtube.
Cloning, expression & purification
The new BM3 gene sequence generated by experimental evolution was synthesized by PCR from the genomic DNA extracted from the B. megaterium variant which had evolved to tolerate oleic acid. This genomic DNA served as a template for the forward primer 5UTR, CATTGAAAGCGGTCTGGCAAACGAGAGA, and the reverse primer 3UTR2, CATGTGAAGGTGGCGCGTGATGGA and the pfu polymerase to generate PCR products to send for sequencing and for cloning procedures. The PCR program for this amplification went as such: 95°C, 5 min / 95°C, 1 min / 60°C, 1 min / 72°C, 4 min, last three steps repeated 29 times followed by 72°C, 10 min. The WT BM3 gene sequence was similarly obtained from the genomic DNA extracted from unevolved WT B. megaterium 14581. The genomic DNA therein acquired served as a template for the forward primer P450-BM3-AS2, ATGACAATTAAAGAAATGCCTCAGCC, reverse primer P450-BM3-AAS2, ACACGTCTTTTGCGTATCGG and pfu polymerase. The PCR program for this amplification went as such: 95°C, 5 min / 95°C, 1 min / 60°C, 1 min / 72°C, 3.5 min, last three steps repeated 29 times followed by 72°C, 10 min. Both evolved and WT PCR products were then further amplified using the forward primer P450-BM3-AS1, CGACTAGTATGACAATTAAAGAAATGCCTCAGCCA, and the reverse primer P450-BM3-AAS1, ATGGATCCTTACCCAGCCCACACGTCTTT. The PCR products were then subcloned into an altered pET-15B vector using SpeI/BamHI restriction sites. As a result, the BM3 enzyme integrated into the vector would now have an octahistidine tag (GHHHHHHHH) followed by a spacer sequence (SSGHHTSM) added to the N-terminus of their sequence. Backcross mutations added to the evolved BM3 variant in relation to its WT parent were performed by DpnI (NEB) site directed mutagenesis. The forward primer I26V/T28A, TTATTAAACACAGATAAACCGGTTCAAGCTTTGATGAAAATTGCGGATGA, and the forward primer I127V/T135A were used with the reverse primer RC1230, GTTGCTTCATGAAGAGCGAACTGCTGACCGATACACGCAC, to generate the corresponding mutations by megaprimer PCR. For mutagenesis of the DE sequence the PCR reactions were carried out with 100 ng of plasmid, 10 nmoles of deoxynucleotides (Bio Basic), 10 pmoles of each primer and 5 units of Pfu polymerase (Bio Basic) which were then added in a 50 µL PCR reaction tube. To bring about a megaprimer, 20 cycles of PCR with an elongation time of 1.5 min/cycle were carried out followed by 30 cycles with a 20min/cycle elongation time to allow full plasmid amplification. Afterwards, 10 units of DpnI restriction enzymes were added to the resulting PCR reactions which were then incubated at 37°C for 1 h. The PCR reactions were then inserted into E. coli dh5a competent cells through a standard heat shock protocol and then sent for sequencing.
The production of the different recombinant BM3 enzymes was accomplished first by transforming the modified pET-15B vectors by heat shock into E. coli strain BL21 (DE3) pLysS on LB plates containing 100 µg/mL ampicillin. A single colony was then picked and grown overnight at 37°C in 100 mL of LB medium with 100 µg/mL of ampicillin. The pre-culture medium would then be replaced by centrifuging it 10 min at 8000 g and resuspending the pellet obtained in 100 mL of fresh LB medium. An inoculum of 22,5 mL of pre-culture would then be added to 0.9 L of modified Terrific Broth (24 g/L Tryptone, 48 g/L yeast extract, 10 g/L NaCl, pH 7.6) containing 100 µg/mL of ampicillin and grown at 37°C. When the culture reached an OD600 = 0.6, induction was initiated by supplementing the culture with 1.1 mL of 1M isopropyl β-D-1-thiogtlactopyranoside (IPTG) to a final concentration of 1 mM. In addition, 150 mL of modified Terrific Broth pH 8, 50 mL of 50% (v/v) 0.48 micron filtered glycerol and 200 µL of 100 mg/mL of ampicillin would be added to the culture, after which the induction temperature was immediately set to 28°C. Cells were incubated for 5–8 hours and were thereafter pelleted by centrifugation at 8000 g for 10 min after which they were stored at -80°C. For purification, centrifuged cells were first thawed on ice, then resuspended in 20 mL of 100 mM lysis buffer (NaCl, 25 mM Tris-HCl pH 8 with 1 mM phenylmethylsulfonyl fluoride) and lysed by sonication using 4 x 15 s bursts separated by 30 s pauses. Sonication and every following step would then be performed on ice or at 4°C. When sonication was complete, the suspension was clarified by centrifugation at 10,000 g, 35 min. The BM3 enzyme was purified by feeding the clarified supernatant onto 25 mL of Ni sepharose 6 fast flow resin (GE Healthcare) packed in a homemade chromatography column. The packed column was first washed with 200 mL of running buffer (40 mM NaH2PO4, 500 mM NaCl, pH 7.4). Then, the column was further washed three times with 100 mL of three different washing buffers identical in composition and pH to the running buffer but with histidine added to the solution. The first washing buffer contained 10 mM histidine, the second 20 mM histidine and the third 40 mM of histidine. The enzyme was eluted from the column with an elution buffer, again identical to the running buffer but also containing 160 mM histidine. The eluate obtained was mixed to 1 volume of glycerol yielding a 50% glycerol enzyme solution which would then be stored at -80°C. In order to measure the P450 concentration, the CO difference spectroscopy method was used with an extinction coefficient of 91 mM− 1cm− 1 to generate the characteristic 450 nm absorption peak of CYP enzymes .
Activity assays with 10-pNCA
To establish the maximum turnover number (TON) the BM3 variants could generate, the spectrophotometric activity assay developed for BM3 based on the release of p-nitrophenolate (pNP) from p-nitrophenoxycarboxylic acids was used . All enzymatic assays were performed overnight with 100 nM enzyme, 600 µM p-nitrophenoxydecanoic acid (10-pNCA) and 1500 µM of either NADPH or NADH in a 100 mM Bis-Tris propane buffer with 5% (v/v) methanol, in a total volume of 1.5 mL, pH 8, 4°C, in darkness. Buffer solutions were always preincubated at 4°C prior to the reaction to ensure the correct temperature would be maintained from the very beginning of the experiment. Noteworthy, 10-pNCA was added before the cofactor (45 µl of 20 mM 10-pNCA and 30 µl of a 75 mM cofactor stock solution). Reactions aimed at investigating thermal stability were preincubated at 37°C for 10 minutes after which reactions were initiated as described above, once 10-pNCA and then NADPH was added, the samples were transferred to a 25°C bath, in darkness. Likewise, the organic solvent tolerance reactions were preincubated for 10 minutes in a 100 mM Bis-Tris propane buffer but with 10% (v/v) methanol at 4°C, in darkness. Reactions were initiated by the addition of 10-pNCA followed by NADPH and also performed at 4°C, in darkness. After an overnight incubation, 1 mL of each reaction solution was mixed with 150 µl of 10 N NaOH in a 2 mL microtube and 850 µl of methanol was added to the mixture up to a final volume of 2 mL. After this, 1 mL of this mixture was added to a 1 cm width cuvette to detect the absorbance of p-nitrophenolate measured at 410 nm in a Genesys 10S UV-Vis spectrophotometer (Thermo Fisher Scientific) and pNP concentration was determined from a calibration curve. To maintain pNP concentration within the range of the calibration, samples could be diluted further in methanol. For each sample set, the standard deviation and mean were calculated from 3 separate, independent experiments, each with three biological replicates. The units of TON in this work are defined as moles of pNP per mole of CYP enzyme or more simply, pNP/CYP.
Similarly, for kinetic measurements, the 100 mM Bis-Tris propane buffer, 5% (v/v) methanol, pH 8 was used at a temperature of 25°C. Concentrations of 100 nM (BM3) and 300 µM (10-pNCA/NADPH) were used. Reactions were performed in a final volume of 300 µl in 96 well plates. To initiate the reactions, 100 µL of 900 µM NADPH stock solution was added to 200 µl of reaction mixture containing both the enzyme and 10-pNCA. To monitor both pNP production and NADPH utilization, absorbance measurements were made at 410 nm and 340 nm, respectively using a Synergy H1 hybrid multi-mode reader (BioTek) every 5 s for 5 min with the first measure taken at t = 30 s. The concentrations of both pNP and NADH (or NADPH) were obtained from a calibration curve. There, the standard deviation and mean for every sample set were calculated from three experimental replicates. The units for the enzymatic activity towards the release of pNP or the consumption of NADPH are defined as moles of pNP or NADPH per moles of CYP per minute which is shortened here to min− 1. Coupling ratio is defined as the ratio between CYP pNP production rate and NADPH consumption rate and is expressed as percentage.