Tyramine Derivatives Catalyze the Aldol Dimerization of Butyraldehyde in the Presence of Escherichia coli

Abstract Biogenic amine organocatalysts have transformed the field of synthetic organic chemistry. Yet despite their use in synthesis and to label biomolecules in vitro, amine organocatalysis in vivo has received comparatively little attention – despite the potential of such reactions to be interfaced with living cells and to modify cellular metabolites. Herein we report that biogenic amines derived from L‐tyrosine catalyze the self‐aldol condensation of butanal to 2‐ethylhexenal – a key intermediate in the production of the bulk chemical 2‐ethylhexanol – in the presence of living Escherichia coli and outperform many amine organocatalysts currently used in synthetic organic chemistry. Furthermore, we demonstrate that cell lysate from E. coli and the prolific amine overproducer Corynebacterium glutamicum ATCC 13032 catalyze this reaction in vitro, demonstrating the potential for microbial metabolism to be used as a source of organocatalysts for biocompatible reactions in cells.


S1.2 Media, Strains and Culturing Conditions
Luria Bertani Lennox (LB) Media was prepared according to the following procedure: bacto-tryptone (10 g/L), yeast extract (5 g/L) and NaCl (10 g/L) was dissolved in ultrapure H2O. LB was autoclaved at 121 ℃ for 20 min, cooled and stored at room temperature. LB agar was made using the same recipe but with the addition of agar (15 g/L). SOC media was prepared according to the following procedure: bacto-tryptone (20 g/L), yeast extract (5 g/L) and NaCl (0.5 g/L) were dissolved in ultrapure H2O. KCl was added to a final concentration of 2.5 mM. The mixture was autoclaved at 121 ℃ for 20 min and cooled before adding glucose (20 mM) and MgCl2 (10 mM). SOC media was then stored at room temperature.
All other reagents used in culturing experiments were filter sterilised using a 0.22 µm filter (Millex).
All chemically competent cells were prepared via treatment with calcium chloride [1]. For use as a culture inoculum, chemically competent E. coli MG1655 or MG1655 RARE were prepared and transformed with a pET22b(+) plasmid (Amp r ) via heat-shock at 42 ℃ for 45 s. Cells were recovered in 1 mL of SOC media for 1 h at 37 ℃. Transformants were selected by plating on LB agar containing appropriate antibiotics and incubating at 37 ℃ overnight. A single colony was picked and grown overnight in 10 mL of LB containing appropriate antibiotics. The resulting overnight culture (0.5 mL) was added to 0.5 mL of 1:1 v/v water:glycerol solution, frozen in liquid nitrogen, stored at -80 ℃ and used as required. For microbiological experiments, antibiotics were used at the following concentrations: ampicillin (Amp), 100 μg/mL. E. coli cultures were prepared by inoculating 10 mL of LB with a -80 ℃ LB:glycerol stock of E. coli MG1655 RARE_pET22b(+) or E. coli MG1655_pET22b(+) and incubating cultures at 30 ℃ (220 rpm) for 18 h. The saturated overnight culture (5 mL) was then inoculated into LB (250 mL in 500 mL Erlenmeyer flask) containing ampicillin and grown aerobically at 220 rpm until the culture reached OD 600 = 0.5−0.6 (ca. 2.5−3 h). E. coli MG1655 was grown at 37 °C, and E. coli RARE was grown at 30 °C.
Cell lysates were prepared according to the following procedure: using a fresh overnight culture of the bacterium, 1 L LB media in a 2 L baffled flask containing appropriate antibiotics was inoculated (1:100) and grown for 24 h at the relevant temperature reaching an OD600 value of 3.0−7.0. Cells were collected using centrifugation (10,000 × g, 10 min, 4 ℃). The supernatants were collected, and a sample taken for use as a media/catalyst source for screening reactions. The cell pellets were resuspended in ice-cold Phosphate Buffered Saline (PBS) to a cell density of OD600 = 50. Keeping samples on ice throughout, an ultrasonic cell disruptor was used to lyse the cells (50% amplitude, 30 seconds on/off, 10 min total).
Half of the crude lysate was centrifugated (20 min, 24,000 × g, 4 ℃). The crude lysate and resulting supernatant (clarified lysate), were used as reaction media/catalyst source for screening reactions.

S1.3 Butanal Toxicity Screen
A fresh 250 mL culture of E. coli MG1655 or MG1655 RARE was grown to OD 0.5−0.6 and 7.5 mL of the resulting culture was added to sterile Hungate tubes. Using a gas tight syringe, a range of butanal concentrations (0.1−100 mM) were added to the cultures. The tubes were then sealed using butyl rubber septa and incubated horizontally at 30 ℃, 220 rpm. After 24 h, 100 µL of each reaction mixture was removed and added to 900 µL of MQ water. These aliquots were subjected to serial 10-fold dilutions (10 1 -10 8 ). Aliquots (100 µL) of each dilution were plated onto individual LB agar plates containing ampicillin (100 μg/mL). The plates were incubated at 30 ℃ overnight and the number of colonies from these plates were used to calculate the number of colony-forming units (CFUs) in each mL of culture.

S1.4 Investigating the mass balance of butanal added to bacterial cultures
Butanal (25 mM) was added to a Hungate tube containing 7.5 mL of a fresh culture (OD = 0.5−0.6) of E. coli MG1655 RARE or C. glutamicum WB2. A control sample was prepared by replacing the bacterial culture with sterile LB medium. The tubes were sealed and incubated horizontally for 24 h at 30 ℃, 220 rpm. The contents of the tubes were extracted using the protocol for catalyst screening the standard deviation of triplicate samples quantified using 1 H NMR as outlined in S1.5.

S1.5 Catalyst Screening Reactions
All catalyst screening reactions were carried out in triplicate using autoclaved 15 mL glass Hungate tubes with butyl rubber septa and screw caps. Tubes contained 7.5 mL reaction volume and 7.5 mL headspace. Reaction media/cultures were preadjusted to the desired pH using either hydrochloric acid (2 M) or NaOH(aq) (2 M). Catalysts were weighed directly into the empty tubes, to which 7.5 mL reaction media was added. Adding catalysts after adjusting the pH to 9.0 resulted in small changes to the pH (< 0.25). Butanal was then added using a gas-tight syringe.
The Hungate tubes were then incubated for 24 h (30 ℃, 220 rpm), after which they were cooled to −20 ℃ for 15 min. Aliquots (0.5 mL) were added to an equal volume of brine in 2 mL microcentrifuge tubes, to which diethyl ether (0.333 mL) containing 2 mM TMB was added. The tubes were vortexed for 3 min, then centrifugated for 2.5 min (4,500 × g, 4 ℃). The organic phase was decanted into clean microcentrifuge tubes using a glass Pasteur pipette. This extraction process was repeated three times in total. Anhydrous sodium sulfate was added to dry the extract, which was then added to vials for GC-FID analysis. For 1 H NMR analysis, samples were extracted using CDCl3 and analyzed directly.

S7 Cell Lysate Screening Experiments
Cell lysates of E. coli MG1655 RARE and C. glutamicum were prepared as outlined in S1.2. Butanal (25 mM) was added to 7.5 mL samples of PBS, LB medium, OD=5 supernatants, crude lysates, clarified lysates and clarified lysates at pH 9. After 24 h incubation at 30 °C, samples were extracted and analysed by 1 H NMR spectroscopy as outlined in S1.5.

S8 Measuring the toxicity of tyramine derivatives
Mid-log cultures of E. coli MG1655 RARE were grown as outlined in S1.2 and added to sterile Hungate tubes containing 25 mM of tyramine derivative. The tubes were sealed with rubber septa and screw caps, then incubated at 30 °C (220 rpm). Optical density measurements were taken regularly for the first 4−5 h. After 24 h, serial dilutions were prepared as outlined in S1.3, and plated on ampicillincontaining agar plates before counting colonies for CFU measurements.   acid (10 mL) was added aqueous HBr (47%, 10 mL) and the mixture was stirred at reflux for 6 h. The reaction mixture was concentrated under reduced pressure and the resulting residue basified to pH 8 with 1 M aqueous NaOH. The solution was washed with diethyl ether (2 x 30 mL). The aqueous fraction was then acidified to pH 2 with aqueous HCl (37%) and then made basic with aqueous ammonium hydroxide (28%). The solution was saturated with NaCl and then extracted with n-butanol (3 x 30 mL), dried over K2CO3 and concentrated to provide the title compound as a brown oil (0.12 g, 24%). 1

3-[2-(methylamino)ethyl]phenol
H N OH To a solution of [2-(3-methoxyphenyl)ethyl](methyl)amine hydrobromide (1.0 g, 4.1 mmol) in acetic acid (10 mL) was added aqueous HBr (47%, 10 mL) and the mixture was stirred at reflux for 3 h. The reaction mixture was concentrated under reduced pressure and the resulting residue basified (to pH 8) with 1 M aqueous NaOH. The solution was washed with diethyl ether (2 x 30 mL). The aqueous fraction was then acidified to pH 2 with aqueous HCl (37%) and then made basic with aqueous ammonium hydroxide (28%). The solution was saturated with NaCl and then extracted with n-butanol (3 x 30 mL), dried over K2CO3 and concentrated to provide the title compound as a brown oil (0.23 g, 37%). 1