Light-driven hydroxylation of testosterone by Synechocystis sp. PCC 6803 expressing the heterologous CYP450 monooxygenase CYP110D1

730 CDW L antibiotic-free The reaction was initiated by mixing of concentrated cell 625 µL DMSO, reach final of 1 The reaction mixtures were incubated within an open glass cylinder with a diameter of and cm high and placed within the photoreactor mentioned above for 42 hours. The product extraction was performed by thoroughly mixing the reaction with an equal volume of ethyl acetate for three times. The organic phase was dried in a rotavapor and at 4 °C prior to


Experimental Preparative-scale biotransformations with Synechocystis
Synechocystis cells were grown as described in the previous sub-section and concentrated to OD 730 =10 (1.8 g CDW L -1 ) in antibiotic-free BG11 medium. The reaction was initiated by mixing 50 mL of concentrated cell suspension with 625 µL of 80 mM testosterone dissolved in DMSO, to reach a final concentration of 1 mM. The reaction mixtures were incubated within an open glass cylinder with a diameter of 9 cm and 11.5 cm high and placed within the photoreactor mentioned above for 42 hours. The product extraction was performed by thoroughly mixing the reaction mixture with an equal volume of ethyl acetate for three times. The organic phase was dried in a rotavapor and stored at 4 °C prior to analysis.

Samples derivatization and gas chromatography/high-resolution mass spectrometry (GC-HRMS)
Samples of 1 mM testosterone, 1 mM 15β-hydroxytestosterone as well as the extracted reaction mixture from the preparative scale biotransformation (with approximately 1 mM of the product) were analyzed via GC-HRMS. The samples were first derivatized using N,Obis(trimethyl-silyl)-trifluoracetamide and trimethylchlorsilane in pyridine to produce the respective OTMS-derivatives before GC-HRMS measurements.
For the GC separation, a Thermo Scientific Trace 1310 gas chromatograph (Thermo Scientific, Bremen, Germany) was equipped with a 30 m analytical column (Phenomenex ZB5-MS, 30 m x 0.25 mm ID, t f = 0.25 µm). A split injection port at 270°C was used for sample introduction and the split ratio was set to 10:1. The temperature program was 50 °C (3 min) -10°C min -1 -310 °C (3 min). The helium carrier gas was set to 1.0 mL min -1 flow rate (constant flow mode). The transfer line was kept at 290°C.
For the mass spectrometry analysis, a Q Exactive TM GC orbitrap mass spectrometer (ThermoScientific, Bremen, Germany) was used. The resolution was set to 60,000 (FWHM; instrument setting at 200 u). Mass range was 50-650 u and 2 micro scans were averaged per data scan. Automated gain control (AGC target) was set to 1 × 10 6 and maximum inject time was set to "auto". Auxiliary temperatures were set to 290 °C for both transfer lines 1 and 2. MS transfer line temperature was set to 290 °C and the temperature of the electron ionization source was set to 220 °C. EI was performed at 70 eV energy in positive mode. Helium (carrier gas) and nitrogen (supply for the C-Trap) were equipped with gas purification cartridges to trap moisture and organic impurities of the gases (Thermo Scientific, Bremen, Germany). Column bleed ion at 207.03235 u was used as lock mass for internal mass calibration of the data. High resolution masses of the derivatized 15β-hydroxytestosterone standard and the respective product peak of the reaction mixture (from the preparative scale biotransformation) after derivatization were m/z = 520.32153 and m/z = 520.32159, respectively, corresponding to the steroid with 3 OTMS groups.

Nuclear magnetic resonance (NMR) analysis
NMR analysis of the extracted reaction mixture from the preparative scale biotransformation were performed with a 300 MHz Bruker spectrometer using deuterated chloroform as solvent. Complete assignment of all 13 C signals was done using literature data, 37 Distortionless Enhancement by Polarization Transfer (DEPT) and Heteronuclear Single Quantum Coherence (HSQC) analysis and reported in Figures S6-S8 and Tables S3 and S4 . This study a Restriction sites are underlined; RBS sequence is in lower case; His tag coding sequence is bold.

Fig. S1
Confirmation of the presence of each of the synthetic modules P psbA2* ::B0032::CYP110D1 (with or without His-tag), or P trc.x.tetO2 ::B0032::CYP110D1 (with or without His-tag) in Synechocystis strains by PCR using the primer pair (CYP110D1_central_FW and pSEVA251_RV) . C -, negative control; C + , positive control using the purified plasmid as a template; #, clone identification number; M, Gene Ruler DNA Ladder Mix (Thermo-Fisher Scientific).     Table S3). The reaction was performed by incubating 1 mM testosterone in 50 mL reaction volume of Synechocystis cells (expressing CYP110D1 under the control of P trc.x.tetO2 promoter) concentrated to OD 730 =10 (1.8 g CDW L -1 ) in BG11 medium, in open glass cylinders, at 30 °C, with a light intensity of 150 µmol photons s -1 m -2 .      Error bars represent the standard error of the mean (SEM) of 3 biological replicates and 2 technical replicates (*** p-value < 0.001).