Photobiocatalytic Oxyfunctionalization with High Reaction Rate using a Baeyer–Villiger Monooxygenase from Burkholderia xenovorans in Metabolically Engineered Cyanobacteria

Baeyer–Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to lactones under very mild reaction conditions. This enzymatic route is hindered by the requirement of a stoichiometric supply of auxiliary substrates for cofactor recycling and difficulties with supplying the necessary oxygen. The recombinant production of BVMO in cyanobacteria allows the substitution of auxiliary organic cosubstrates with water as an electron donor and the utilization of oxygen generated by photosynthetic water splitting. Herein, we report the identification of a BVMO from Burkholderia xenovorans (BVMOXeno) that exhibits higher reaction rates in comparison to currently identified BVMOs. We report a 10-fold increase in specific activity in comparison to cyclohexanone monooxygenase (CHMOAcineto) in Synechocystis sp. PCC 6803 (25 vs 2.3 U gDCW–1 at an optical density of OD750 = 10) and an initial rate of 3.7 ± 0.2 mM h–1. While the cells containing CHMOAcineto showed a considerable reduction of cyclohexanone to cyclohexanol, this unwanted side reaction was almost completely suppressed for BVMOXeno, which was attributed to the much faster lactone formation and a 10-fold lower KM value of BVMOXeno toward cyclohexanone. Furthermore, the whole-cell catalyst showed outstanding stereoselectivity. These results show that, despite the self-shading of the cells, high specific activities can be obtained at elevated cell densities and even further increased through manipulation of the photosynthetic electron transport chain (PETC). The obtained rates of up to 3.7 mM h–1 underline the usefulness of oxygenic cyanobacteria as a chassis for enzymatic oxidation reactions. The photosynthetic oxygen evolution can contribute to alleviating the highly problematic oxygen mass-transfer limitation of oxygen-dependent enzymatic processes.


Experimental Procedures 1. Reagents
All chemicals were purchased either from Sigma-Aldrich (Steinheim, Germany) or Carl-Roth GmbH (Karlsruhe, Germany) unless otherwise mentioned and were used without further purifications.  Table S2. The primers used during PCR for Gibson assembly.

Correlation of Cell Dry Weight and Optical Density at 750 nm (OD750)
An OD750 of 10 corresponds to 2.4 gDCW L -1 as previously determined for Synechocystis sp. PCC6803. 4,5

Media for Cultivation and whole-cell biotransformations For E. coli
Lysogeny Broth (LB, 1 L: Tryptone 10 g, Yeast Extract 5 g, NaCl 5 g) was utilized for 5 mL overnight cultures which were then transferred to Terrific Broth (TB, 1L: Peptone 12 g, Yeast extract 24 g, Glycerol 4 mL, KH2PO4 2.2 g, K2HPO4 9.4 g) media for protein production biotransformation. Liquid media were autoclaved at 121 °C for 21 minutes and kept at room temperature. Solid media were prepared in LB agar plates (15 g Agar L -1 ). Ampicillin (Amp, 100 µg mL -1 ) was utilized as selection marker.

For Synechocystis
Standard BG-11 4 was utilized for the cultivation of Syn::PcpcBVMOXeno and pH was adjusted to 7 using NaOH and autoclaved as described above.
BG-11/MOPS buffer (900 mL) was prepared by combining 90 mL of MOPS buffer (10X) with standard BG-11 (without HEPES and EDTA). The solution was autoclaved prior usage and has a resulting pH of 6.8-7.

Selection of BVMOs from High-Throughput screening
The sequences of 28 experimentally confirmed type I BVMOs were used for a blast-based screening against 1500 sequences from the public databases (Genoscope, CEA, Evry, France -Dr. V. de Bérardinis). 3,6 The gene candidates were chosen depending on their accessibility in the Genoscope strain collection. After clustering, 450 putative bvmo genes were cloned in E.
coli BL21(DE3) then 370 candidate enzymes were produced and sixty active enzymes were identified, selected out of a high throughput screening (HTS) over twenty substrates. In this study, eleven enzymes from this screening were tested via whole cell experiments against cyclohexanone.

Construction of Synechocystis mutants
SynRekB_Pcpc_chmo and SynRekB_PPsbA2_chmo plasmids were kindly provided by Prof.
Dr. Robert Kourist (Graz University of Technology, Graz, Austria). BVMOParvi (accession number: A7HU16) and BVMOXeno (accession number: Q13I90) are coded by the genes bvmoParvi and bvmoXeno, respectively. The genes were obtained from high throughput cloning and were available in a modified pET22b plasmid (Genescope). 3 The two bvmo genes were inserted into the SynRekB::Pcpc and SynRekB::PPsbA2 vectors resulting in SynRekB His-BVMOParvi and SynRekB His-BVMOXeno plasmids. All of the constructs contain a 6-His tag in the N-terminus. Cloning into the SynRekB plasmids was performed using the Gibson assembly user manual.

Cultivation and Maintenance of Synechocystis strains
Seed cultures (in BG-11 liquid media or on BG-11 agar plates) were maintained in a plant growth chamber (SWGC-1000, WISD) at 30 °C under atmospheric CO2 conditions and 50% humidity. Continuous illumination was provided by fitted white fluorescent lamps with an intensity between 40 to 60 µE m -2 s -1 . Cells were cultured in 100 mL or 300 mL Erlenmeyer flasks with 50 mL and 100 mL of working volumes, respectively on rotary shakers at 140 rpm.
For higher light cultivations, Synechocystis strains were cultivated in standard BG-11, supplemented with the appropriate antibiotics. The cultures were placed on a rotary shaker at 140 rpm and illuminated by a tunable LED lamp (CellDEG, Berlin, Germany) emitting red and blue light at an intensity of 150 µE m -2 s -1 as previously described. 5 Synechocystis cells harboring BVMOXeno and BVMOParvi were grown until an OD750= 0.80-1.20 and OD750= 1-2, respectively. On the other hand, DFlv1 variants were grown until an OD750 of 1.5-2.0.

Transformation of mutants and segregation check
Wild-type cells were cultivated and harvested at an optical density of OD750 = 0.5-1. The cells were then re-suspended in fresh BG-11 medium (500 µL). Plasmid DNA (5 µg) containing the vector was added to the cell suspension and were incubated at 30 °C for 6 h in darkness with shaking (140 rpm). Afterwards, the cells were transferred onto a sterile nitrocellulose membrane (GE Healthcare) and placed on a BG-11 agar plate without antibiotics.

Achiral compounds
Samples (100 µL) were extracted with dichloromethane (300 µL) containing 2 mM of ndecanol as internal standard. The suspension was mixed by inverting up and down for 1 min and the organic layer was dried using one spatula tip of anhydrous MgSO4. After centrifugation (4 °C, 5 min, 13 000 g), the organic phase was separated and measured directly on achiral GC-FID using nitrogen as the carrier gas with a split ratio of 20 as previously described. 5 All compounds stemming from the oxidation of cyclohexanone (1a) to its corresponding lactone (1b) and side-product (1c) was determined using GC equipped with a Flame Ionization Detector (FID, GC-2010 Plus, Shimadzu, Japan). The GC

Statistical Analysis
Statistical analysis was performed using GraphPad Prism version 8.0. Shapiro-Wilk's test was used to assess the normality of the data sets. Unpaired Welch's t test was then utilized for comparison of two data sets following a Gaussian distribution. A minimum of three independent (N=3) measurements or calculations were utilized for the statistical analyses (α = 0.05).

Purification of BVMOXeno and CHMOAcineto
For purification of the 6xHis-tagged BVMOXeno and CHMOAcineto, the cells were grown at 37˚C and 160 rpm until OD 600 = 0.8. After induction with 1 mM IPTG (final), the production of BVMO Xeno and CHMO Acineto were performed overnight at 18 ˚C and 15 ˚C, respectively. Protein purification was performed under native conditions according to Protino®-Macherey-Nagel Ni-NTA purification protocol. All buffers were prepared accordingly and stored at 4 ˚C.
Purifications were performed at 4 ˚C and the enzymes were kept on ice throughout the whole process. After Ni-NTA affinity purification, imidazole was removed from the purified enzymes by utilizing desalting columns (GE healthcare) using Tris-HCl buffer, pH 8.0. The samples were then loaded to 10% SDS gel and were ran at 100 V. The images were taken following the Coomassie staining/destaining steps.  Table 1.

Determination of degradation temperatures (T m ) of BVMO Xeno
The apparent melting point (Tm) of both purified enzymes were determined by an adapted ThermoFAD method. 8   Potassium buffer (20 mM, pH = 6.5) was degassed for 10-15 min by sparging with nitrogen.
A solution containing 1a (1 mM), NADPH (125 µM) in potassium buffer was prepared and placed in a glass cuvette with cover. The reaction was started with the addition of the CFE (100 µL) and the absorption was monitored at 340 nm for 15min. Background reactions were subtracted by performing the reaction without the substrate. The enzyme's in vitro activity was calculated using the slope and the extinction coefficient for NADPH (ɛ = 0.0045 L µmol -1 cm -1 ) normalized to the protein content determined from the BCA Assay. Figure S6 show a representative diagram from the in vitro assays.

Application of substituted cyclic ketones with Syn::PcpcBVMOXeno
Biotransformation with 2-Phenylcyclohexanone (2a) is performed with Syn::PcpcBVMOXeno, resulting in full conversion of both ketones and E>200. The ee values were calculated from GC-FID analysis. Figure S7 shows the whole-cell biotransformation of 2-Phenylcyclohexanone using Syn::PcpcBVMOXeno. To be able to compare the specific activities with the same promoter, experiments were carried out using Syn::PcpcCHMOAcineto. Figure

Whole cell biotransformation of 1a using BVMO Xeno with different promoters
Synechocystis cells harboring BVMO Xeno controlled by the P psbA2 promoter were cultivated similarly and utilized in whole-cell biotransformations of 1a. Figure S8 show the specific activity comparison and time course of 1b formation using Syn::P cpc BVMO Xeno and Syn::P psbA2 BVMO Xeno .

Inhibition studies in Syn::P cpc BVMO Xeno
To be able to study the extent of product inhibition, we ran whole cell biotransformations in Syn::P cpc BVMO Xeno containing different concentrations of the product from the beginning of the reaction, ɛ-caprolactone (1b). The activity was calculated relatively to reactions without 1b. Figure S9. Effect of ɛ-caprolactone concentration during whole cell biotransformation of 1a in Syn::PcpcBVMOXeno. Reaction conditions: V=1 mL, T= 30 °C, 300 µE m -2 s -1 , 160 rpm, N = 3, CDW= 2.4 g L -1 , initial concentration of 10 mM 1a.