Transmembrane Shuttling of Photosynthetically Produced Electrons to Propel Extracellular Biocatalytic Redox Reactions in a Modular Fashion

Abstract Many biocatalytic redox reactions depend on the cofactor NAD(P)H, which may be provided by dedicated recycling systems. Exploiting light and water for NADPH‐regeneration as it is performed, e.g. by cyanobacteria, is conceptually very appealing due to its high atom economy. However, the current use of cyanobacteria is limited, e.g. by challenging and time‐consuming heterologous enzyme expression in cyanobacteria as well as limitations of substrate or product transport through the cell wall. Here we establish a transmembrane electron shuttling system propelled by the cyanobacterial photosynthesis to drive extracellular NAD(P)H‐dependent redox reactions. The modular photo‐electron shuttling (MPS) overcomes the need for cloning and problems associated with enzyme‐ or substrate‐toxicity and substrate uptake. The MPS was demonstrated on four classes of enzymes with 19 enzymes and various types of substrates, reaching conversions of up to 99 % and giving products with >99 % optical purity.


Source of organisms
Synechocystis sp. PCC 6083 wild-type (substrain Kazusa, geographical origin in California (USA)) was received from Prof. Tamagnini at the University of Porto. [1][2][3] The wild-type and recombinant Synechococcus elongatus PCC 7942 (alcohol dehydrogenase from L. kefir under the PpsbA1 promoter and spectinomycin resistance cassette integrated into the neutral site 1 (NS1) of the chromosome) were received from Prof. Waginkar at the Indian Institute of Technology Bombay. [4] 3

.4 Enyzmes
The enzymes used in this study are enlisted in Table S11. S16 Table S11. Enzymes used in this study.

Expression of enzymes in E. coli
The enzymes used in this study were expressed in E. coli BL21(DE3) or its derivatives (Table S13).

Transformation
Plasmids (100 ng) were mixed with chemically competent E. coli BL21 (DE3) cells (100 µL), rested on ice for 30 minutes and heat-shocked for 30 seconds at 42 °C. SOC medium (200 µL) was added, and the transformed cells were incubated for 1 h at 37 °C and 300 rpm. The cells were then plated on a LBagar plate supplemented with corresponding antibiotic (ampicillin, 100 µg mL -1 or kanamycin, 50 µg mL -1 ) and incubated overnight at 37 °C.

Cultivation
Overnight cultures (ONC) were prepared in LB-medium (10 mL) supplemented with the corresponding antibiotic at 30 °C and 120 rpm. The ONCs were then used for the inoculation of sterile medium (1% v/v) supplemented with the corresponding antibiotic. Precultures were incubated until OD600 of 0.6 was reached, then protein expression was induced, and cells incubated further. Detailed conditions for every enzyme can be found in Table S13.

Harvesting
To harvest the cells, cultures were centrifuged at 3184 g, 20 min, 4 °C, the cell pellet suspended in wash buffer (1 -2.5 g cells per 10 mL phosphate buffer, 10 mM, pH 7), and then centrifuged again under the same conditions. S17

Preparation of cell-free extracts
Cell pellets were suspended in lysis buffer and sonicated on ice (for conditions see Table S12). The sonicated cells were centrifuged for 25 minutes at 17 000 g and 4 °C. The supernatant (cell-free extract) was shock-frozen (liquid nitrogen) inside a round bottom flask, lyophilized, and stored at -20 °C. The pH of the buffers was adjusted by using hydrochloric acid (HCl) and sodium hydroxide (NaOH).

SDS-PAGE
Protein concentrations of cell pellets and supernatants were determined via a Bradford Assay.
Volumes equivalent to 15 µg of protein were mixed with Laemmli sample buffer (1:1) and heated to 95 °C for 5 minutes. Prepared samples and a marker (PageRuler Prestained Protein Ladder; 7 µL) were loaded onto the 10 % SDS-PAGE gel (100 V, MOPS buffer). The gel was stained overnight using Coomassie Quick Stain and afterwards destained with deionized water. S18

Preparation of purified enzymes
Buffers and sonication conditions used are listed in Table S14.

Cell lysis
Harvested cell pellets were resuspended in binding buffer (ene-reductases were supplemented with a spatula tip of FMN) and sonicated on ice. (Digital sonifier, BRANSON). The cell suspension was centrifuged (20 min, 18 000 g, 4 °C) and the supernatant was filtered (0.45 µm syringe filter) and stored on ice. hydroxide (10 mM NaOH in water). NaOH was removed immediately by washing the column two times with four column volumes binding buffer. The column was stored at 4 °C.

His-tag purification
LkADH and the ene-reductases containing the His6-tag were purified by an immobilized metal ion affinity chromatography (HisTrapTM FF, 5 mL, GE HEALTHCARE). The purification was performed at 4 °C and at a flow rate of 5 mL min -1 . Prior to loading the soluble fraction to a HisTrap FF column (GE Healthcare, 5mL) equilibrated in binding buffer it was filtered through a 45 μm syringe filter. After loading the column was washed with binding buffer according to the manual. Then, the enzyme was eluted using elution buffer. All purification steps were verified by SDS PAGE.

Storage
The volume of the fractions containing the enzyme (colored yellow or visualized with Bradford reagent) was reduced to 2.5 mL using a Vivaspin® 20 mL Ultrafiltration Unit (Satorius). Then, the buffer was changed to storage buffer using a Sephadex G-25 PD10 desalting column (GE Healthcare). The final enzyme solution was aliquoted and stored at -20 °C. The pH of the buffers was adjusted by using hydrochloric acid (HCl) and sodium hydroxide (NaOH). Buffers used for purification were degassed using ultrasonication and filtered with a Steritop® Filter (Millipore Express® PLUS, 0.22 µm PES Membrane).

Cultivation of cyanobacteria
Cyanobacteria were cultivated in BG11 medium [1]  Seed cultures of recombinant Synechococcus elongatus were grown in the presence of spectinomycin (100 µg mL -1 ).

Determination of the cell dry weight and chlorophyll a content
The dry cell weight and the amount of chlorophyl a were determined from samples originating from at least three independent cultivations under growth conditions for working cultures, each measured in triplicates.

Cell dry weight
Working cultures grown and harvested as above were shock-frozen in liquid nitrogen, lyophilized overnight and weighed in three independent experiments.
The chlorophyll a was determined as described. [32] A sample of the cell culture (100 μL) was mixed with cold methanol (900 μL) and incubated in darkness at 4 °C for 2-3 hours or overnight. Then, the samples were centrifuged for 3 min at 14000 g and the absorption of the supernatant was measured at 665 nm. The amount of chlorophyll was determined using the extinction coefficient ε = 78.74 L g -1 cm -1 according to Eq. S 1, where the dilution factor corresponds to 10.

S22
The vials were incubated in a custom photoreactor [33] equipped with cool white LEDs (LED stripes, 5200 K) for the indicated reaction time (16 h) at 600 rpm shaking and at room temperature at an average light intensity of 215 µE m -2 s -1 (photoreactor settings: frequency 100 Hz, duty range 100, duty cycle 5) or covered with aluminum foil for dark reactions. The workup and analytics are described in section 4.  (Table S17).  (Table S 18).
The reaction mixtures containing different shuttle-molecule pairs were extracted twice with ethyl acetate (400 μL, 250 μL), dried over anhydrous sodium sulphate and measured on GC-MS.