One-Pot De Novo Synthesis of [4Fe-4S] Proteins Using a Recombinant SUF System under Aerobic Conditions

Fe–S clusters are essential cofactors mediating electron transfer in respiratory and metabolic networks. However, obtaining active [4Fe-4S] proteins with heterologous expression is challenging due to (i) the requirements for [4Fe-4S] cluster assembly, (ii) the O2 lability of [4Fe-4S] clusters, and (iii) copurification of undesired proteins (e.g., ferredoxins). Here, we established a facile and efficient protocol to express mature [4Fe-4S] proteins in the PURE system under aerobic conditions. An enzyme aconitase and thermophilic ferredoxin were selected as model [4Fe-4S] proteins for functional verification. We first reconstituted the SUF system in vitro via a stepwise manner using the recombinant SUF subunits (SufABCDSE) individually purified from E. coli. Later, the incorporation of recombinant SUF helper proteins into the PURE system enabled mRNA translation-coupled [4Fe-4S] cluster assembly under the O2-depleted conditions. To overcome the O2 lability of [4Fe-4S] Fe–S clusters, an O2-scavenging enzyme cascade was incorporated, which begins with formate oxidation by formate dehydrogenase for NADH regeneration. Later, NADH is consumed by flavin reductase for FADH2 regeneration. Finally, bifunctional flavin reductase, along with catalase, removes O2 from the reaction while supplying FADH2 to the SufBC2D complex. These amendments enabled a one-pot, two-step synthesis of mature [4Fe-4S] proteins under aerobic conditions, yielding holo-aconitase with a maximum concentration of ∼0.15 mg/mL. This renovated system greatly expands the potential of the PURE system, paving the way for the future reconstruction of redox-active synthetic cells and enhanced cell-free biocatalysis.


Supporting Methods.
Heterologous protein overexpression and purification.Heterologous protein overexpression and purification were performed based on the established protocols published previously with some modifications 1 .Briefly, the cultures were grown under aerobic conditions at 37℃ in a shaker using Terrific Broth (200 rpm) to the optical density of 0.4-0.6 at OD600 nm, followed by the induction using isopropyl β-D-1-thiogalactopyranoside (IPTG) (0.4 mM) and overnight incubation at 16℃.
For Thermococcus profundus ferredoxin (Fd) and Escherichia coli aconitase A (AcnA) expression, after 2 h of the IPTG induction, the cultures were transferred to 1-L screw-capped glass bottles and were supplemented with cysteine (0.2 mM) and FeCl3 (0.1 mM), followed by overnight incubation at 37℃ for Fd and at 16℃ for AcnA, respectively, with constant stirring.Recombinant proteins were purified to at least 95% homogeneity using Ni-sepharose-based immobilized affinity chromatography as previously described 2 , and protein purity was analyzed by SDS-PAGE (5-20% SDS-PAGE gels; Supercep TM Ace; Fujifilm Wako Pure Chemical, Osaka, Japan) followed by Coomassie staining (Figure S1).Briefly, cells were collected by centrifugation at 8,000 × g for 10 min at 4℃, suspended in the binding buffer: HEPES-Na (pH 7.5; 25 mM), NaCl (0.5 M), glycerol (5%, v/v), and imidazole (5 mM), and the cells were homogenized via sonication in an ice bath for 10 min (1 s on and 2 s off).For Fd, the lysate was subsequently boiled for 20 min at 92℃ to denature the endogenous proteins.The lysate was centrifuged at 20,000 × g for 30 min at 4℃ and the supernatant was passed through a glass gravity column containing approximately 0.5 mL Ni Sepharose™ High Performance resin (GE Healthcare, IL, USA).After washing with the wash buffer: HEPES-Na (pH 7.5; 25 mM), NaCl (0.5 M), glycerol (5%, v/v), and imidazole (25 mM), recombinant proteins were eluted using the elution buffer: HEPES-Na (pH 7.5; 25 mM), NaCl (0.5 M), glycerol (5%, v/v), and imidazole (250 mM).For Fd and holo-AcnA, FeSO4 (0.1 mM) and Na2S (0.8 mM) were supplemented to the lysate and wash buffer to chemically reconstitute Fe-S cluster on the proteins.The protein stock solutions were flash-frozen in liquid N2 and stored at −80℃.The N-terminal His6-tag was retained for all experiments except the experiment for purification of the holo-aconitase A synthesized by the PUREfrex.

Purification of holo-aconitase A.
Holo-AcnA was purified from the one-pot cell-free reaction under anaerobic conditions.The one-pot cell-free reaction mixture was passed through a glass gravity column containing approximately 0.1 mL of Ni-sepharose resin.After washing with a wash buffer: HEPES-Na (pH 8.0; 25 mM), NaCl (0.1 M), FeSO4 (40 µM), 2-mercaptoethanol (0.1%, v/v), and glycerol (5%, v/v), HRV 3C Protease (1 U, Takara) was added to the resin to cleave the tag sequence from AcnA and elute the protein.The protein cleavage reaction was performed at 4℃ for 16 h and later the eluate was collected by centrifugation.The eluate was flash-frozen in liquid N2 and stored at −80℃.
Briefly, in an ice bath, solution I (amino acids, NTPs, tRNAs and substrates for enzymes, etc.; 10 µL), solution II (enzyme mixtures; 1 µL), and solution III (ribosome; 2 µL) were mixed with the mRNAs (0.4-0.7 µM) to a final volume of 20 µL.Cell-free protein synthesis was performed at 37℃ for up to 6 h and the reaction mixture was kept on ice after incubation before further SDS-PAGE analysis or reconstitution experiments.

In vitro transcription. In vitro transcription was performed using ScriptMax® Thermo T7
Transcription Kit (TOYOBO, Osaka, Japan) following the manufacturer's standard protocols.
Briefly, DNA templates (0.7 µg) were added to the RNA transcription reaction mixtures (40 µL) and were incubated at 40℃ for 4 h.The RNA transcripts were then purified using NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany) and the concentrations were measured by NanoDropTM2000c spectrophotometer (Thermo Fisher Scientific, MA, USA).
Cysteine desulfurase activity assays.Total sulfide was measured based on a previously published protocol 3 .Reactions were carried out anaerobically in a buffer containing HEPES-Na (pH 7.5; 50 mM), NaCl (0.1 M), SufE (3 µM), SufS (0.5 µM), and glycerol (5%, v/v).Pyridoxal 5′-phosphate was added to 10 μM, and reactions were initiated by dilution of an L-cysteine/DTT stock (40 mM) to a 1 mM final concentration of each in a total reaction volume of 1 mL.Reactions were allowed to proceed for 15 min at 37℃ and then were quenched by mixing 100 µL of the reaction with 100 µL of N, N-dimethyl-p-phenylenediamine (17 mM), and FeCl3 (22.2 mM) in HCl (6 M).
Subsequently, the quenched reactions were incubated for 20 min, leading to the formation of methylene blue.Precipitated protein was removed by 30 s centrifugation at 20,000 × g, and methylene blue was measured with a UV-Vis scanning at λ670 nm on a microplate reader (Epoch 2 microplate reader, Agilent, CA, USA).

Cytochrome C reduction optical assays.
Oxidized ferredoxin is reduced enzymatically by spinach ferredoxin-NADP + reductase (Sigma-Aldrich, MO, USA) using NADPH.Reduced ferredoxin was used to reduce cytochrome C from the equine heart (Sigma-Aldrich, MO, USA) in a nonenzymatic manner.The formation of reduced cytochrome C is monitored by the time-dependent increase in λ550 nm.The reaction mixtures (300 µL) contained HEPES-Na (pH 8.1; 50 mM), NaCl (0.1 M), glycerol (5%, v/v), NADPH (1 mM), cytochrome C (0.1 mM), ferredoxin-NADP + reductase (0.25 µg/mL; Sigma-Aldrich, MO, USA), and reconstituted holo-Fd.The holo-Fd was diluted and assayed at concentrations ranging from 0.5 to 4.0 µM.The reaction was initiated by the addition of holo-Fd.Under aerobic conditions, assays were performed in an optical 96-well microplate at 30℃ for 25 min with a UV-Vis scanning at λ550 nm every 5 s on a microplate reader (EnSpire®, PerkinElmer, MA, USA).All enzyme assays were conducted in duplicates or triplicates as indicated in the figures.
Aconitase A and isocitrate dehydrogenase-coupled activity assays.Aconitase activity was assayed using the procedure previously described 4 by monitoring the formation of NADPH through the increase in λ340 nm.The reaction mixture (100 µL) contained HEPES-K (pH 7.6; 50 mM), MnCl2 (0.5 mM), citrate (10 mM), NADP + (1 mM), 2-mercaptoethanol (0.1%, v/v), and isocitrate dehydrogenase (IDH) (0.2 U; Sigma-Aldrich, MO, USA) with different concentrations of recombinant holo-AcnA.Note that the stock solution for the citrate (1 M) was adjusted to pH 7.6 in HEPES-K buffer (50 mM) to prevent a pH shift in the reaction mixture.The reaction was initiated by the addition of holo-AcnA.Under anaerobic conditions, assays were performed in an optical 96-well microplate at 37°C for 10 min with a UV-Vis scanning at λ340 nm every 10 s on a microplate reader (Epoch 2 microplate reader, Agilent, CA, USA).The λ340 nm was later converted to NADPH concentration based on the standard curves derived from the different concentrations of NADPH.All enzyme assays were conducted in duplicates or triplicates as indicated in the figures.The concentration of reconstituted holo-AcnA was estimated using a standard curve generated by serial diluted holo-AcnA overexpressed and purified from E. coli.Concentration of AcnA in the enzyme assays were estimated from the band intensity in the SDS-PAGE by using AcnA purified from E.coli as a standard.

LC-MS analysis
The completed reactions of the AcnA and IDH-coupled activity assays were collected and filtered through the Amicon® Ultra-0.5 centrifugal filter (cut-off: 3 kDa) (Merck KGaA, Darmstadt, Germany), and the flowthrough fractions were used for an LC-MS system consisting of an Acquity UPLC plus system (Waters, Milford, MA, USA) connected to XEVO G2-XS QTof Mass Spectrometry (Waters, Milford, MA, USA).The citrate and alpha-ketoglutarate were separated and detected as described previously 5 .Briefly, the separation of underivatized carboxylates was achieved by injecting 5 µL of a sample onto an Atlantis Premier BEH C18 AX column (100×2.1 mm, 1.7 mm; Waters, Milford, MA, USA).The mobile phase was composed of (A) 10 mM ammonium formate in the pH 2.6 water and (B) 10 mM ammonium formate in 50/50 water/methanol (v/v) of pH 9.4 (pH of aqueous buffer before mixing with methanol).The mobile phase was delivered to the column at a flow rate of 0.35 mL/min with the following gradient: 0.0 min (0% B), 0.5 min (0% B), 3.0 min (70% B), 6.0 min (70% B), 7.0 min (0% B), and 10.0 min (0% B).The column was thermostated at 30°C.Analytes were ionized in an electrospray ion source operated in the negative mode.The source and gas parameters were set as follows: capillary 2.50 kV, source temperature 100 °C, cone gas 100 L/h, desolvation temperature 300°C, and desolvation gas 490 L/h.The experiment was conducted with and without Apo-AcnA.TIC, total ion current spectrum.The experiment was conducted with and without Apo-AcnA.TIC, total ion current spectrum.

Supporting Tables
Figure S1.SDS-PAGE gel images of purified recombinant proteins and apo-Fd/AcnA synthesized by the PURE system.All recombinant proteins (SufABCDSE, FDH.FRE, CAT,Holo-Fd and Holo-AcnA) are overexpressed and purified from E.coli.Apo-Fd was expressed using PUREfrex2.0and purified by boiling and centrifugation.Apo-AcnA was ran on the gel along with the protein components in the PUREfrex reaction mixture.We did not purify Apo-AcnA due to its application for the one-pot de novo synthesis of holo-AcnA.

Figure S2 . 4 Figure S3 .
Figure S2.Images of the mature SufBC 2 D complex at different concentrations of FeSO 4

Figure S4 .
Figure S4.UV-Vis analysis of the recombinant proteins purified from the anaerobically cultivated E. coli BL21 cells.The two recombinant proteins (A) holo-Fd and (B) AcnA were purified in an anaerobic chamber under strictly anaerobic conditions.The UV-Vis analysis was

Figure S5 .
Figure S5.Standard curve to estimate the holo-AcnA concentration using the aconitase A

Figure S6 .
Figure S6.NADH and FADH 2 production using formate dehydrogenase and the O 2scavenging system, respectively.(A) The production of NADH was monitored for 10 min using the absorbance at λ340 nm in formate and formate dehydrogenase (FDH) supplemented PUREfrex buffer.(B) Reaction mixture subjected to UV-Vis analysis (300-450 nm) after 10 min incubation.(C) UV-Vis analysis of the PUREfrex buffer containing the formate, NADH and FDH, supplemented with/without flavin reductase (FRE), catalase (CAT) and FAD.The absorbances of NADH λ340 nm and FADH 2 λ 445 nm were measured after 10 min incubation.

Figure S7 .
Figure S7.LC-MS spectra of citrate.(A) LC-ESI-MS extracted ion chromatograms and (B) MS spectrum of the completed reaction obtained from the aconitase A (AcnA) and isocitrate dehydrogenase-coupled assay.Apo-AcnA was produced in the PURE system and then the reactions were combined with the [4Fe-4S]-charged SUF under aerobic or anaerobic conditions.

Figure S8 .
Figure S8.LC-MS spectra of alpha-ketoglutarate. (A) LC-ESI-MS extracted ion chromatograms and (B) MS spectrum of the completed reaction obtained from the aconitase A and isocitrate dehydrogenase-coupled assay.Apo-AcnA was produced in the PURE system and then the reactions were combined with the [4Fe-4S]-charged SUF under aerobic or anaerobic conditions.