Strains, medium, and cultivation
The strains used in this study are listed in Table 2. E. coli DH5α was used for plasmid cloning and cultivated in liquid modified lysogeny broth (1 % tryptone, 1 % NaCl, 0.5 % yeast extract)  while shaking or on respective agar plates at 37 °C. When required medium was supplemented with 250 µg mL-1 erythromycin. E. limosum NG-6894 was cultivated in modified DSM 135 medium under strictly anaerobic conditions at 37 °C. The modified DSM 135 medium contained 12.9 mM KH2PO4, 48.5 mM K2HPO4, 18.7 mM NH4Cl, 49.6 mM NaCl, 58.9 mM KHCO3, 2.8 mM L-cysteine-HCl, 1.5 mM MgSO4, 4.4 µM resazurin, 0.2 % (wt/vol) yeast extract, 0.1 % (vol/vol) trace element solution SL-9 , 0.1 % (vol/vol) selenite-tungstate solution , and 0.2 % (vol/vol) vitamin solution DSM 141. If necessary, the medium was supplemented with antibiotics (5 µg mL-1 clarithromycin) after autoclaving. Heterotrophic cultivations of E. limosum was carried out in 50 mL medium in 125-mL Müller-Krempel flasks and were supplemented with 30 mM glucose or 50, 100 or 200mM methanol. Autotrophic cultivations were also carried out in 50 mL medium in 500-mL Müller-Krempel flasks, which were pressurized to 1.1 bar overpressure with H2 + CO2 (67 % H2 + 33 % CO2) or syngas (10 % CO2, 40 % CO, 10% N2, 40 % H2) and repressured when below 0.5 bar. Before growth experiments were carried out, cells were grown from DMSO stock cultures in 5 mL of the respective medium. These 5-mL cultures were used to prepare 50-mL precultures supplemented with glucose or methanol for subsequent growth experiments. For autotrophic growth experiments, cells from 5-mL cultures were adapted to growth conditions by transferring them two times to fresh 50 mL medium in 500-mL Müller-Krempel culture flasks with the respective gas atmosphere.
All plasmids and primers used in this study are listed in Tables 3 and 4, respectively. DNA fragments for cloning purposes were amplified using the KAPA Hifi polymerase (Kapa Biosystem, Sigma-Aldrich Chemie GmbH, Munich, Germany). Primers used in this study were synthesized by biomers.net GmbH (Ulm, Germany) and designed to have a 15 - 25 nucleotide overlap with the respective vector DNA. Vector DNA for cloning purposes was linearized using Fast digest enzymes (Thermo Fisher Scientific Inc., Waltham, USA). PCR and digested vector DNA fragments were purified using the NucleoSpin® Gel and PCR clean up kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany). All plasmids were constructed by assembling PCR and vector DNA fragments using the NEBuilder Hifi DNA Assembly Cloning Kit according to the manufacturers protocol (New England Biolabs, Ipswich, MA, USA). Assembled plasmids were used for the transformation of chemically competent E. coli DH5α cells and verified via Sanger sequencing (Eurofins Genomics GmbH, Luxemburg).
To establish FAST as a reporter protein in E. limosum, plasmids harboring feg were constructed. Therefore, feg was amplified together with the promoter Pthlsup from plasmid p95thlsupFAST  using primers FW_Pthlsup_FAST_NdeI and RV_Pthlsup_FAST_XhoI (Table 4). The amplified PCR fragment was cloned via NdeI and XhoI recognition sites into vector pMTL83251 resulting in the plasmid pMTL83251_Pthlsup_FAST. The promoter region of Pthlsup from plasmid pMTL83251_Pthlsup_FAST was further exchanged with the lactose inducible bgaR-PbgaL system from C. perfringens  and PthlA from C. acetobutylicum . Therefore, the bgaR-PbgaL-encoding DNA fragment was amplified from plasmid pMTL83151_gusA_PbgaL  using the primers FW_PbgaL_NdeI and RV_PbgaL_BamHI. The DNA fragment containing the promoter region of PthlA was amplified from plasmid pJIR750_act  using primers FW_Pthl_NdeI and RV_Pthl_BamHI (Table 4). Both promoter containing DNA fragments were assembled with pMTL83251_Pthlsup_FAST (excision of Pthlsup) resulting in plasmids pMTL83251_PthlA_FAST and pMTL83251_PbgaL_FAST.
The gene feg of pMTL83251_PbgaL_FAST was exchanged with the gene encoding tdFAST2. The sequence of tdFAST2 was obtained from Tebo et al.  and codon-optimized for E. limosum using the GENEius tool (Eurofins Genomics GmbH, Luxemburg) and synthesized by Eurofins Genomics Germany GmbH (Ebersberg, Germany). The gene tdFAST2-opt was amplified from the delivered plasmid pEX-A128_tdFAST2-opt using the primers FW_tdFAST2-opt_BamHI and RV_tdFAST2-opt_XhoI (Table 4) and ligated with BamHI and XhoI digested pMTL83251_PbgaL_FAST (excision of feg) vector DNA resulting in plasmid pMTL83251_PbgaL_tdFAST2.
For butanol production with E. limosum the plasmid pMTL83251_PbgaL_AdhE2 was constructed. The gene adhE2 was amplified from genomic DNA of C. acetobutylicum using primers FW_C1_adhe2 and RV_N2_adhE2 (Table 4). The gene was assembled under the control of the lactose-inducible bgaR-PbgaL promoter using the plasmid DNA of pMTL83251_PbgaL_FAST, which was digested using BamHI and XhoI (excision of feg).
For acetone production with E. limosum the plasmid pMTL83251_PthlA_act was constructed encoding the genes of the APO under the control of promoter PthlA. The origin of these genes as well as their locus tags are listed in Table 3. These genes as well as the DNA fragment containing the promoter region of PthlA were amplified from plasmids pJIR750_act using primers FW_act_NdeI and RV_act_XhoI (Table 4). The amplified PCR fragments were cloned via the NdeI and XhoI restriction sites of pMTL83251 resulting in the plasmids pMTL83251_PthlA_act.
Moreover, plasmids were constructed to produce C- and N- terminal FAST-tagged fusion proteins of AdhE2 and Adc. FAST was C- and N-terminal fused to AdhE2 or Adc using a glycine linker (GGGGS) with the sequence ggtggtggtggttct. For the construction of C-terminal tagged fusion proteins, the 3’ end of the gene of interest was fused to the 5’ end of feg. Therefore, the gene of interest was amplified without its stop codon while feg was amplified without its start codon. To construct N-terminal FAST-tagged fusion proteins, the 3’ end of feg was fused to the 5’ end of the gene of interest. The gene of interest was amplified without its start codon, while feg was amplified without its stop codon.
The plasmid pMTL83251_PbgaL_C-FAST-AdhE2 was constructed to produce the C-terminal FAST-tagged AdhE2 fusion protein. adhE2 was amplified from plasmid pMTL83251_PbgaL_AdhE2 using primers FW_C1_adhE2 and RV_C1_adhE2-FAST (Table 4), feg was amplified from plasmid pMTL83251_PbgaL_FAST using primers FW_C2_adhE2-FAST and RV_C2_FAST (Table 4). Primers RV_C1_adhE2-FAST and FW_C2_adhE2-FAST contain the sequence for the glycine-linker. The plasmid pMTL83251_PbgaL_N-FAST-AdhE2 was constructed to produce the N-terminal FAST-tagged AdhE2 fusion protein. adhE2 was amplified from plasmid pMTL83251_PbgaL_AdhE2 using primers FW_N1_adhE2 and RV_N1_FAST-adhE2 while feg was amplified from plasmid pMTL83251_PbgaL_FAST using primers FW_N2_FAST-adhE2 and RV_N2_adhE2 (Table 4). Primers RV_N1_FAST-adhE2 and FW_N2_FAST-adhE2 contain the sequence for the glycine-linker.
The plasmid pMTL83251_PthlA_C-FAST-Adc was constructed to produce the C-terminal FAST-tagged Adc fusion protein. adc was amplified from plasmid pMTL83251_PthlA_act using primers FW_C1_adc and RV_C1_adc-FAST, and feg was amplified from plasmid pMTL83251_PbgaL_FAST using primers FW_C2_adc-FAST and RV_C2_FAST (Table 4). Primers RV_C1_adc-FAST and FW_C2_adc-FAST contain the sequence for the glycine-linker. The plasmid pMTL83251_PthlA_N-FAST-Adc was constructed to produce the N-terminal FAST-tagged Adc fusion protein. The gene adc was amplified from plasmid pMTL83251_PthlA_act using primers FW_N1_adc-FAST and RV_N1_FAST-adc and feg was amplified from plasmid pMTL83251_PbgaL_FAST using primers FW_N2_FAST-adc and RV_N2_adc (Table 4). Primers RV_N1_FAST-adc and FW_N2_FAST-adc contain the sequence for the glycine-linker.
Electroporation and preparation of electrocompetent E. limosum cells were performed according to the protocol of Leang et al.  with several modifications. All steps were carried out under anaerobic conditions in an anaerobic chamber (gas atmosphere 95 % N2 and 5 % H2). Plastic materials were placed 24 h before use into the anaerobic chamber to remove any traces of oxygen. For the preparation of electrocompetent cells, E. limosum was cultivated at 37 °C in 100 mL modified DSM 135 medium supplemented with 100 mM methanol and 40 mM DL-Threonine. Cells were cultivated until the early exponential growth phase (OD600 0.3 to 0.5) and harvested by centrifugation at 7,690 x g for 10 min at 4 °C. Subsequently, cells were washed two times with anaerobic SMP-buffer (270 mM sucrose, 1 mM MgCl2, 7 mM NaH2PO4, pH 6) by centrifugation at 7.690 x g for 10 min at 4 °C. Afterwards the cell pellet was suspended in 648 µL SMP-buffer and 72 µL DMSO, distributed into cryotubes, and stored at -80 °C until use.
Transformation of E. limosum cells was performed using 3-5 µg plasmid DNA, which were added to 25 µL of electrocompetent cells into a pre-cooled 1 mm electroporation cuvette (Biozym Scientific GmbH, Oldendorf, Germany). Cells were pulsed (625 V, 25 μF, 600 Ω; Gene Pulser XcellTM, Bio-Rad Laboratories GmbH, Munich, Germany) and immediately transferred into 5 mL fresh medium. E. limosum cells were recovered at 37 °C in 5 mL modified DSM 135 medium supplemented with 100 mM methanol. The OD600 of transformed cells was monitored. After one or two doublings, 5 µg mL-1 clarithromycin was added to the medium for selection of recombinant strains. After a further increase of the OD600, which indicates a successful transformation, cells were transferred two more times into fresh medium supplemented with 5 µg mL-1 of clarithromycin. Successfully transformed cells were verified by isolation of plasmid DNA using the ZyppyTM Plasmid Miniprep Kit (Zymo Research, Irvine, CA, USA). This preparation was used to transform chemically competent E. coli DH5α cells to amplify the plasmids. After transformation plasmids were isolated and checked by restriction analysis. Recombinant strains were stored in cryotubes with 10 % DMSO at -80 °C. All recombinant strains constructed in this study are listed in Table 5.
Fluorescence of FAST and FAST-tagged fusion protein-producing recombinant E. limosum strains, cultivated anaerobically until the stationary growth phase, were determined using a microplate reader, fluorescence microscopy, or flow cytometry. Therefore, 2 mL culture broth was taken anaerobically during growth and harvested by centrifugation at 7,711 x g for 15 minutes at 4°C. The supernatant was discarded and harvested cells were washed with anaerobic PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) followed by centrifugation at 7,711 x g for 15 minutes at 4°C. Cell pellets were suspended in anaerobic PBS buffer (end OD600 1).
100 µL of suspended cells were transferred to black flat bottomed 96-well microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Deutschland) and supplemented with 10 µM fluorogen of the green dye TFLime (ex. 480/em. 541) or the red dye TFCoral (ex. 516/em. 600) (The Twinkle Factory, France, Paris). Excitation and emission wavelengths both correspond to the respective maximum of the fluorogen. Fluorescence intensities of the whole population were determined using the SYNERGY H1 microplate reader (BioTek, Bad Friedrichshall, Germany) located in an anaerobic chamber and finally normalized to the OD600 of PBS-washed cells.
Washed cells were stained with 10 µM TFLime and transferred onto an 1 % agarose pad on a microscopy slide, covered with a glass coverslip, and sealed with nail polish. Cells were viewed with a 63x objective, using a Zeiss Axio Observer Z1 (Zeiss, Oberkochen, Germany). Green fluorescence of the FAST:fluorogen complex was detected using the Zeiss filter set 38 HE (ex. BP 470/40, em. BP 525/50). Data were analyzed using the Zeiss Zen 2.6 blue edition software.
Washed cells were stained with 10 µM TFLime diluted in PBS buffer (end OD600: 0.01). Green fluorescence of the FAST:fluorogen complex was assessed using an excitation wavelength of 488 nm and a 528/46 nm emission filter. For analysis, at least 10,000 events were recorded using an Amnis® CellStream® flow cytometer (Luminex Corporation, Austin, TX, USA). Acquired data were analyzed using the CellStreamTM Analysis tool version 1.2.152 (Luminex Corporation, Austin, TX, USA).
2-mL samples were withdrawn from cell cultures during the growth of E. limosum to analyze OD600, product spectrum as well as substrate consumption. After OD600 of the withdrawn culture broth was determined at 600 nm using the GENESYS 10vis photometer (Thermo Scientific, Waltham, MA, USA), the remaining culture was centrifuged at 17,968 x g for 20 minutes at 4 °C to remove cell debris. The supernatant was used for HPLC and GC analysis.
High-performance liquid chromatography
The concentration of glucose, acetate, and butyrate from culture supernatant was determined using the Agilent 1260 Infinity II HPLC system (Agilent Technologies, Santa Clara, CA, USA), equipped with a diode array detector and a refractive index detector. To achieve separation, 20 μL of the supernatant was injected into the organic acid resin 150 x 8 mm column (CS-chromatographie-Service GmbH, Langerwehe, Germany) packed with polystyrene divinylbenzene copolymer operating at a constant temperature of 40 °C. As mobile phase, 5 mM H2SO4 was used with a flow rate of 0.7 mL min-1. The software OpenLAB CDS ChemStation Edition A.01.03 (Agilent Technologies, Santa Clara, CA, USA) was used for data analysis.
The concentration of methanol, ethanol, butanol, and acetone in the culture broth was analyzed via gas chromatography. A PerkinElmer Clarus 680 GC system (Perkin Elmer LAS GmbH, Waltham, MA, USA) equipped with an Elite-FFAP capillary column (length 30 m x inner diameter 0.32 mm, film thickness 0.25 µm) (Perkin Elmer LAS GmbH, Waltham, MA, USA) and FID detector was used. Supernatants were acidified with 2 M HCL. H2 was used as the carrier gas. The injector and detector were operated at 225 and 300 °C, respectively. 0.5 µL of the culture broth was injected into the gas chromatograph and analyzed using the following temperature profile: 40 °C for 2.5 minutes; 40 °C to 250 °C with 40 °C min‑1; 250 °C for 2 minutes.