Materials and methods

Bacterial strains and growth conditions

E. coli NEB5-alpha (NEB) was used for plasmid construction and was grown in lysogeny broth (LB) at 37°C with rotary shaking at 250 rpm or on LB agar plates. E. coli strains transformed with plasmids (Table S1) were cultured in LB broth or on LB plates supplemented with carbenicillin (100 μg·mL−1), tetracycline (10 μg·mL−1) or chloramphenicol (12.5 μg·mL−1). E. coli blue/white colony screening was performed on LB agar plates supplemented with ChromoMax™ IPTG/X-Gal Solution (Fisher BioReagents). Clostridium acetobutylicum ATCC 824 was maintained at 37°C under an anaerobic atmosphere of N2:H2:CO2 (80:10:10, vol:vol:vol) in a Whitley A35 Anaerobic Workstation (Don Whitley, UK) in Clostridium Basal Medium (CBM) static cultures or on CBM agar plates [39]. Before inoculation, media were prereduced overnight in the anaerobic workstation. Cultures were supplemented with thiamphenicol (15 μg·mL−1) as required for plasmid selection and with erythromycin (40 μg·mL−1) for selection of genomic integrants.

Plasmid construction and combinatorial assembly

Plasmid construction was carried out using standard molecular biology methods [40]. Construction of the new Start-Stop Assembly plasmids (pPM900-pPM910, pPM64-lacZ and pPM65-lacZ) and Level 0 plasmids encoding the synthetic RBS library (plasmids pPM926-pPM932), FLAG_-gusA_ (pPM911), adhE2 (pPM921), bktb (pPM924), and ter (pPM934) is described in the Supplementary Materials.

Plasmid libraries pPM71 and pPM76 were assembled by assembly reactions containing 20 fmol of destination plasmid, 60 fmol of each insert, T4 DNA Ligase (400 U; NEB), T4 DNA Ligase buffer (1×; NEB) and the appropriate restriction enzyme (SapI or BsaI, 10 U; NEB). Reactions were incubated in a thermocycler for 30 two-step cycles of 37°C for 5 min then 16°C for 5 min, before a final digestion step at 42°C for 5 min and a denaturation step at 65°C for 20 min.

Plasmid library pPM71 (pPM71-O1, O2, -O4, -O11, -O20, -O24, and -O28) was generated by performing individual assembly reactions for each plasmid. Each Level 1 reaction contained the appropriate RBS part (pPM926-pPM932), FLAG_-gusA_ donor plasmid (pPM911) and the recipient plasmid pPM903 (1AZ, β-δ). Reaction mixtures were used to transform E. coli. Level 1 plasmids purified from cultures originating from single colonies from the transformation plates were used in individual Level 2 reactions with the recipient plasmid pPM64-lacZ.

Plasmid library pPM76 was generated by a combinatorial assembly of RBS parts for each coding sequence. Level 1 reaction AB contained an equimolar mixture of six RBS donor plasmids (pPM927-pPM932), adhE2 CDS (pPM921) and the recipient plasmid pPM902 (1AB, β-δ). Reaction BC contained an equimolar mixture of six RBS donor plasmids (pPM927-pPM932), bktb CDS (pPM924) and the recipient plasmid pPM904 (1 BC, β-δ). Reaction CZ contained an equimolar mixture of six RBS donor plasmids (pPM927-pPM932), ter CDS (pPM934) and the recipient plasmid pPM907 (1CZ, β-δ). Reaction mixtures were used to transform E. coli and plated on selection plates supplemented with X-Gal and IPTG. White colonies from each transformation plate were collected using an inoculation loop and pooled together for plasmid purification to generate three Level 1 plasmid libraries. Then, the three Level 1 plasmid libraries were used in a single assembly reaction with the recipient plasmid pPM65-lacZ and the reaction mixture was transformed to E. coli. White colonies from the transformation plate were collected using an inoculation loop and pooled together for plasmid purification.

Clostridium acetobutylicum transformation and chromosome integration

Plasmids or plasmid libraries (3 μg) were methylated in vitro using GpC methyltransferase M.CviPI (NEB), transformed to C. acetobutylicum by electroporation [4] and plated on 2YTG agar plates (tryptone, 16 g/L; yeast extract, 10 g/L; NaCl, 5 g/L; glucose, 20 g/L; pH 5.2) supplemented with thiamphenicol (15 μg·mL−1). Single colonies from transformation plates were streaked on CBM agar plates supplemented with erythromycin (40 μg·mL−1) to select for genomic integrants. Erythromycin-resistant colonies were re-streaked on the same medium to confirm the phenotype.

Glucuronidase assay

Glucuronidase activity in E. coli and C. acetobutylicum was determined as described by Dupuy and Sonenshein [41]. Briefly, strains expressing gusA were grown in CBM with erythromycin to OD 600 nm of 1 (Eppendorf BioSpectrometer kinetic). Samples (1.5 mL) were harvested by centrifugation and pellets were frozen at −80°C. Before testing, pellets were resuspended in 0.8 mL of buffer Z (60 mM Na2HPO4·7H2O, 40 mM NaH2PO4·H2O, 10 mM KCl, 1 mM MgSO4·7H2O, pH adjusted to 7.0, and 50 mM 2-mercaptoethanol added freshly). 0.2 mL of each sample was used for OD 600 nm measurement. To the remaining sample (0.6 mL), toluene (6 μL) was added, tubes were vortexed for 1 min and incubated on ice for 10 min. Tubes were transferred into a 37°C heating block and preincubated for 30 min with caps open. Reactions were started by addition of 120 μL of 6 mM p-nitrophenyl-β-D-glucuronide (Merck Millipore) solution in buffer Z. After incubation at 37°C (5–30 min), reactions were stopped by addition of 1 M Na2CO3 (300 μL) and reaction time was recorded. Cell debris was removed by centrifugation at 10 000× g for 10 min. Supernatants were transferred into polystyrene spectrophotometer cuvettes and absorbance at 405 nm was determined (Eppendorf BioSpectrometer kinetic). Relative glucuronidase activity was calculated by dividing the absorbance at 405 nm by sample OD 600 nm and incubation time and normalization to the positive control sample (O20, RBS_thl_).

Extended carbon chain alcohol fermentation and strain genotyping

Cultures in CBMS [42] (10 mL) supplemented with erythromycin (40 μg·mL−1) were inoculated with fresh colonies from integration plates and incubated at 37°C for 72 h. Samples (1 mL) were collected and cells were separated by centrifugation at 10 000× g for 3 min. Supernatants (0.5 mL) were filtered through a Nylon syringe filter (0.22 μm, 13 mm; Thames Restek UK) and extracted with ethyl acetate (0.5 mL) containing an internal standard tert-butylbenzene (10 mM). Ethanol, butanol, hexanol, and octanol were quantified using a gas chromatograph (Agilent 7890B) equipped with a DB-624 Ultra Inert capillary column (30 m by 0.25 mm by 1.4 μm; Agilent) and a mass selective detector (Agilent 5977A). Ultrapure helium was used as carrier gas at 0.8 mL min−1 flow rate. Samples (0.2 μL, split ratio 100:1) were injected at 240°C. The initial oven temperature was held at 35 °C for 6 min, then increased at 10°C min−1 to 260°C, and held for 1 min. Fermentation products were quantified by comparing their peak areas normalized to the peak area of the internal standard with calibration curves of authentic standards (Sigma). Standards were used to verify extraction. Cell pellets separated before ethyl acetate extraction were used for purification of genomic DNA [43]. Extracted DNA (50 ng) was used for PCR with primers oligoPM326 and oligoPM455 (amplification of the left integration junction, including the RBS of adhE2 gene), oligoPM446 and oligoPM327 (3′ end of adhE2, bktb and 5′ end of ter, including RBSs of bktb and ter genes) and oligoPM456 and oligoPM324 (right integration junction). Gel-purified PCR products were sequenced with primers oligoPM550 (RBS_adhE2_), oligoPM446 (RBS_bktb_), and oligoPM447 (RBS_ter_).