All chemicals, solvents and reagents were purchased from commercial suppliers, and were of analytical grade or better. The gas calibration standard was a custom blend of 1% each of propane, butane and isobutane in nitrogen (Thames Restek, Saunderton, UK). Media components were obtained from Formedium (Norfolk, UK). The E. coli strains used for propagating all plasmids were Stellar™ (Clontech) or NEB®5α (New England Biolabs). Expression studies were carried out using E. coli strains BL21(DE3) and NiCo21(DE3) (New England Biolab). E. coli strain BL21(DE3) was modified by chromosomal deletion of two aldehyde reductase genes yqhD and yjgB (BL21(DE3)∆yqhD/∆yjgB/KanR) , and the kanamycin selection gene was removed (BL21(DE3)ΔΔ) as described previously . Halomonas strain TQ10-MmP1 and modified pSEVA plasmids have been described previously .
Gene sequencing and oligonucleotide synthesis were performed by Eurofins MWG (Ebersberg, Germany). Details of all the plasmid and chromosomal constructs used in this study are found in Additional file 1: Tables S5–S7. pSBR1Ks-i-SceI is a hybrid of pSEVA and Biobrick (pBb-type) plasmids, containing oriT, I-SceI meganuclease gene under trc control, pRO1600-ColE1 double origin and both kanamycin and spectinomycin resistance markers. Plasmids pSH-N3 and pSH-N15 are based on the CRISPR/Cas9 editing Halomonas genome donor DNA pSEVA241 plasmid of Quin et al. , except the gRNA and antibiotic resistances were removed, and the insert contained the target DNA with a pKIKO-derived chloramphenicol resistance gene flanked by FRT sequences . Node 3 and node 15 refer to Halomonas chromosomal loci for insertion of target DNA . The modified pPorin-like constitutive promoter plasmids pHc102-RFP, pHc69-RFP and pHc59-RFP were kindly supplied by Duangthip Trisrivirat (Vidyasirimedhi Institute of Science and Technology; VISTEC). These plasmids are a modified pHal2-based plasmid  containing RFP with the T7-like promoter swapped for a modified pPorin-like constitutive promoter. The BglBrick series of vectors were obtained from Addgene (https://www.addgene.org) . Gene synthesis was performed by GeneArt (ThermoFisher, Germany) or GenScript (USA). The sequences of all the oligonucleotides used in cloning and mutagenesis can be found in Additional file 1: Tables S8–S12.
The mounted high-power blue LEDs and LED drivers were from Thorlabs (Ely, U.K.), with spectra centred at 470 nm (FWHM 25 nm, 710 mW typical output). The custom-built LED blue light array had area of 396 cm2 of relatively consistent light intensity and a fixed average culture-to-LED distance of 8 cm. The photobioreactor was a thermostatic flat panel FMT 150 (500 mL; Photon Systems Instruments, Czech Republic) with integral culture monitoring (OD 680/720 nm), pH and feeding control and an LED blue light panel (465 nm; maximum PPFD = 1648 μE photons).
Single and multi-gene construct synthesis
The propane synthesis plasmids pTPC7 (YciA-sfp-CAR; ), ADOA134F, petF (Ferr) and pCvFAPG462V (fatty acid photodecarboxylase from Chlorella variabilis) were assembled as described previously [4, 6]. A second IPTG-inducible CvFAPG462V construct was generated by sub cloning the variant gene into pBbA1c-RFP (pTrCvFAPG462V), eliminating the RFP gene. The following genes were designed, synthesised and sub-cloned into pET21b, incorporating a C-terminal His6-tag: leucine 2-oxoglutarate transaminase from E. coli (ilvE; UniProt: P0AB80); human branched-chain α-keto acid dehydrogenase (BCKDHAB; P12694 and P21953; His6-tag on subunit B only), phenylacetaldehyde dehydrogenase 17 from E. coli (PadA; P80668), threonine dehydratase from E. coli (ilvA; P04968) and the E. coli leuABCD complex composed of 2-isopropylmalate synthase (LeuA; P09151), 2-isopropylmalate dehydrogenase (LeuB; P30125) and isopropyl malate isomerase complex (LeuC/LeuD; P0A6A6/P30126). Additional synthesised genes sub-cloned into pETM11 were α-ketoglutaric semialdehyde dehydrogenase from Azospirillum brasilense (αKGSDH; Q1JUP4) and 3-hydroxypropionaldehyde dehydrogenase from E. coli (Hpad; P23883), while branched-chain keto acid decarboxylase from Lactococcus lactis (KdcA; Q6QBS4) was sub cloned into pET28b. These latter genes contained a vector-derived N-terminal His6-tag. Genes were codon optimised to remove rare codons for optimal expression in E. coli. For LeuABCD, the native E. coli operon sequence was synthesised (no His6-tags), with gene expression controlled by a single T7 promoter (Additional file 1: Table S5).
The multi-gene construct pYSCAP (YciA-sfp-CAR-ADOA134F-Ferr) contained the genes encoding non-His6-tagged versions of acyl-CoA thioester hydrolase from Haemophilus influenza (YciA; P44886); maturation factor phosphopantetheinyl transferase from Bacillus subtilis (sfp; P39135); carboxylic acid reductase from Mycobacterium marinum (CAR; B2HN69); aldehyde deformylating oxygenase variant A134F from Prochlorococcus marinus (ADO; Q7V6D4) and ferredoxin from Synechocystis sp PCC6803 (Ferr; P27320) [5, 6]. This construct was synthesised as a complete operon with codon-optimised genes, synthetic Shine–Dalgarno (SD) sequences and the constitutive promoters R0011 (http://2015.igem.org/) and proD upstream of YciA and ADOA134F, respectively .
Multi-gene constructs assembly in E. coli
In most cases, the assembly of multi-gene constructs was performed by In-Fusion cloning, according to the manufacturer’s protocols . Vector linearisation and insert(s) amplification were performed by polymerase chain reaction (PCR), using the CloneAmp™ HiFi PCR Premix kit (Clontech), incorporating 15–25 bp overhangs necessary for subsequent ligations. In some cases, overlap extension PCR (OEP) was performed to ligate two or more DNA fragments generated by PCR to simplify subsequent construct assembly. In this method an initial 5 PCR cycles were performed with the template DNA fragments only, followed by the addition of the forward primer of the first DNA fragment and the reverse primer of the last insert. Following In-Fusion cloning, each construct was transformed into the E. coli strain Stellar or NEB5α for plasmid recovery, and the correct assembly was confirmed by DNA sequencing. The oligonucleotide sequences and template DNA used in each PCR reaction is shown in Additional file 1: Tables S8–S12. The following sections will detail the general approaches taken for the assembly of each plasmid.
ADO-containing CoA-dependent multi-gene E. coli constructs assembly
A dual construct was assembled in pBbE2k  containing ADOA134F and its electron transfer partner Ferr , under the control of a tetracycline-inducible promoter. Both ADOA134F and Ferr genes were PCR amplified from their respective constructs in pCDFDuet-1 and pRSF-Duet1 . The PCR products included the existing vector-derived 5′-SD sequence for ADOA134F and a non-native SD sequence (GGAGGACAGCTAA) for Ferr. In-Fusion cloning was performed with the linearised destination vector pBbE2k-RFP, minus the RFP gene and its SD sequence, to generate pTetADOA134FFerr (Additional file 1: Table S8).
The assembly of a butyryl-CoA to propane pathway construct pAFYSC (T7-ADOA134F-Ferr-YciA-sfp-CAR) was performed using the vector pETDuetT-1. PCR linearisation of construct TPC7 (T7-YciA-sfp-CAR) occurred between the T7 promoter and the initial SD sequence, while the two genes from pADOA134FFerr were amplified with both SD sequences. In-Fusion cloning between the two PCR products generated the IPTG-inducible pAFYSC pathway (Additional file 1: Table S8).
To eliminate the need for IPTG induction, a constitutive expression system was constructed using the BglBrick plasmid pBbE7k-RFP as the backbone . The plasmid was linearised by reverse PCR, eliminating the T7 promoter-RFP cassette (lacIq retained), and the proD-ADOA134F insert was amplified from the multi-gene construct pYSCAP. Following In-Fusion cloning, the new ADO-containing constitutive expression vector (pPrADOA134F) was used as the backbone for the construction of a series of ADO-dependent pathways from butyryl-CoA to propane. The first constitutive pathway assembled was pPr*AFYSC (proD*-ADOA134F-Ferr-YciA-sfp-CAR), via the ligation of the pAFYSC pathway genes (SD-ADOA134F-Ferr-YciA-sfp-CAR) into the linearised proD-containing empty plasmid (pPrADOA134F minus SD-ADOA134F; Additional file 1: Table S8). This was followed by linearisation of pPr*AFYSC by reverse PCR to eliminate the now redundant lacIq repressor. The new construct (pPrAFYSC) was re-circularised by In-Fusion cloning in the absence of any insert.
The generation of ADO-dependent pathways to propane, butane and isobutane from the amino acids valine, isoleucine and leucine, respectively, requires the addition of genes ilvE and BCKDHAB to the existing AFYSC constructs (Fig. 1). Initially a constitutively controlled dual enzyme construct (pPr*IB; proD*-ilvE-BCKDHAB) was assembled in the same modified BioBrick plasmid as used for pPr*AFYSC construction. The individual genes (ilvE and BCKDHAB) were amplified by PCR from their respective synthesised constructs. This was followed by OEP to generate a dual enzyme insert, followed by ligation to the linearised empty vector (pPr*AFYSC minus AFYSC). A complete pathway from amino acid to gaseous hydrocarbon was generated by the inclusion of ilvE-BCKDHAB with AFYSC to form pPr*AFYSCIB (proD*-ADOA134F-Ferr-YciA-sfp-CAR-ilvE-BCKDHAB). This was performed by linearising pPrAFYSC after CAR, and ligating it to the ilvE-BCKDHAB insert amplified from pPr*IB. To increase the expression of ilvE and BCKDHAB, a second proD promoter was inserted downstream of CAR in pPrAFYSC by In-Fusion cloning, to generate pPrAFYSCPr (Additional file 1: Table S8). Insertion of the ilvE-BCKDHAB fragment of pPr*IB after the second proD generated pPr*AFYSCPrIB (proD*-ADOA134F-Ferr-YciA-sfp-CAR-proD-ilvE-BCKDHAB.
IPTG-inducible constructs catalysing gaseous hydrocarbon production from amino acids were generated by amplifying the seven pathway genes from pPr*AFYSCIB and ligating them into pBbE1k, linearised downstream of the trc promoter (pTrAFYSCIB; trc-ADOA134F-Ferr-YciA-sfp-CAR-ilvE-BCKDHAB). Similarly, the addition of a second trc promoter upstream of ilvE was performed by PCR coupled to In-Fusion cloning to generate pTrAFYSCTrIB (trc-ADOA134F-Ferr-YciA-sfp-CAR-trc-ilvE-BCKDHAB; Additional file 1: Table S8).
FAP-containing CoA-dependent multi-gene E. coli constructs assembly
Simplified gaseous hydrocarbon producing constructs from amino acids were generated by substituting the four genes CAR, sfp, ADOA134F and Ferr for a single gene CvFAP variant G462V or G462I. An initial construct pTrFG462VYIB was generated (trc-CvFAPG462V-YciA-ilvE-BCKDHAB) using pTrAFYSCIB as the backbone. This latter plasmid was linearised, eliminating the genes encoding AFYSC (Additional file 1: Table S9). A dual gene insert was constructed (CvFAPG462V-YciA) by PCR amplification of each individual gene, followed by OEP. This was ligated to the backbone plasmid upstream of ilvE, generating an IPTG-inducible construct. A similar constitutive construct pPrFG462VYIB (proD-CvFAPG462V-YciA-ilvE-BCKDHAB) was generated as above, except the backbone template was the proD-containing plasmid pPrAFYSCIB.
KdcA-dependent multi-gene E. coli constructs assembly
Alternative CvFAPG462V-dependent pathways were constructed by the substitution of six genes (BCKDHAB, YciA, CAR, sfp, ADOA134F and Ferr) for KdcA, an alcohol dehydrogenase and CvFAPG462V. These pathways were constructed in pBbE1k, including the insertion of a second trc promoter upstream of the latter two genes (trc-KdcA-CvFAPG462V). Each individual gene and second trc promoter were amplified by PCR, and OEP was performed between trc and KdcA DNA fragments (Additional file 1: Table S10). In-Fusion cloning was performed generating three KdcA-dependent and IPTG-inducible constructs (pTrIA*TrKFG462V, where A* = αKGSDH, PadA or Hpad; Additional file 1: Table S5). Each of these three constructs underwent site-directed mutagenesis of the CvFAP gene to produce the equivalent pathway with the variant G462I (pTrIαKTrKFG462I, pTrIΠTrKFG462I and pTrIΗTrKFG462I, respectively) as described previously .
Halomonas KdcA-dependent construct assembly
Six KdcA-dependent pathways were constructed in the Halomonas-compatible plasmid pHal2 , which varied by the type of promoter used. This was performed by multi-step In-Fusion cloning, where PCR was used to amplify the inserts, eliminating the His6-tags, and/or linearise the vectors (Additional file 1: Table S11). Each construct was propagated in the E. coli conjugative donor strain S17-1 . Plasmid transformation into Halomonas was performed by conjugation according to the method described previously . Plasmid content of each trans-conjugant was confirmed by DNA isolation, restriction mapping and sequencing.
The initial construct was generated under control of the IPTG-inducible MmP1 T7-like promoter (pHT7LIHKFG462I; ), which later underwent LacIq elimination (T7LΔL) to generate the respective constitutive construct (pHT7LΔLIHKFG462I). A second constitutively expressed construct was generated by substituting the T7-like promoter for a truncated trc promoter, which was deficient in both trc and lacI (pHΔLIHKFG462I). Three constitutive promoters were generated (c102, c69 and c59) based on the major outer membrane protein porin constitutive expression system in Halomonas (Additional file 1: Fig. S11; [30, 31]). The IHKFG462I operon was inserted downstream of each promoter, generating a further three Halomonas constructs (pHc102IHKFG462I, pHc69IHKFG462I and pHc59IHKFG462I).
Chromosomal integration of pathway genes into Halomonas
Chromosomal insertion of the KdcA-dependent pathways into Halomonas TQ10 was performed using a novel suicide vector (pSH) protocol (Additional file 1: Fig. S7) based on previously published methods [4, 34, 35]. The pSH insertion plasmids (pSH-N3 and pSH-N15) contained the biocatalytic and pKIKO-derived FRT flanked  chloramphenicol resistance genes surrounded by homology arms (node 3 or 15; ), an I-SceI restriction site and a colE1 ori (incompatible) with replication in Halomonas (Additional file 1: Fig. S10 inset). This plasmid was co-conjugated into Halomonas TQ10-MmP1 with a second spectinomycin-resistant plasmid (pSBR1Ks-i-SceI), the latter expressing the restriction enzyme I-SceI. In vivo expression of I-SceI linearised pSH plasmids facilitates chromosomal integration [34, 35]. Successful integration was seen as growth of Halomonas on chloramphenicol-selective medium, as the pSH plasmid is not replicated in Halomonas. Integration was confirmed by colony PCR, genomic sequencing and in vivo propane production after pSceI plasmid curing [34, 35].
Eight KdcA-dependent constructs (ilvE-Hpad-kdcA-CvFAPG462I) were integrated into Halomonas strain TQ10 (Additional file 1: Table S12). The constructs varied by the chromosomal loci (node 3 or 15) and the promoter type (inducible vs constitutive). The inducible system was the MmP1 T7-like promoter (T7L), while the constitutively expressed constructs were controlled by pPorin-like 69 (c69), lacIq-deficient MmP1 T7-like promoter (T7LΔL) or the truncated pTrc promoter minus trc and lacI (ΔL; Additional file 1: Table S12). The insertion of inducible T7LIHKFG462I or constitutive c69IHKFG462I constructs into pSH-N3 or pSH-N15 was performed via In-Fusion cloning using PCR linearised destination vectors (between one homology arm and upstream of the chloramphenicol gene; Additional file 1: Fig. S5) and amplified multi-gene constructs with their own promoters (N3- or N15T7LIHKFG462I; N3- or N15c69IHKFG462I). For the ΔL-containing plasmids, a similar protocol was performed as above, except the T7LIHKFG462I construct for each node was used as the template, and the only DNA eliminated/inserted was the promoter (N3- or N15cΔLIHKFG462I). To generate the constructs with the T7LΔL promoter, PCR elimination of the laciq gene was performed on the equivalent T7L-containing constructs followed by self In-Fusion cloning to re-circularise the plasmid (N3- or N15cT7LΔLIHKFG462I). Successful integration of the constructs at the correct loci was confirmed by colony PCR and genome sequencing.
Protein expression and lysate production
IPTG-dependent expression of proteins YciA, CAR, sfp, CvFAPG462V, ADOA134F and Ferr in E. coli has been demonstrated previously [4, 6]. The remaining proteins ilvE, BCKDHAB, KdcA, αKGSDH, PadA, Hpad ilvA and LeuABCD were transformed into E. coli strain BL21(DE3) for protein overexpression studies. Cultures (1 L) were grown in LB Broth Miller (Formedium) containing the required antibiotic (50 μg/mL ampicillin or 30 μg/mL kanamycin) at 37 °C with 180 rpm shaking until OD600nm = 0.6. Recombinant protein induction was performed with IPTG (0.1 mM), followed by a further 12–16 h incubation at 25 °C. Cells were harvested by centrifugation at 3320×g for 30 min at 4 °C.
Cells were resuspended in lysis buffer (5 mL/g pellet; 50 mM Tris pH 7.0 containing 1 mM MgCl2, 1 mM β-mercaptoethanol, 10% glycerol, 2X protease inhibitors, 50 μg/mL DNAse and 50 μg/mL lysozyme) and freeze-thawed in liquid nitrogen. Cells were lysed by sonication, and clarified using centrifugation (48,000×g). Protein content was determined using 12% SDS-PAGE gels (Mini-Protean TGX Stain-Free Precast Gels, Bio-Rad). Protein gels were imaged using a BioRad Gel Doc EZ Imager and relative protein band intensity was determined using the BioRad ImageLab software. Identification of His6-tagged proteins was performed by Western blots using the Trans-Blot® Turbo™ Transfer system (PVDF membranes; BioRad) and the Western Breeze Chemiluminescent Immunodetection kit (alkaline phosphatase; Life Technologies) with mouse (His tag monoclonal antibody) and alkaline phosphatase-containing (Anti-C-My) primary and secondary antibodies, respectively.
Gaseous hydrocarbon production
In vivo gaseous hydrocarbon production by recombinant E. coli was performed using the following general protocol: cultures (10 mL) were incubated for 4–6 h (OD600 ~ 1.6–2) at 37 °C and 180 rpm in LB or TB medium containing 50 µg/mL ampicillin, 30 µg/mL kanamycin or 50 µg/mL chloramphenicol, dependent on the antibiotic resistance (Additional file 1: Table S5). Supplemental valine, leucine or isoleucine (up to 30 mg/L) were included in the medium where required. Protein induction (0.1 mM IPTG) was performed for trc or T7-containing constructs, and triplicate samples (1 mL) each of 3 biological replicate cultures were sealed into glass vials (3 mL) and incubated at 30 °C for 16–18 h at 200 rpm, illuminated continuously with a blue LED (455 nm or 470 nm).
For propane production in Halomonas, LB60 medium (1% tryptone, 0.5% yeast extract, 6% NaCl) pH 9.0 was used containing spectinomycin (50 μg/mL). Cultures were agitated (180 rpm) at 37 °C for 5 h incubation (OD ~ 1.6–2) prior to induction. IPTG (0.1 mM) was added (where necessary), and the remaining methodology was performed as described above for E. coli cultures.
General photobioreactor cultivation (400 mL) was performed with high salt glycerol medium (30–32% seawater or Instant Ocean, NaCl to 6%, 0.1% glycerol and 0.5% yeast extract) pH 6.8 containing 0.5 mL/L antifoam and antibiotic (50 μg/mL spectinomycin or 34 μg/mL chloramphenicol for plasmid-borne and constitutive constructs, respectively). Alternative growth media were based on LB60 pH 6.8, which were supplemented with 1.5% valine (LB60Val) or casamino acids (LB60Cas). Cultivation was performed in batch mode, pre-equilibrated at 30 °C with 60% stirring output. An overnight starter culture (10–15 mL) of Halomonas TQ10 expressing pHT7LIHKFG462I was added, to achieve a starting OD680 nm of ~ 0.2, and the culture was maintained at 30 °C with an airflow rate of 1.21 L/min, automated pH maintenance, culture optical density monitoring and ambient room lighting until mid-log phase (4–5 h). Protein induction by IPTG (0.1 mM) was performed for T7L-promoter systems with continual monitoring for 2–10 days with blue light exposure (1656 or 600 μE for plasmid-borne and chromosomal systems, respectively). Alkane gas production was monitored at 20-min intervals by automated headspace sampling using a Micro GC, while aqueous amino acid and glycerol depletion were quantified by HPLC.
Fermentations of Halomonas TQ10 containing chromosomally integrated N3T7LIHKFG462I or N3cΔLIHKFG462I was performed as above with LB60Val pH 6.8, except culture medium feeding was employed to maintain an optical density of 0.8 and to replenish the carbon source. Cultures were maintained for about 240 h, with alkane gas production monitored at 2–3 times daily by manual sampling using a Micro GC.
Propane levels were determined by manual headspace injection or automated (fermentation off gas monitoring) using an Agilent 490 Micro GC, containing an Al2O3/KCl column and a thermal conductivity detector (TCD). Aqueous culture metabolites (VFAs and glycerol) were analysed by HPLC using an Agilent 1260 Infinity HPLC with a 1260 ALS autosampler, TCC SL column heater, a 1260 refractive index detector (RID) with an Agilent Hi-Plex H column (300 × 7.7 mm; 5 mM H2SO4). The running conditions for both the Micro GC and HPLC were the same as described previously . For amino acid quantitation, analysis was performed according to the method of Bartolomeo and Maisano . Each analyte concentration was calculated by comparing the peak areas to a standard curve generated under the same running conditions. Error bars indicate one standard deviation of the data obtained for the replicates (biological and/or technical triplicates).