All strains, plasmids, and primers constructed and used in this study are listed in the Supplementary Tables. B. methanolicus MGA3 was used as the expression host, Escherichia coli strain DH5α (Stratagene) was used as the general cloning host.

All standard recombinant DNA procedures were performed as described by Sambrook and Russell (2001). Plasmid DNA was introduced into chemically competent E. coli cells (Higa and Mandel, 1970; Hanahan, 1983). Total DNA was isolated from B. methanolicus using the MasterPure^TM Gram Positive DNA Purification Kit (Epicenter) or as previously described (Eikmanns et al., 1994). The NucleoSpin^® Gel and PCR Clean-up kit (Machery-Nagel) and the Qiaquick PCR Purification and Gel Extraction kits (Qiagen) were used for PCR purification and gel extraction. Plasmids were isolated using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific) or the Wizard^® Plus SV Minipreps (Promega). Plasmid backbones were amplified with PfuTurbo DNA polymerase (Agilent), inserts with ALLin^TM HiFi DNA Polymerase (highQ) or the Expand^TM High Fidelity PCR System (Roche). Dephosphorylation of plasmid DNA was performed using Antarctic Phosphatase or Calf Intestinal Alkaline Phosphatase (New England Biolabs). The DNA fragments were joined either with Rapid DNA Ligation Kit (Roche), T4 DNA ligase (New England Biolabs) or by the means the isothermal DNA assembly (Gibson et al., 2009). For colony PCR the Taq polymerase (New England Biolabs) was used. Site-directed mutagenesis was performed essentially as described by Liu and Naismith (2008) using Pfu polymerase (Agilent). All cloned DNA fragments and introduced mutations were verified by sequencing. B. methanolicus competent cells were prepared according to Jakobsen et al. (2006). SOBsuc plates [1% (w/v) agar] supplemented with suitable antibiotics were used instead of regeneration plates. SOBsuc medium is SOB medium (Difco) supplemented with 0.25 M sucrose. Electroporation was performed as previously described (Jakobsen et al., 2006).

Escherichia coli strains were cultivated at 37°C in Lysogeny Broth (LB) or on LB–agar plates supplemented with antibiotics (ampicillin 200 μg/mL, chloramphenicol 30 μg/mL, kanamycin 50 μg/mL) when relevant. Unless otherwise stated, B. methanolicus strains were cultured at 50°C in MVcMY minimal medium with 200 mM methanol as previously described (Brautaset et al., 2004). When appropriate, media were supplemented with kanamycin 50 μg/mL (or 10 μg/mL) and/or chloramphenicol 5 μg/mL. Inducers were used at the following concentrations: mannitol [2.5, 5.0, 12.5, 25, 50, and 55 mM (1%)], arabitol (50 mM), ribitol (50 mM), xylitol (50 mM), xylose [0.01, 0.05, 0.1, 0.5, or 1% (w/v)], or CuSO₄ (10, 20, 50, 100, and 200 μM). All experiments were performed in triplicates.

For LacZ enzymatic assays, overnight cultures of B. methanolicus strains MGA3 (pTH1mp-lacZ), MGA3 (pTH1xp-lacZ), MGA3 (pTH1cup-lacZ), MGA3 (pTH1mtlAp-lacZ), or MGA3 (pHP13), were diluted to OD₆₀₀ 0.2 in fresh medium with appropriate antibiotics. When the cultures reached OD₆₀₀ = 0.5, they were split in two equal halves. Inducer (50 μM CuSO₄, 1% (w/v) xylose, or 1% (w/v) mannitol) was added to one of the two and growth was continued until OD₆₀₀ 1–1.5. Cells were harvested by centrifugation (5000 g, 10 min, 4°C) and the pellets were stored at -80°C. Cells were thawed, resuspended in potassium phosphate buffer (100 mM, pH 7.0) (10% of the original volume) and sonicated on ice/water for 10-15 min (Branson Sonifier 250, output control = 3 and duty cycle = 30%). Cellular debris was removed by centrifugation (10000 g, 45 min, 4°C) followed by filtration through a 0.2 μm sterile filter. Enzymatic activities were measured by monitoring the liberation of o-nitrophenol from o-nitrophenyl β-D-galactopyranoside (ONPG) at 410 nm. 100 mM potassium phosphate buffer pH 7.0 (910 μl), 68 mM ONPG (30 μl), and 30 mM MgCl₂ (30 μl) were mixed and the catalysis started by the addition of cell extract (30 μl). The molar extinction coefficient used for o-nitrophenyl at 410 nm, pH 7.0 used for calculation is 3500 M^-1 cm^-1 and the light path 1 cm. One unit (U) is defined as the amount of enzyme able to convert 1.0 μmol of ONPG per min.

For the fluorescent activated cell scanning analysis, overnight cultures were diluted to an initial OD₆₀₀ of 0.15 and cultivated for 6 h at 50°C prior to incubation at 37°C for two hours. Samples were centrifuged at 13,000 g, 5 min, 4°C, washed twice with cold phosphate-buffered saline (PBS) and resuspended therein to a final OD₆₀₀ of 0.3. The fluorescence was determined in a flow cytometer (Becton Dickinson) using the Kaluza for Gallios Acquisition Software 1.0. The fluorescence emission signal was collected with a 450/50 BP bandpass filter (FL9) for GFPuv and with a 620/30 BP, bandpass (FL3) for mCherry. The following data analysis was performed using Kaluza Analysis Software 1.3.

To test for stability of plasmid segregation, overnight cultures were diluted in 50 mL fresh medium with and without relevant antibiotics to an initial OD₆₀₀ of 0.05, grown for 12 h (six generations) and then diluted again into fresh medium to an initial OD₆₀₀ of 0.05. This was repeated over the course of the whole experiment. After 6 h from inoculation, 10 mL of the cultures were aliquoted to 100 mL shaking flasks and incubated for 2 h at 37°C, 200 rpm, after which the flow cytometry analysis was carried out. This procedure was repeated every 24 h (every 12 generations) for a total of 5 days (60 generations). The stability is presented as the ratio of cells fluorescent in absence of antibiotics to the cells fluorescent in presence of antibiotics.

Overnight cultures of MGA3 (pHCMC04), MGA3 (pHP13), MGA3 (pNW33Nkan), or MGA3 (pUB110Smp-lacZ) were diluted to 2% in fresh medium (supplemented with 5 μg/ml chloramphenicol or 10 μg/ml kanamycin) and cultivated until the mid-exponential growth phase (OD₆₀₀ 2-4). Cell pellets were harvested from 5 ml cultures by centrifugation and total DNA was extracted using the MasterPure^TM Gram Positive DNA Purification Kit (Epicenter), followed by an additional purification step using the Agencourt^® AMPure XP system (Beckman Coulter). DNA concentrations were determined on a Qubit^® 2.0 Fluorometer using the Qubit^® dsDNA BR Assay Kit (ThermoFischer Scientific). Twenty microliter ddPCR reaction mixtures containing EvaGreen Supermix (Bio-Rad), primers (0.2 μM) and gDNA template (8 or 20 pg) were prepared according to the manufacturer’s instructions and used for droplet generation (QX200 droplet generator, Bio-Rad). Forty microliter of sample was manually transferred to a 96-well plate and heat-sealed prior to amplification initiated by enzyme activation at 95°C for 5 min, followed by 40 cycles of amplification (95°C for 30 s, 60°C 1 min) and signal stabilization (4°C 5 min, 90°C 5 min), temperature ramp 2.5°C/s. Following amplification, fluorescence intensity was measured in a QX200 Droplet Reader (Bio-Rad) and the signal data were analyzed with QuantaSoft, Version 1.5.38 (Bio-Rad). Primer sequences are listed in the Supplementary Material.

Fed-batch fermentation was performed at 50°C in UMN1 medium using Applikon 3 L fermenters with an initial volume of 0.75 L medium essentially as previously described (Jakobsen et al., 2009; Brautaset et al., 2010). Kanamycin (50 μg/mL) or chloramphenicol (5 μg/mL) was added to the initial batch growth medium, the pH was maintained at 6.5 by automatic addition of 12.5% (w/v) NH₃ solution, and the dissolved oxygen level was maintained at 30% saturation by increasing the agitation speed and using enriched air (up to 60% O₂). The methanol concentration in the fermenter was monitored by online analysis of the headspace gas with a mass spectrometer (Balzers Omnistar GSD 300 02). The headspace gas was transferred from the fermenters to the mass spectrometer in insulated heated (60°C) stainless steel tubing. The methanol concentration in the medium was maintained at a set point of 150 mM by automatic addition of methanol feed solution containing methanol, trace metals and antifoam 204 (Sigma), as previously described (Brautaset et al., 2010). All fermentations were run until the carbon dioxide content of the exhaust gas was close to zero (no cell respiration). Bacterial growth was monitored by measuring OD₆₀₀. Dry cell weight was calculated using a conversion factor of one OD₆₀₀ unit corresponding to 0.24 g dry cell weight per liter (Jakobsen et al., 2009). Due to significant increase in the culture volume throughout the fermentation, the biomass, cadaverine, and amino acid concentrations were corrected for the increase in volume and subsequent dilution. A volume correction factor of 1.8 was used for values presented in Table 2. The actual concentrations measured in the bioreactors were therefore accordingly lower as described previously (Jakobsen et al., 2009). Samples for determination of volumetric cadaverine and amino acid yields were collected from early exponential phase and throughout the cultivation (10–47 h).

Samples were analyzed by RP-HPLC as described previously by Skjerdal et al. (1996) using pre-column derivatization with o-phtaldialdehyde and a buffer containing 0.02 M sodium acetate +2% tetrahydrofuran at pH 5.9.

For the detection of α-amylase activity, overnight cultures were diluted to an initial OD₆₀₀ of 0.15 and cultivated for 6-8 h at 50°C. The cultures were diluted to OD₆₀₀ of 1 and 15 μL of the diluted cultures were placed on the appropriate plates in the form of a drop. The plates were incubated for 12 h at 50°C to allow the cell growth and then placed at 37°C for next 24 h for in order to support activity of the heterologous α-amylase. Ten milliliter of iodine solution were placed on the plate in order to visualize the formation of the halo in the starch.

Genetic engineering of B. methanolicus has until now relied on only two plasmids, pNW33N and pHP13. Therefore, we decided to analyze a range of different replicons with regard to their applicability for gene overexpression in B. methanolicus MGA3. We compared four different plasmids that were able to replicate: pTH1mp (derived from pHP13), pUB110Smp, pNW33Nmp, and pBV2mp (derived from pHCMC04). As shown in Table 1, we have chosen plasmids differing in the copy number, original host organism and the replication mechanism. All rolling circle (RC) plasmids used belong to the pC194/pUB110 family, which is characterized by similarity in Rep protein and the sequences of sites involved in the replication with pNW33N and pUB110 sharing identical Rep protein sequences. The pUB110 plasmid is reported to be a high copy number plasmid in B. subtilis, pNW33N – medium, pHP13 and pBV2mp – low copy number, both of the low copy number plasmids originate from B. subtilis.

Our initial goal was to characterize the copy number, expression levels and stability of the chosen plasmids in B. methanolicus MGA3. To analyze the expression levels, gfpuv (Chalfie et al., 1994; Crameri et al., 1996) was used as a reporter controlled by the mdh promoter from B. methanolicus MGA3. Fluorescence intensity was evaluated during growth in methanol minimal medium by flow cytometry. Using the ddPCR, plasmid copy numbers were estimated for the selected plasmids and, in comparison, for the native MGA3 plasmids pBM19 and pBM69 (Table 1). The plasmid pUB110Smp-gfpuv showed the highest fluorescence levels among the plasmids tested (Table 1), followed by pNW33Nmp-gfpuv, pTH1mp-gfpuv, and pBV2mp-gfpuv, respectively, which was in accordance to the plasmid copy number results (Table 1).

Next, we compared the plasmid stability for gfpuv-expressing RC plasmids transferred to B. methanolicus MGA3. The strains were grown for 60 generations in media with and without antibiotic selection and plasmid-containing cells emitting a fluorescence signal were counted every 12 generations. As shown in Figure 1 only the pTH1mp plasmid was lost at a significant level over the course of the experiment.

The plasmids pTH1mp and pBV2mp, containing the mdh promoter were used to study cadaverine production in B. methanolicus during fed-batch methanol fermentation. We have previously reported a methanol-based cadaverine production titer of 6.5 g/L by B. methanolicus MGA3 (pTH1mp-cadA), a strain overexpressing the lysine decarboxylase cadA gene from E. coli (corrigendum to Nærdal et al., 2015). We compared cadaverine production in the strain overexpressing cadA from a theta-replicating plasmid during high cell density fed-batch fermentation. The B. methanolicus strain MGA3 (pBV2mp-cadA) was tested in duplicates under comparable fermentation conditions. Samples for cadaverine and amino acid analysis, cell dry weight and OD₆₀₀ were taken throughout the cultivation. As presented in Table 2, we obtained a cadaverine production titer of 10.2 g/L based on the alternative theta-replicating pBV2mp plasmid. A substantial 55% production increase compared to the previously reported (pTH1mp-cadA)-based strain was observed. While biomass and by-product levels were similar between the two strains, the specific growth rate of MGA3 (pBV2mp-cadA) was lower than that of MGA3 (pTH1mp-cadA) (Table 2).

In order to establish a two plasmid-based gene expression system, we analyzed the compatibility of the chosen RC (pTH1mp and pUB110Smp)- and theta (pBV2mp)-replicating plasmids in B. methanolicus. pTH1mp and pUB110Smp share high identity (42%) of their replication protein Rep and of the origin of replication sequence (95%) and for this reason it was not clear whether they can coexist in the same cell. Similarly, we did not analyze the pUB110mp/pNW33N plasmid pair which display 100% identity of Rep protein sequences. Plasmids for expression of either gfpuv or mcherry (Shaner et al., 2004) were constructed to simultaneously analyze gene expression from two vectors. The following plasmid combinations were applied: pTH1mp-mcherry with pUB110Smp-gfpuv or pTH1mp-mcherry with pBV2mp-gfpuv. Overexpression of mcherry from pTH1mp led to red fluorescence (depicted on the y-axis in Figure 2). Similarly, overexpression of gfpuv from pUB110Smp or from pBV2mp yielded green-fluorescent cells (x-axis of Figure 2). Cells transformed with pTH1mp-mcherry and pUB110Smp-gfpuv or with pTH1mp-mcherry and pBV2mp-gfpuv showed simultaneous red and green fluorescence (Figure 2) providing evidence for two plasmid-based gene expression in B. methanolicus.

In order to choose a suitable system for inducible gene expression we screened several inducible promoter systems using the thermostable LacZ from B. coagulans as a reporter (Kovács et al., 2010). We have tested the B. megaterium xylose inducible system from plasmid pHCMC04 (Nguyen et al., 2005), a native mannitol inducible promoter from MGA3, and a copper inducible promoter from Lactobacillus sakei (Crutz-Le Coq and Zagorec, 2008). As shown in Figure 3, the xylose inducible promoter system was functional in B. methanolicus MGA3 and, when fully induced, yielded higher expression levels than the hitherto used mdh promoter. Very low expression was observed from both the mannitol-inducible promoter present in the upstream region of the mtlA gene of B. methanolicus MGA3, and the copper inducible promoter. The copper-inducible promoter showed a dose-response where the activity in cultures induced by 100 μM CuSO₄ was approximately threefold higher than in cultures induced by 50 μM CuSO₄ (data not shown). The inducer, however, had a toxic effect on the cells, reducing the growth rate considerably at concentrations above 50 μM CuSO₄ (data not shown), making it not suitable for industrial applications.

Since only very low expression was observed from the mannitol-inducible mtlA promoter, we used DNA microarray data (Heggeset et al., 2012) and RNA-seq data (Irla et al., 2015) to identify other mannitol-inducible genes. Figure 4 presents the genomic and transcriptomic organization of four genes which belong to the mannitol utilization pathway: mtlA coding for PTS system mannitol-specific EIICB component, mtlR encoding a transcriptional regulator, mtlF coding for mannitol-specific phosphotransferase enzyme IIA and mtlD encoding mannitol-1-phosphate 5-dehydrogenase. Genes mtlF and mltD are co-expressed as an operon. Transcription start sites (TSSs) were not detected either for mtlA or for mtlF-mtlD; however, a TSS was found for mtlR (Irla et al., 2015). This 5′ untranslated region (5′ UTR) of mtlR is 80 nt in length and its upstream sequence contains conserved -10 and -35 regions (bold): 5′-TTGTATTAAGGGATATAAACGTTTTATGATAAATATG-3′, furthermore the putative ribosome binding site (RBS) sequence is AGTGGAG, which differs in two positions from the B. methanolicus consensus RBS motif AGGAGG (Irla et al., 2015). We cloned the upstream sequence of the mtlR gene into the plasmid pTH1 containing the gfpuv gene and exchanged the RBS sequence to the consensus motif, which resulted in plasmid pTH1m2p-gfpuv.

At first, several sugar alcohols were tested as potential inducers (Figure 5A). Supplementation with 50 mM mannitol induced gfpuv expression; however, neither arabitol, ribitol, nor xylitol induced reporter gene expression (Figure 5A). Subsequently, a titration experiment with different concentrations of mannitol was performed. While the addition of 5 mM mannitol did not increase reporter gene expression, high GFPuv fluorescence intensities were observed upon addition of 12.5, 25, and 50 mM mannitol (Figure 5B). It has to be noted that GFPuv fluorescence intensities in the presence of 12.5, 25, and 50 mM mannitol were comparable suggesting that full induction has been achieved (Figure 5B). The expression level of the mannitol-inducible promoter in the presence of 50 mM mannitol was similar to that obtained with the conventionally used mdh promoter. To test whether higher gene expression is possible in the mannitol inducible system, we decided to exchange the sequences of the -10 and/or the -35 region for the previously described consensus sequences (Irla et al., 2015). As shown in Table 3, the exchange of the -35 region or the -35 region together with the -10 region led to higher fluorescence levels in comparison to the native promoter. However, the double exchange caused a 3.5-folds increased background expression.

MtlR has been characterized as a mannitol-dependent transcriptional activator in several species (Joyet et al., 2015). The alignment of the B. methanolicus MtlR protein sequence with the sequences of characterized regulators from L. casei BL23, B. subtilis ssp. subtilis str. 168 and Geobacillus stearothermophilus ATCC 7954 (Figure 6) revealed the conserved residues important for the regulatory activity of MtlR. The high similarity to the characterized proteins suggested that MtlR of B. methanolicus most probably serves as a transcriptional activator. For this reason, we decided to test whether the plasmid-borne overexpression of mtlR increased mtlR promoter activity. As shown in Figure 7, the overexpression of this gene increased the reporter gene expression from the mannitol inducible promoter m2p by more than 2.5-fold while maintaining a low level of background expression in the absence of mannitol. Taken together, evidence is provided for a versatile mannitol inducible system for the thermophilic B. methanolicus on the basis on the previously obtained RNA-seq data.

Based on our screening experiments (Figure 3), we decided to further develop the xylose inducible system for gene expression in B. methanolicus. We have subcloned the xylR regulator gene together with the promoter and the RBS sequence of the B. megaterium xylA gene into pTH1 to drive expression of gfpuv. The resulting plasmid was named pTH1xpx-gfpuv. Figure 8 shows the expression levels of gfpuv transcribed from the xylose inducible promoter in media with different xylose concentrations. Reporter gene expression increased linearly in the concentration range between 0.01% (w/v) and 0.1% (w/v) and reached a plateau at 0.5% (w/v). The fluorescence from fully induced xpx promoter is around 15-fold higher in comparison to the conventionally used mdh promoter (Table 1). Furthermore, the background gene expression with uninduced MGA3 (pTH1xpx-gfpuv) was very low (0.17 ± 0.01 a.u.) as compared to the background fluorescence (0.12 ± 0.00 a.u.) obtained for wild type B. methanolicus MGA3. Notably, mannitol did not induce expression of gfpuv from the xylose inducible promoter (data not shown).

α-Amylases degrade starch to glucose and expression of heterologous α-amylase genes in glucose-positive, but starch-negative species enabled starch utilization as for example shown for C. glutamicum expressing α-amylase gene (amy) from Streptomyces griseus IMRU3570 (Seibold et al., 2006). A BLAST search of the Bacillus methanolicus genome revealed two genes putatively encoding α-amylases (BMMGA3_04340, BMMGA3_04345) and one coding for an α-glucosidase (Heggeset et al., 2012; Irla et al., 2014). For heterologous expression of amy from S. griseus plasmid pTH1xpx was used for xylose inducible expression in B. methanolicus. Starch degradation by the control strain B. methanolicus MGA3 (pTH1mp) on LB agar plates supplemented with 0.5% soluble starch and 0.05% xylose at 37°C was not observed (Figure 9A). By contrast B. methanolicus MGA3 (pTH1xpx-amy) showed a halo on starch LB plates containing xylose as an inducer and incubated at 37°C (Figure 9B) indicating that expression of amy from S. griseus plasmid allowed for starch degradation by recombinant B. methanolicus.