GFP screen of expression vectors in R. palustris CGA009
Different combinations of promoter and vector sequences were screened for highest levels of expression within R. palustris. The endogenous ppckA promoter of R. palustris is one of the few promoter sequences that had been previously validated to be active in R. palustris under gluconeogenic conditions [23]. However, we only observed a minimal increase in fluorescence for both the pMG105 and pBBR1MCS-2 ppckAp GFPmut2 constructs compared to the empty vector controls (Fig. 2). While the pBBR1MCS-2 arap GFPmut2 construct yielded slightly higher levels compared to ppckAp, the pBBR1MCS-2 lacp GFPmut2 construct offered the best expression overall and was selected as the vector for all further expressions in R. palustris (Fig. 2). Expression from the lac promoter in R. palustris was not affected by the addition of the inducer IPTG and catabolite repression only showed a minor decrease in fluorescence (Additional file 3). This suggests that the lac regulatory response system utilized by E. coli is not functional in R. palustris. Although the pBBR1MCS-2 lac promoter system provided the best expression in R. palustris of all the combinations tested (Fig. 2), the fluorescent signal was still ~13 times lower than the signal from E. coli pBBR1MCS lacp GFPmut2 (data not shown).
Engineering adhE
BisB18 in R. palustris
As determined by the lack of growth under restrictive conditions, the absence of butanol production (data not shown), and the lack of a signal in a western blot under permissive conditions (Fig. 3a, AdhEBisB18 Aerobic), direct introduction of adhE2
824 in the pBBR1MCS-2 lacp construct did not result in the production of AdhE2824 in R. palustris. This is likely due to the large GC discrepancy (30% vs. 65%) and codon utilization preference between C. acetobutylicum and R. palustris [24]. To address this, we looked for homologous proteins within R. palustris. Indeed, endogenous alcohol/aldehyde dehydrogenases with homology to AdhE2824 have been found to function in the production of butanol from butyrate when overexpressed. This was previously demonstrated with the fucO gene in E. coli, where overexpression allowed production of butanol from an engineered reverse β-oxidation pathway where no butanol production was observed previously [25]. In an attempt to enable butanol production in R. palustris, a BLAST search for an endogenous enzyme yielded the identification of RPC_4481 (AdhEBisB18) in R. palustris strain BisB18, which is an aldehyde/alcohol dehydrogenase with high homology to both domains of AdhE2824.
Before expressing adhE
BisB18 as a candidate butanol-pathway gene, R. palustris strain BisB18 was screened for its ability to grow by producing butanol from butyrate under restrictive electron sink conditions (without HCO3
−). Although no growth or butanol production occurred (data not shown), it is possible that wild-type production of this protein under these conditions is either inactive or insufficient for an observable effect, similar to the FucO protein in E. coli [25]. Because no experimental studies have previously characterized the activity of AdhEBisB18, its highly conserved primary sequence was further investigated for its structural homology to the verified butanol-producing AdhE2824.
Using Swiss-Pdbviewer/DeepView [26], three-dimensional structures of the individual protein domains for both AdhE2824 and AdhEBisB18 were generated. The predicted tertiary structure of both aldehyde/alcohol dehydrogenases appeared highly conserved with regard to overall shape, location, active site residues, channel volume, and surface charge (Additional file 4). This suggested that the two proteins likely recognized similar substrates. We, therefore, expressed adhE
BisB18 in R. palustris using the pBBR1MCS-2 lacp construct. Transformants were inoculated into RM with butyrate and with and without HCO3
− to screen for viability and butanol production, respectively. Although transformants grew up with HCO3
−, no growth was observed without HCO3
− condition (data not shown), suggesting that either AdhEBisB18 was not successfully produced by R. palustris CGA009 or did not have butyryl-CoA-to-butanol conversion activity.
To investigate this further, a crude cell extract of the culture with HCO3
−, along with E. coli harboring the same construct, was analyzed via western blotting. Although the strong signal at the expected size of 94.5 kD for AdhEBisB18 for E. coli pBBR1MCS-2 lacp adhE
BisB18 demonstrated that the protein was produced (Fig. 3a, AdhEBisB18), the lack of a signal from R. palustris pBBR1MCS-2 lacp adhE
BisB18 suggested that the protein was either not produced or was produced at levels below detection (Fig. 3a, AdhEBisB18 Aerobic). Aerobic conditions were preferred since anaerobic cultures often had photosynthetic pigments overlapping the expected size for AdhE (Fig. 3a, AdhE2opti Anaerobic). The lack of growth and butanol production under restrictive conditions, however, demonstrated conclusively that R. palustris pBBR1MCS-2 lacp adhE
BisB18 was not endowed with butyrate-to-butanol conversion functionality (data not shown).
Engineering adhE2
opti in R. palustris
Another route to circumvent the large GC discrepancy and codon utilization preference between C. acetobutylicum and R. palustris was to perform codon optimization on adhE2
824 to enable expression. The codon utilization table for R. palustris CGA009 was determined by analysis of all genomic coding sequences and was retrieved from the Kazusa database [27]. The adhE2
opti gene resulted in an increase of the average codon utilization frequency in R. palustris from 23% for the original adhE2
824 to 64% for the optimized adhE2
opti (Additional file 5). Codon optimization resulted in a shift of the GC content from 32.5% for adhE2
824 to 62% for adhE2
opti. In addition, the presence of rare codons (<10% utilization frequency), which previously composed 47.4% of all codons in adhE2
824, was entirely eliminated. One disadvantage to this approach of codon optimization is that rare codons are known to induce ribosomal pausing, which has been demonstrated important for proper folding of proteins. Thus, with the elimination of all rare codons from adhE2
opti, the elimination of codon-dependent ribosomal pausing could lead to improper folding and aggregation of the target protein [28]. For comparison with adhE2
opti, the codon utilization frequency of adhE
BisB18 averaged 55% (Additional file 5), the gene possessed an overall 63% GC content, and the frequency of rare codons within the gene was 5%, demonstrating the utilization of rare codons for proper translation of proteins in endogenous alcohol/aldehyde genes (Additional file 6).
To test whether adhE2
opti would be expressed in R. palustris CGA009 and enable growth by conversion of butyrate to butanol, the gene was introduced using the pBBR1MCS-2 lacp construct. Transformants were then inoculated into RM with butyrate and with and without HCO3
− for growth screening and western blot analysis. This engineered strain was able to grow without HCO3
− (restrictive conditions) and it produced a measureable amount of butanol, demonstrating the successful production of AdhE2opti with a functional activity. While a detectable signal was observed on a western blot for the E. coli cloning culture, no signal was initially detected from R. palustris pBBR1MCS-2 lacp adhE2
opti (Fig. 3a, AdhE2opti Anaerobic and AdhE2opti Aerobic). We performed a second western blot using a more sensitive X-ray film imaging method. For aerobically grown R. palustris pBBR1MCS-2 lacp adhE2
opti, we observed a single signal at approximately 94 kD, demonstrating successful expression (Fig. 3b, AdhE2opti Aerobic). As stated above, GFPmut2 production from the pBBR1MCS lac promoter is much lower in R. palustris than in E. coli, so the lack of a signal in the majority of the samples is likely due to low protein production in R. palustris.
Butanol production rates and selectivity for R. palustris pBBR1MCS-2 lacp adhE2
opti
Because of the rescued growth and butanol production observed, R. palustris pBBR1MCS-2 lacp adhE2
opti was further investigated for its ability to grow under both permissive and restrictive conditions and these results were compared to R. palustris pBBR1MCS-2 lacp GFPmut2 as a control. R. palustris pBBR1MCS-2 lacp adhE2
opti was capable of growth without HCO3
− (restrictive conditions) (Fig. 4b), while this growth did not occur for the control strain. The growth for R. palustris pBBR1MCS-2 lacp adhE2
opti occurred on a long time scale and reached a relatively low maximum cell density of ~0.06 OD600 (Fig. 4b) at neutral pH levels (Fig. 4d). Following the onset of the stationary phase, consumption of butyrate and production of butanol still occurred (Fig. 4f, h), but the cultures only reached a maximum butanol concentration of <0.4 mM after extended incubation (Fig. 4h).
Although only low levels of butyrate were consumed (Fig. 4f), butanol production occurred with a 39.7 ± 7.6% selectivity based on moles of carbon. The theoretical maximum for butanol production is based on all electrons derived from complete oxidation of one molecule of butyrate reducing five molecules of butyrate to butanol (6 butyrate → 5 butanol + 4 CO2) for 83% conversion efficiency. However, based on previous empirical measurements of excess reducing equivalents of R. palustris growing with butyrate by McKinlay et al. [3], only an estimated 0.45 mol of butanol should be produced per mole of butyrate consumed for an effective 45% selectivity (6 butyrate → 2.7 butanol + biomass). Therefore, almost all reducing equivalents predicted to be in excess were being converted into butanol by R. palustris pBBR1MCS-2 lacp adhE2
opti (40% vs. 45%), which we had anticipated for an obligatory reaction.
To further investigate why R. palustris pBBR1MCS-2 lacp adhE2
opti showed low growth rates, R. palustris was screened for product (butanol) toxicity. However, no inhibition of growth was observed in R. palustris at butanol concentrations up to ~30 mM (Additional file 7), removing our concerns about a concentration of ~0.4 mM (Fig. 4h). In addition, we operated a bioreactor with product removal to investigate whether this would increase growth and butanol production rates by removing butanol from the substrate pool, thereby, improving the thermodynamics for butanol formation and eliminating any end-product inhibition. Sparging of N2 has been previously used to remove butanol from fermentation broths [29]. Here, N2 gas was continuously sparged into 350-mL anaerobic bioreactors to remove butanol produced by R. palustris pBBR1MCS-2 lacp adhE2
opti in batch-mode for 6 consecutive weeks. The R. palustris pBBR1MCS-2 lacp adhE2
opti culture grew for that entire period up to an OD600 of 0.12 (Additional file 8: Fig. S6A), and achieved an average maximum butanol production rate of 0.0017 g L−1 day−1 (Additional file 8: Fig. S6D) with a maximum butanol concentration of 0.35 mM (Additional file 8: Fig. S6C) without depleting butyrate (Additional file 8: Fig. S6B). The lack of a considerable improvement compared to the earlier experiment showed that neither butanol toxicity nor thermodynamics nor end-product inhibition was a main reason for low growth rates. Finally, we found that the extraction system removed a majority of the butanol without removing any of the butyrate (Additional file 8: Fig. S6E, F).
The intermediate butyraldehyde within microbial cells is another metabolite that could have inhibited R. palustris, even though the maximum concentration in the supernatant was measured to be only 50 μM. However, due to the fusion nature of AdhE2opti, we anticipate that butyraldehyde would rapidly be converted into butanol with a high flux rate, resulting in a low effective concentration [30]. Without butanol and butyraldehyde toxicity, we can only speculate about why growth and butanol production rates of our engineered strain are so low. Since AdhE2opti is provided as the sole route to maintaining redox balance, all excess electrons must be funneled through this pathway. When the capacity of the AdhE2opti pool is outweighed by the demand for regenerating oxidized reducing equivalents, this would introduce a bottleneck in the maximum rate of metabolism and growth. However, more work is required to be conclusive.
Some butanol production did also occur for R. palustris pBBR1MCS-2 lacp adhE2
opti with HCO3
− (permissive conditions) when exogenous CO2 was available for maintaining redox balance (Fig. 4a, g) at a neutral pH level (Fig. 4c). Because the preculture had originally been maintained without HCO3
−, a long lag phase and carryover of butanol producing activity was observed for R. palustris pBBR1MCS-2 lacp adhE2
opti (Fig. 4a), resulting in a maximum concentration of 0.37 mM with 11.5 ± 2.4% selectivity (Fig. 4e, g). Though the transient production of butanol was a surprise, this lower selectivity for the permissive condition was expected because more reducing equivalents could be used in central metabolism following the introduction of exogenous HCO3
−, resulting in much higher growth (~10× OD at 200 h), the complete depletion of butyrate (Fig. 4e), no production of acetate, and ultimate consumption of all butanol produced (Fig. 4g). This contrasts with the restrictive condition where all growth proceeds through the obligate engineered pathway, butyrate is not depleted, and all butanol produced remains in solution.
Optimizing butanol production by pregrowing R. palustris pBBR1MCS-2 lacp adhE2
opti
In an attempt to improve the maximum butanol concentrations produced by the engineered strain, 50 mL aerobic cultures was grown to mid-log phase, washed 3 times with sterile RM, resuspended in 0.5 mL RM, and used as an inoculum into 20-mL anaerobic cultures under restrictive conditions. This procedure ensured that the butyrate-to-butanol conversion was not catalyst limited as the initial OD600 of these cultures was >1. By using a concentrated inoculum, R. palustris pBBR1MCS-2 lacp adhE2
opti produced butanol concentrations of greater than 1.5 mM at volumetric butanol production rates of 0.034 g L−1 day−1 (Fig. 5d, f). All butanol was produced within 100 h of inoculation (Fig. 5d, f). Following 100 h, however, the R. palustris pBBR1MCS-2 lacp adhE2
opti culture continued to grow and consume butyrate (Fig. 5a, c) with the main carbon product switching from butanol to acetate (Fig. 5b). Acetate had not been detected before in any of the previous anaerobic experiments and in terms of maintaining redox balance, acetate is an unfavorable redox product compared to butanol. This metabolism reduced the butanol selectivity (Fig. 5e). We do not know exactly why this switch occurred, but it was likely a result from having a high biomass culture that underwent a rapid switch from aerobic to anaerobic conditions.