Abstract
Methanol is one of the most widely produced organic substrates from syngas and can serve as a bio-feedstock to cultivate acetogenic bacteria which allows a major contribution to reducing greenhouse gas. Acetobacterium woodii is one of the very few acetogens that can utilize methanol to produce acetate as sole product. Since A. woodii is genetically tractable, it is an interesting candidate to introduce recombinant pathways for production of bio-commodities from methanol. In this study, we introduced the butyrate production operon from a related acetogen, Eubacterium callanderi KIST612, into A. woodii and show a stable production of butyrate from methanol. This study also reveals how butyrate production by recombinant A. woodii strains can be enhanced with addition of electrons in the form of carbon monoxide. Our results not only show a stable expression system of non-native enzymes in A. woodii but also increase in the product spectrum of A. woodii to compounds with higher economic value.
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Introduction
In times of global warming, there is an increasing need to reduce CO2 emissions into the atmosphere. Therefore, petroleum-based technologies for the production of added-value compounds need to be replaced by sustainable biotechnological approaches. Various bacteria and archaea are known to convert CO2 but strictly anaerobic, acetogenic bacteria have gained much interest in recent times for they grow in the dark in the absence of oxygen, are easy to cultivate, and are metabolically very diverse (Drake 1994; Müller 2003; Drake et al. 2008). They can grow lithotrophically on H2 + CO2, some also on syngas (H2 + CO2 + CO), on C1 compounds such as formate or methanol but also organotrophically on various sugars, carbonic acids, aldehyde, primary, and secondary alcohols (Drake et al. 1997; Schuchmann and Müller 2016). Many of these metabolic pathways go along with the fixation of CO2. For example, acetogenesis from methanol according to:
requires half a mol of CO2 per mol of methanol converted.
Methanol is a promising feedstock for acetogens and it can be produced from syngas chemically and then used to feed acetogens without the problems inherent to gas fermentation (Cotton et al. 2019; Satanowski und Bar-Even 2020; Kremp und Müller 2021). Butyrate is such an added value compound that is the starter molecule for butanol production, a biofuel with a much higher energy density than ethanol and some acetogens like Clostridium carboxidivorans, Clostridium drakei (Liou et al. 2005), Eubacterium callanderi KIST612 (Chang et al. 1997; Litty and Müller 2021), and few intestinal acetogens like Butyvibrio crossotus and Eubacterium rectale (van den Abbeele et al. 2013) have been shown before to produce butyrate naturally. Butyrate was also produced from syngas in co-culture of Clostridium kluyveri and Clostridium autoethanogenum (Diender et al. 2016). Here, we have metabolically engineered one of the most robust and well-studied acetogens, Acetobacterium woodii, as a new production platform for butyrate formation from fructose or methanol.
Results
Generation of recombinant A. woodii
To generate a recombinant A. woodii strain able to produce butyrate, we chose to clone the butyrate production genes from E. callanderi KIST612, an acetogen known to produce butyrate naturally. E. callanderi KIST612 harbors the genes encoding thiolase (thlA, ELI_0537), 3-hydroxybutyryl-CoA dehydrogenase (hbd, ELI_0538), crotonase (crt, ELI_0539), and the electron bifurcating butyryl-CoA dehydrogenase complex (bcd/etfAB, ELI_0540-0542). While the genes of the butyrate pathway are clustered in a single gene cluster, the phosphobutyryl transferase (ptb, ELI_0834) is encoded elsewhere in the genome. Unfortunately, there is no annotated butyrate kinase gene in the genome. First, we cloned the genes thlA, hbd, crt, and bcd/etfAB of the butyrate cluster in plasmid pMTL84211 (Purdy et al. 2002; Heap et al. 2007) under the control of pta-ack (phosphotransacetylase-acetate kinase) promoter from Clostridium ljungdahlii giving rise to the plasmid pMTL84211pAck_NP_But6. A second plasmid was also created which contained the Ptb gene along with its 137 bp upstream region forming the plasmid pMTL84211pAck_NP_But7 (Fig. 1).
The plasmids were transferred into A. woodii and the recombinants grew well on fructose with doubling times of 9.8 h (Aw_But6 strain) and 9 h (Aw_But7 strain) to similar final optical densities (Fig. 2). Each of these strains produced acetate, as expected, but Aw_But6 and Aw_But7 also produced butyrate. In Aw_But6, the yield was low but clearly above the control and the yield was increased by 400% up to a concentration of 1.5 mM in Aw_But7 (Fig. 2). In total, the carbon recovery was 78–83%, not accounting for CO2.
The increased production of butyrate in Aw_But7 argued for the ptb gene being transcribed in addition resulting in an active phosphobutyryl transferase leading to the production of butyryl phosphate which is further dephosphorylated to butyrate. Indeed, this was observed; genes encoding butyrate production like thlA and hbd were expressed in A. woodii during growth on fructose in Aw_But6 and Aw_But7 strain and in addition, ptb was also transcribed in Aw_But7 strain (Fig. 3). Apparently, co-transcription of ptb led to higher butyrate yields. Although butyrate kinases are not annotated in the genomes of A. woodii or E. callanderi, cell-free extract of fructose-grown cells of A. woodii and E. callanderi catalyzed butyrate kinase activity with 0.43 and 0.57 U/mg, respectively. Acetate kinase activity was sevenfold higher in A. woodii (3.1 U/mg) and twice as high in E. callanderi (1.38 U/mg). Apparently, butyrate kinase activity is present in wild type A. woodii and E. callanderi.
Butyrate production is carbon source dependent
As shown above, butyrate was produced during growth on fructose as carbon source. A. woodii also grows lithotrophically on H2 + CO2, but butyrate was not detected under these conditions in neither Aw_But6 nor Aw_But7 strains (data not shown). Like the wild type, Aw_But6 and Aw_But7 grew well on methanol, although the doubling time was increased (24 h) compared to the wild type (16 h). Methanol consumption was accompanied by the formation of acetate and a total of around 38 mM of acetate was formed from 60 mM methanol, corresponding to an acetate methanol ratio of 0.63. Interestingly, parallel to the formation of acetate, butyrate production started in Aw_But7 strain at the mid exponential growth phase and reached a maximum of 0.25 mM in the stationary phase (Fig. 4). Butyrate formation could not be detected in Aw_But6 strain.
Butyrate production in non-growing cells
Non-growing, resting cells have the advantage of not losing carbon to biosynthetic pathways. Therefore, we analyzed butyrate formation in resting cells. Cells grown on fructose and resuspended in buffer immediately started to produce acetate from fructose. Aw_ctrl strain, not carrying the butyrate synthetic genes, produced 37 mM acetate from 17 mM fructose. As expected, butyrate was not produced. Interestingly, in the recombinant strain, unlike in growing condition, fructose consumed to acetate formed was lower (1:2.1) compared to growing cells (~ 1:2.5). Aw_But6 produced less acetate (36 mM), but when production started to reach the plateau, butyrate production started. Finally, 0.3 mM butyrate was produced, similar to observed under growing conditions. In comparison to Aw_But6 strain, Aw_But7 carrying ptb, in addition, produced similar amounts of acetate (35 mM) but butyrate formation was increased drastically by 400% to a final concentration of 1.2 mM (Fig. 5).
Cells of Aw_But7, grown on methanol and resuspended in buffer, also metabolized methanol but in contrast to growing cells, only acetate was produced and butyrate could not be detected. However, when carbon monoxide (10% v/v) was added to the headspace, acetate formation increased and concomitantly butyrate was produced up to a maximum concentration of 0.35 mM (Fig. 6).
Discussion and conclusion
The global-interest in reducing levels of CO2 and increase carbon re-cycling led the scientific community to find several effective methods to fix CO2 artificially and also use microorganisms to convert CO2 into biochemicals. In recent years, a lot of success has been achieved where acetogens like Clostridium autoethanogenum (Köpke et al. 2014; Liew et al. 2016a, b; Heffernan et al. 2020), Clostridium ljungdahlii (Ueki et al. 2014), and Moorella thermoacetica (Kita et al. 2013) had been engineered with genes from other clostridia to utilize H2 + CO2 or syngas to produce higher carbon chain biochemicals. In a proof-of-concept study, it was shown that A. woodii can produce acetone under autotrophic conditions when the thiolase, CoA transferase, and acetoacetate decarboxylase genes from Clostridium acetobutylicum were expressed (Hoffmeister et al. 2016). While most biochemical production pathways are energy invasive, in a very recent study, recombinant A. woodii (Beck et al. 2020) was shown to conserve additional energy when the arginine deiminase pathway from C. autoethanogenum was heterologusly produced in A. woodii. The production of longer carbon-chain compounds in A. woodii would also require additional supply of energy and indeed this approach will find its merit.
Here in this study, we rather ask a very simple question. Can we can transfer metabolic pathways within related acetogens and induce them to produce a chemical that the other does not produce? It has been reported before that under certain circumstances, E. callanderi KIST612 naturally produces butyrate along with acetate as sole end products (Jeong et al. 2015; Dietrich et al. 2021; Litty and Müller 2021). The results presented here clearly demonstrate that transferring the butyrate formation pathway in a related acetogen like A. woodii that does not produce butyrate naturally leads to production of butyrate in the recombinant A. woodii strains. However, the maximal amount of butyrate produced during growth on 60 mM methanol was rather low (0.25 mM) in comparison to E. callanderi KIST612 (3.7 mM butyrate on 20 mM methanol). While the butyrate formation pathway requires the eight proteins thiolase, hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase/electron transferring flavoprotein complex, phosphotransbutyrylase, and butyrate kinase, introduction of only seven of the protein encoding genes into A. woodii led to continued formation of butyrate from both C6 and C1 compounds. Though the terminal gene of the butyrate pathway encoding for a butyrate kinase was missing, it is likely that the highly active acetate kinase (ack, Awo_c21260) synthesizes butyrate from butyryl-phosphate in recombinant A. woodii (Eden und Fuchs 1983; Schuchmann and Müller 2014). Indeed, cell-free extract of A. woodii showed butyrate kinase activity. Whether this is done by the acetate kinase or by an unknown butyrate kinase remains to be established. The same is true for E. callanderi. It should also be noted that the acetate kinase from Methanosarcina thermophila, which is 60% identical to the acetate kinase from A. woodii, can utilize butyrate, although with a 50-fold higher km value (Ingram-Smith et al. 2005).
Also, we could show that recombinant A. woodii could produce a four-carbon product (C4) from a C1 compound like methanol. The metabolism of methanol in recombinant A. woodii likely involves the methanol-specific methyltransferase system (Kremp et al. 2018; Kremp and Müller 2021) to generate methyl-THF which further condenses with an incoming CO using CODH/ACS enzyme to generate the central metabolic intermediate, acetyl-CoA. In recombinant strains, acetyl-CoA is further metabolized downstream to produce acetate or butyrate and conserve energy. The anaerobic utilization of methanol via the WLP provides NADH but needs an input of reduced ferredoxin for reduction of CO2 to CO (Fig. 7A). Reduced ferredoxin is generated from NADH by reverse electron transport catalyzed by the Rnf complex, energized by ATP hydrolysis (Kremp and Müller 2021). However, the production of butyrate from methanol requires NADH (Song et al. 2017) by hydroxybutyryl-CoA dehydrogenase and the electron bifurcating Bcd/EtfAB complex (Buckel und Thauer 2013; Jeong et al. 2015; Katsyv and Müller 2020) (Fig. 7A), which are generated by methanol oxidation. Importantly, the electron bifurcating butyryl-CoA dehydrogenase provides reduced ferredoxin, the fuel for electron transport phosphorylation thus increasing the ATP yield. In theory, from 5 methanol and 1 CO2, 1.5 butyrate could be produced leading to an ATP yield of 0.64 mol ATP/mol methanol consumed according to Eq. 2 (Fig. 7A):
In recombinant strains, the effective utilization of NADH is supposed to shift the acetate:butyrate ratio in favor of butyrate when cells are grown on methanol + CO2. This was true for Aw_But7 strain during growth on methanol (60 mM). However, the recovery of carbon in form of butyrate was relatively low in comparison to acetate, which is attributed either by the missing butyrate kinase or electron imbalance. In such a scenario, during cell suspension assays, with reduced electron pressure in form of lower concentration of methanol (20 mM), Aw_But7 strain did not synthesize butyrate.
Our results show that this electron imbalance could be partially surpassed by introduction of electron rich CO gas into the gaseous phase which significantly enhanced butyrate formation in the recombinant strain. In theory, butyrogenesis from methanol + CO is simple and involves condensation of 2 methanol with 2 CO leading to the formation of 1 butyrate. Also, owing to the reduced ferredoxin generated by CO oxidation, in this modular branch, an increase in the energy efficiency by almost 140% can be obtained with an ATP yield of 1.55 mol ATP/mol methanol consumed according to Eq. 3 (Fig. 7B):
However, in this case, addition of CO also increased acetate formation and this stresses the necessity of tailoring the pathway by specific genetic deletion to redirect the carbon flux towards butyrate instead of acetate. One such approach would be to delete the acetate kinase or phosphotransacetylase gene and introduce a clostridial butyrate kinase to complete the butyrate synthetic pathway. Indeed, it was reported that deletion of pta in C. ljungdahlii led to a decrease in acetate production by > 80% and improved ethanol formation via aldehyde ferredoxin oxidoreductase (AOR) (Lo et al. 2020). We could imagine that a similar deletion in A. woodii would lead to an accumulation of acetyl-CoA and in the presence of a recombinant butyrate pathway, the carbon flux would also be pushed towards the ATP generating butyrate synthesis. Finally, this study shows that A. woodii can be made to produce butyrate from methanol or methanol + CO which makes methanol a promising feedstock for an alternative bioeconomy using acetogens as biocatalyst.
Materials and methods
Cultivation of A. woodii and E. callanderi KIST612
A. woodii DSMZ 1030 and transformants were cultivated under strict anoxic conditions at 30°C in carbonate buffered complex medium. The medium was prepared as described previously (Heise et al. 1989). Fructose (20 mM), methanol (60 mM), or H2 + CO2 (80:20 [v/v]) served as a sole carbon and energy source for butyrate production studies. Transformed A. woodii cells harboring butyrate pathway genes were first selected on Heise media containing 20 mM fructose and 15 µg/ml erythromycin. E. callanderi KIST612 was cultivated at 37°C in anoxic carbonate-buffered basal medium (CBBM) (Chang et al. 1999) with glucose under a N2/CO2 (80/20% [v/v]) atmosphere. Growth was followed by measuring the optical density at 600 nm. All growth experiments were performed in 115-ml serum flasks containing 50 ml of media.
Construction of plasmids for butyrate production
For the construction of a synthetic pathway, the genes necessary for synthesis of butyrate in E. callanderi KIST612 were selected and amplified by PCR. The genes clustered in the butyrate operon, ELI_0537 – ELI_0542, consisting of thiolase (thlA), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd), and two subunits of electron transferring flavoprotein (EtfA/B) were amplified and subcloned into pMTL84211 backbone (Purdy et al. 2002) together (upstream) with PCR-amplified Ppta-ack promoter from C. ljungdahlii. The resulting plasmid was called pMTL84211Ack_NP_But6. Furthermore, the phosphobutyryl transferase (ptb, ELI_0834) with its 137 bp upstream region was also amplified by PCR and subcloned into the pMTL84211_6kb plasmid, downstream of etfA (ELI_0542). The resulting plasmid had 7 genes (thl, hbd, crt, bcd/EtfAB, and ptb) of the butyrate synthetic pathway and was called pMTL84211Ack_NP_But7. A control plasmid was also created by fusing PCR-amplified Ppta-ack promoter into the pMTL84211 vector with no other genes. All subcloning procedures were performed using fusion cloning strategy using NEBuilder HiFi DNA Assembly Kit (New England Biolabs, USA). Transformation was performed according to an earlier described procedure (Westphal et al. 40). In both the plasmids, the butyrate synthetic genes were under the direct control of the strong Ppta-ack promoter. The plasmids were used to electro-transform A. woodii WT cells to generate Aw_ctrl (control), Aw_But6 (6 gene variant), and Aw_But7 (7 gene variant) strains, respectively. The transformants were grown in a volume of 5 ml of carbonate-buffered complex medium (Heise et al. 1989) containing 20 mM fructose and 15 µg/ml erythromycin.
Semi-quantitative PCR for gene expression analysis
To analyze transcript levels of butyrate synthetic genes in A. woodii, RNA was prepared from recombinant A. woodii strains (Aw_But6 or Aw_But7) grown on fructose or H2 + CO2 to mid‐exponential growth phase as described earlier (Chowdhury et al. 2020). A total of 1 μg of RNA from each sample was converted into cDNA by using M‐MLV Reverse Transcriptase according to the manufacturer’s protocol (Promega, Mannheim, Germany). Transcript levels of representative genes of the butyrate operon (thlA and hbd) and ptb were analyzed using gene-specific primers (see Supplementary, Table 1). PCRs were performed using Phusion DNA polymerase (NEB, USA) with 10 ng cDNA as template and 500 nM of gene-specific primers in a final reaction volume of 25 μl. Confirmation of gene expression on different substrates was done by analyzing PCR products on an agarose gel.
Preparation of cell suspensions and analysis
For cell suspension analysis, recombinant A. woodii strains were adapted on either fructose, methanol or H2 + CO2 and cells were grown in 500/1000 ml volumes to mid-exponential growth phase. Cells were harvested by centrifugation (10,000 × g; 10 min) and washed two times with imidazole buffer A (50 mM imidazole–HCl, 20 mM MgSO4, 20 mM KCl, 2 mM dithioerythritol (DTE), 1 mg ∙ l−1 resazurin, pH 7.0) under strictly anoxic conditions in an anaerobic chamber (Coy Laboratory Products, Grass Lake, MI) filled with 95–98% N2 and 2–5% H2 as described previously (Heise et al. 1992). Cells were resuspended in 115-ml glass bottles in resuspension buffer (imidazole buffer supplemented with 20 mM NaCl and 60 mM KHCO3, pH 7.2) either under a N2/CO2 or H2/CO2 atmosphere (80:20 [v/v]).
For determination of the conversion of methanol in cell suspension experiments, 10% CO was added to the N2/CO2 headspace with no overpressure. For acetogenesis from H2 + CO2 by recombinant A. woodii, a cell concentration corresponding to 1 mg total cell protein per ml and a gas atmosphere of H2 + CO2 (80:20 [v/v]) at 1 bar overpressure were used. The suspensions were incubated at 30°C in a shaking water bath and substrate/product analyses were done as earlier mentioned. Substrate/product analyses were done from 500 µl samples withdrawn with a syringe at different time points. The concentrations of acetate or butyrate were determined by gas chromatography as described previously (Litty and Müller 2021). The peak areas were proportional to the concentration of each substance and calibrated with standard curves. A total of 5 mM isopropanol was used as the internal standard for all measurements.
Determination of acetate and butyrate kinase activities
A. woodii and E. callanderi were grown on 20 mM fructose to mid-exponential growth phase, harvested, and washed twice in buffer A (50 mM Tris/HCl, 5 mM MgCl2, pH 7.5). Cells were broken in a French pressure cell at 110 MPa and the resulting cell-free extract was freed of cells by low speed centrifugation (8 min, 8000 Upm; JA 25.50 rotor, Beckmann Coulter, Krefeld, Germany). Acetate kinase was measured by an NADH-coupled enzyme assay as described previously (Lindley et al. 1987). The assay mixture contained Tris/HCl (pH 7.5), 4 mM ATP, 1.7 mM PEP, 0.3 mM NADH, pyruvate kinase (1 U), lactate dehydrogenase (1 U), and 750 mM of acetate. The reactions were started with acetate after addition of acetate kinase-containing samples. NADH oxidation was followed at 340 nm. The same assay was used to determine butyrate kinase activity by omitting acetate and adding butyrate (750 mM) instead.
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Open Access funding enabled and organized by Projekt DEAL. The research was funded by an Advanced Grant of the European Research Council under the European Union’s Horizon 2020 Research and Innovation Program (grant agreement no. 741791).
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NPC and VM designed the research. NPC and DL performed the experiments, analyzed the data, and prepared the figures. NPC, DL, and VM wrote the manuscript.
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Chowdhury, N.P., Litty, D. & Müller, V. Biosynthesis of butyrate from methanol and carbon monoxide by recombinant Acetobacterium woodii. Int Microbiol 25, 551–560 (2022). https://doi.org/10.1007/s10123-022-00234-z
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DOI: https://doi.org/10.1007/s10123-022-00234-z