Acetogenesis can be defined as the ability to convert two molecules of CO₂ into acetyl-CoA through the WLP (Schuchmann and Müller, 2016). Although the WLP is present in methanogens, this review focuses on homoacetogens which utilize this metabolic pathway for energy conservation. In these organisms, the WLP, described in details elsewhere (Ragsdale, 2008), consists of two converging branches, the methyl and carbonyl branches (Figure 1), which provide the methyl and carbonyl groups, respectively, for acetyl-CoA formation. This pathway requires an essential enzyme, the CO dehydrogenase/acetyl-CoA synthase (CODH-ACS) to form acetyl-CoA that is needed for biomass formation. Additionally, acetyl-CoA is converted into acetate, releasing one molecule of ATP. As formate conversion in the methyl branch requires one molecule of ATP, the net gain of ATP in the WLP is zero, placing acetogenic metabolism at the thermodynamic limit of life. Under autotrophy, the WLP is vital for carbon fixation, but this pathway is also active during heterotrophic growth as it participates into energy conservation and acts as a crucial electron sink.

Energy-conserving mechanisms have evolved to regenerate cofactors and are highly species-specific, as reviewed elsewhere (Basen and Müller, 2017). Briefly, most thermophilic acetogens rely on a membrane-bound energy-converting hydrogenase (Ech) to create a proton gradient across the membrane, which is utilized for ATP synthesis by an F₁F₀ ATPase (Figure 1). This proton translocation is also coupled to the oxidation of reduced ferredoxin, provided by the electron-bifurcating hydrogenase HydABC (Wang et al., 2013). NADPH, involved in the carbonyl branch of the WLP, is provided by the electron-bifurcating transhydrogenase NfnAB, with the concomitant conversion of reduced ferredoxin and NADH. Some of these processes and their stoichiometry remain unclear in thermophilic acetogens. In addition, acetogens can utilize a variety of electron carriers, enabling them to conserve energy by metabolizing diverse substrates such as pentoses, alcohols or organic acids (Basen and Müller, 2017). This metabolic flexibility is believed to be advantageous in their natural environments, where they compete with methanogens and sulfate-reducing bacteria. Acetogenic thermophiles important for industrial biotechnology applications are described in more details in the following sections.

To date, only a few acetogenic thermophiles have been isolated (Table 1). Most of these acetogens belong to the Moorella genus, with the first species, M. thermoacetica, isolated in the 1940s (Fontaine et al., 1942). M. thermoacetica has become the model Moorella species, and was key to describing and characterizing the WLP. Additional Moorella strains have since been isolated (Jia et al., 2023), and continuous discovery of new species highlights their diversity. Notably, the taxonomy of Moorella species still remains uncertain, as previously distinct species have recently been proposed to be the same species based on sequencing data (Redl et al., 2020).

Moorella sp. grow optimally at 55–60°C (Table 1) although pH and salinity parameters differ between species. They catabolize a variety of substrates, with several species reported to utilize methanol (Santaella et al., 2023). Both M. thermoacetica and M. thermoautotrophica are capable of microbial electrosynthesis (Yu et al., 2017; Chen et al., 2018; Ha et al., 2022), converting electricity and CO₂ into high-value organic acids. This ability has, for example, been harnessed for acetate formation by M. thermoautotrophica, with the supply of electricity through metal electrodes, and further improved by embedding cells with carbon nanoparticules (Yu et al., 2017) and increasing cell permeability (Chen et al., 2018).

In addition to the energy-conserving mechanisms described above, the presence of quinones and cytochromes in Moorella sp. is unique among acetogens (Rosenbaum and Müller, 2021); however their roles remain unclear. Although a possible function as electron carriers in the electron transport chain, for example during lactate metabolism, has been proposed (Rosenbaum and Müller, 2021, 2023; Rosenbaum et al., 2021), further evidence is required to elucidate the role and importance of quinones and cytochromes for energy conservation in Moorella sp.

Among Thermoanaerobacter species, only T. kivui has been reported to fix CO₂ through the WLP as other Thermoanaerobacter sp. do not contain CODH/ACS and hydrogen-dependent carbon dioxide reductase (HDCR) enzymes, essential to the WLP (Basen and Müller, 2017). Evolutionary emergence of acetogenic capabilities in this species remains unclear. In contrast to other thermophilic acetogens, T. kivui has a fast doubling time (~2 h) under H₂ + CO₂ conditions (Weghoff and Müller, 2016) and is naturally competent (Basen et al., 2018), simplifying laboratory cultivation and DNA uptake, which makes it particularly promising for industrial applications. It has been adapted for growth on CO and syngas (Weghoff and Müller, 2016), later shown to be supported by the presence of cooS, coding for a monofunctional CO dehydrogenase and essential for growth on CO (Jain et al., 2022). Recently, it has been reported that T. kivui can utilize mannitol in a CO₂-dependent manner through expression of a mannitol-1-phosphate dehydrogenase (Moon et al., 2019, 2020).

While many unknowns remain regarding energy-conserving mechanisms and electron carriers involved in the WLP in T. kivui, genome analysis suggests that this organism relies on a proton (H⁺) gradient created by the Ech hydrogenase to drive ATP synthesis, similar to Moorella sp. (Hess et al., 2014). Electron carriers necessary for several enzymes involved in the WLP and energy conservation have been elucidated in cell-free extracts (Katsyv et al., 2021a), identifying, for example, NADP⁺-specificity of the methylene-THF dehydrogenase involved in the carbonyl branch of the WLP. However, the identity of electron carriers for other enzymes such as the electron-bifurcating hydrogenase HydABC remains unclear. In addition, the structure of the hydrogen-dependent carbon dioxide reductase (HDCR), which converts H₂ and CO₂ into formate in a high-turnover reaction, has now been elucidated with cryo-electron microscopy (Dietrich et al., 2022), providing a strong fundamental knowledge for this key enzyme. Interestingly, the formation of long HDCR filaments at the plasma membrane in T. kivui cells significantly enhanced enzymatic activity. Unsurprisingly, a Δhdcr mutant was unable to grow autotrophically without formate supplementation in the medium (Jain et al., 2020). However, this phenotype was also observed under heterotrophy, highlighting the importance of the HDCR enzyme and the WLP for both autotrophy and heterotrophy.

Although Thermacetogenium phaeum is able to produce acetyl-CoA from CO₂ with the WLP (Basen and Müller, 2017), this species exhibits poor growth in axenic cultures, reaching low maximal cell densities (Keller et al., 2019a). Instead, it preferentially grows in syntrophic cultures with the methanogen Methanothermobacter thermautotrophicus. In this syntrophic scenario, T. phaeum reverts the WLP for acetate consumption, a unique property not observed in other acetogens (Hattori et al., 2005). Genome analysis suggests that T. phaeum’s energy-conserving mechanisms and autotrophic metabolism differ significantly from other acetogens to accommodate for its bidirectional WLP (Oehler et al., 2012). In particular, ATP synthesis under both acetate formation and consumption raises several thermodynamic questions that are yet to be elucidated. A periplasmically oriented and quinone-dependent formate dehydrogenase has been proposed to allow WLP reversibility in T. phaeum (Keller et al., 2019a) although additional work is needed to elucidate energy-conserving mechanisms during both metabolic processes. Recently, the presence of pathways for methanol and ethanol degradation in T. phaeum have been proposed from proteomics and enzymatic activities, suggesting similar stoichiometries to the mesophilic acetogen Acetobacterium woodii (Keller et al., 2019b). This work also identified bacterial microcompartments involved in ethanolamine utilization in this thermophile. While progresses have been made toward understanding T. phauem’s metabolism, much more work is needed to uncover how this acetogen can revert the WLP, which has potential for industrial acetate valorization.

The new thermophilic acetogenic species Aceticella autotrophica was recently isolated from a Russian terrestrial hot spring (Frolov et al., 2023). This species is the first obligate autotroph identified among acetogens and is unable to grow under heterotrophic conditions. Comparative genomics suggested that this strict autotrophic requirement results from the loss of genes involved in carbohydrate metabolism and sugar transport. Interestingly, while the species belongs to the Thermoanaerobacterales order, it has evolved unique features contrasting to its evolutionary counterparts. It shares the most similarity with T. kivui and contains a WLP gene cluster. Much more work is needed to characterize this new acetogen.

In addition to the aforementioned bacterial species, several mesophilic archaea (Loh et al., 2020; Schöne et al., 2022) are also acetogens. Archaeoglobus fulgidus is, to date, the only thermophilic archaeon reported to grow as an acetogen (Henstra et al., 2007). This species is primarily studied for its sulfate-reducing ability and piezophilic lifestyle (Oliver et al., 2020) but has been adapted to grow on CO (Henstra et al., 2007). CO adaptation eliminated A. fulgidus’s long lag-phase and was later investigated through transcriptomics analysis upon growth on CO (Hocking et al., 2014, 2015). This work proposed a scheme for energy conservation during the acetogenic growth of A. fulgidus by a F₄₂₀H₂:quinone oxidoreductase complex. Other Archaeoglobus species were explored but could not grow as acetogens, although key genes required for acetogenesis were present. Further work is thus needed to understand why A. fulgidus has the unique ability to grow as an acetogen and better-characterize its associated mechanisms.

Native acetogenic properties are extremely valuable for climate mitigation by fixing high CO₂ concentrations in sustainable bioprocesses. However, to further expand their potential, reliable and efficient genetic tools must be developed to enable targeted strain engineering efforts that are crucial to rewire the metabolism for maximized compound bioproduction and heterologous pathway expression. Currently, genetic methods available for thermophilic acetogens are extremely limited. Successful genetic engineering has only been reported in M. thermoacetica and T. kivui although other acetogenic thermophiles have unique valuable properties.

In M. thermoacetica, genetic insertions have been performed using uracil/5-fluoroorotic acid (5-FOA) counterselection through deletion and reinsertion of pyrF (Iwasaki et al., 2013; Kita et al., 2013; Rahayu et al., 2017; Kato et al., 2021, 2024), encoding an orotodine 5′-phosphate decarboxylase. The ΔpyrF mutant becomes auxotrophic for uracil and resistant for 5-FOA, allowing transformant selection without antibiotic pressure. Reintroduction of pyrF, concurrently with the gene(s) of interest, restores uracil biosynthesis in the resulting mutant strain. This strategy has been applied to establish ethanol (Rahayu et al., 2017), acetone (Kato et al., 2021) and isopropanol (Kato et al., 2024) production in M. thermoacetica through expression of an aldehyde dehydrogenase, an acetone operon (consisting of a CoA transferase, a thiolase and an acetoacetate decarboxylase) and a secondary alchohol dehydrogenase, respectively. As these pathways branch from acetyl-CoA, disruption of acetate formation through deletion of the phosphotransacetylases PduL1 and PduL2 has been beneficial to redirect carbon flux toward compound biosynthesis (Kato et al., 2021). In addition to uracil auxotrophy, antibiotic selection by expressing a thermostable kanamycin resistant gene has also been reported (Iwasaki et al., 2013). This strategy has allowed the development of a self-replicating plasmid, harboring the pRKU1 replicon from Thermotoga maritima (Bourgade et al., 2022) and subsequently applied for ethanol production in proof-of-concept experiments. This self-replicating plasmid offers the possibility to rapidly and transiently test genetic constructs, ideal for, for example, CRISPR-Cas tools. Furthermore, to date, only one promoter, the strong constitutive promoter for glyceraldehyde-3-phosphate dehydrogenase (Kita et al., 2013) has been used for heterologous gene expression in M. thermoacetica. Thus, additional promoters are needed to expand the genetic toolbox to tailor target gene expression in this industrially important host.

In naturally competent T. kivui, a similar uracil/5-FOA counterselection technique has been adapted by deleting pyrE, encoding an orotate phosphoribosyltransferase involved in uracil biosynthesis (Basen et al., 2018). This method has primarily been used in fundamental studies to explore enzymatic functions of metabolic relevance. For example, Δhdcr, ΔcooS and ΔmtlD mutants enabled to investigate formate formation, CO metabolism and mannitol consumption, respectively in T. kivui (Moon et al., 2019; Jain et al., 2020, 2022). pyrE-mediated genetic insertions have also allowed overexpression of the native pfor1 (Katsyv et al., 2021b) and mtlD (Moon et al., 2019) genes, coding for a pyruvate:ferredoxin oxidoreductase and a mannitol-1-phosphate dehydrogenase, respectively, for protein purification from T. kivui cells. However, this method has not been reported for insertion and expression of heterologous genes yet but should allow successful pathway implementation in T. kivui in a similar manner to M. thermoacetica. A recent study successfully expressed the thermostable fluorescent reporter pFAST from a self-replicating plasmid, establishing a reporter assay for genetic part testing (Hocq et al., 2023). This tool was applied for promoter characterization to identify new strong constitutive promoters, such as the novel promoter pPta_Tkv for target gene expression in T. kivui. Interestingly, promoters from mesophilic acetogens were also functional in T. kivui, suggesting genetic part transferability. The authors isolated more stable versions of the replicon to promote plasmid propagation through adaptive laboratory evolution under antibiotic selection, significantly expanding T. kivui genetic toolbox.

Genetic tools are currently not available for other thermophilic acetogens but the development of such tools would greatly expand their industrial potential. It is worth noting that beyond microbial cell factories, thermophilic acetogens can also offer promising thermostable enzymes of industrial interest. For example, the recently characterized T. kivui HDCR (Dietrich et al., 2022) stands out as a promising biocatalyst for H₂ storage. An alternative approach to using whole-cell biocatalysis has recently been reported to store H₂ into formate (Schwarz and Müller, 2020). Additionally, the pyruvate:ferredoxin oxidoreductase can also be purified directly from T. kivui and is a promising auxiliary enzyme for enzymatic assays requiring reduced ferredoxin (Fd²⁻) difficult to provide otherwise (Katsyv et al., 2021b). Extensive research efforts on A. fulgidus have focused on characterizing various enzymes such as Argonaute (Manakova et al., 2024) or ferritin (Palombarini et al., 2021), highlighting its potential for enzymatic and therapeutic applications.

As described above, only two thermophilic acetogens have been genetically modified to date. While these efforts mark a significant step toward unlocking their potential, more complex genetic tools are needed to support extensive strain engineering of these thermophiles. Additionally, developing genetic methods for the other isolated thermophilic acetogens is crucial to accelerate their industrial potential for sustainable bioprocesses. However, several challenges remain for efficient genetic engineering of thermophilic acetogens. Successful transformation is currently hindered by multiple factors, such as difficulty of transformant selection and DNA entry into the host. Growth on plates has been reported as problematic for several acetogens (Sanford and Woolston, 2022). For instance, M. thermoacetica seems unable to grow on plates under antibiotic selective pressure (Bourgade et al., 2022). Instead, a rolling strategy in Hungate tubes has been used (Kita et al., 2013) which could result in high proportion of false transformants. In contrast, T. kivui is well adapted for growth on plates and can even tolerate brief exposure to oxygen at room temperature (Basen et al., 2018). The ability of other thermophilic acetogens to grow on plates is unknown but may restrict isolating positive transformants.

Many prokaryotes employ restriction-modification (RM) systems to protect themselves against invading foreign DNA (Vasu and Nagaraja, 2013). These systems recognize specific DNA sequences with associated methylation patterns to induce DNA cleavage; thereby, preventing foreign DNA entry into the host. Many acetogens possess these RM systems (Bourgade et al., 2021), which must be circumvented for a successful DNA entry into these hosts. For M. thermoacetica, bypassing of native RM systems has been reported by expressing three native genes encoding RM systems in an E. coli strain to protect cargo DNA prior to transformation into M. thermoacetica (Kita et al., 2013; Jia et al., 2023). This method may be applied for DNA insertion into other Moorella species or thermophilic acetogens.

Although genomic integration tends to enhance strain stability, it is often time-consuming and unsuitable for rapid construct testing or transient expression for CRISPR-based methods. Instead, self-replicating plasmids, able to propagate independently of chromosomal replication, are valuable genetic tools. However, these plasmids require compatible replicons for plasmid replication with the host’s machinery, often difficult to identify. Recently, a self-replicating shuttle vector was developed for M. thermoacetica using the pRKU1 replicon from Thermotoga maritima (Bourgade et al., 2022), which may be compatible with other closely related Moorella sp. However, additional work is needed to better understand this plasmid behavior in M. thermoacetica. T. kivui has previously been transformed with pMU131 replicon from Thermoanaerobacterium saccharolyticum (Basen et al., 2018). This replicon was later shown to be unstable at higher temperatures and subsequently improved through adapted laboratory evolution to increase its stability (Hocq et al., 2023).

In addition to replicons, genetic parts that are essential for achieving tuneable gene expression levels in thermophilic acetogens are poorly characterized. To date, only one promoter, from the glyceraldehyde-3-phosphate dehydrogenase has been used in M. thermoacetica to drive strong constitutive expression of heterologous genes (Kita et al., 2013). More promoters of varying strengths are needed to precisely control heterologous expression and metabolic output. Promoter characterization may prove difficult under thermophilic and anaerobic conditions, which render many fluorescent reporters non-functional. However, a reporter assay was recently developed for T. kivui with the O₂-independent FAST system, allowing promoter characterization at high temperatures (Hocq et al., 2023) and is possibly compatible with other thermophilic acetogens. Ribosome-binding sites have yet to be characterized in these organisms but would be useful for achieving predictable translation levels.

Thermophily, while advantageous for metabolic efficiency and industrial applications, complicates genetic engineering work by limiting the pool of candidate enzymes that are functional at high temperatures. A thermostable kanR gene from Streptococcus faecalis has allowed kanamycin selection in M. thermoacetica (Iwasaki et al., 2013). Similarly, a thermostable acetone operon was engineered for M. thermoacetica by selecting candidate enzymes from other thermophilic prokaryotes (Kato et al., 2021). As exemplified by FAST (Hocq et al., 2023), exploring enzyme thermostability is crucial when working with thermophilic acetogens.

Finally, acetogenic metabolism is highly constrained by energy limitations and cofactor availability. These constraints significantly limit metabolic engineering possibilities, preventing expression of ATP-demanding pathways in these hosts. In particular, most pathways successfully implemented in acetogens stem from acetyl-CoA, therefore competing with ATP-yielding acetate formation. However, increasing target compound biosynthesis by abolishing acetate formation poses a problem for ATP synthesis during autotrophy and is often unviable for the host. For example, a M. thermoacetica Δpdul1Δpdul2::aldh strain, producing ethanol instead of acetate, was unable to grow autotrophically on H₂:CO₂ (Takemura et al., 2021). Instead, ethanol was produced autotrophically with Δpdul2::aldh strain under CO supplementation, allowing ATP synthesis by decreased acetate formation using alternative routes. Another engineered M. thermoacetica strain required an additional electron acceptor to produce acetone autotrophically (Takemura et al., 2023). This work identified dimethyl sulfoxide as the strongest electron acceptor by enhancing ATP synthesis under H₂:CO₂ conditions.