Depending on the electron acceptors, Mdhs in methylotrophs are classified into three groups: NAD⁺-dependent Mdh, PQQ (pyrrolo-quinoline quinone)-dependent Mdh, and O₂-dependent AOX (alcohol oxidase) (Figure 2).

NAD⁺-dependent Mdh in thermophilic Gram-positive methylotrophs uses NAD⁺ as the cofactor for the methanol oxidation. The first NAD⁺-dependent Mdh was reported in 1989 (Arfman et al., 1989). NAD⁺-dependent Mdhs also obtained from non-methylotrophic bacteria. To date, several NAD⁺-dependent Mdhs have been isolated from Bacillus sp. (such as B. methanolicus (Arfman et al., 1989; Arfman et al., 1991; Müller et al., 2015; Witthoff et al., 2015; Price et al., 2016) and B. stearothermophilus (Whitaker et al., 2017)), Lysinibacillus sp. (such as L. xylanilyticus (Lee et al., 2020)), and Cupriavidus sp. (such as C. necator (Wu et al., 2016)). In particular, their NAD⁺-dependent Mdhs have been focused and reported for studies of recombinant E. coli as synthetic methylotrophs (Müller et al., 2015; Wu et al., 2016; Whitaker et al., 2017; Lee et al., 2020; Le et al., 2021). Three NAD⁺-dependent Mdhs have been found in B. methanolicus MGA3 (Mdh, Mdh2, and Mdh3). Moreover, the activity of all three Mdhs is modulated by an endogenous Mdh activator protein (ACT). In vitro studies suggest that ACT enhances the methanol affinity, oxidation rate, and catalytic activity of Mdhs; however, the detailed mechanism for activation is currently unclear (Hektor et al., 2002; Witthoff et al., 2015) and no effect has been shown in vivo in a synthetic methylotrophy (Müller et al., 2015) because detail research for the activator protein functions in native host has not been tested. To enable the assimilation of methanol as the carbon source in metabolic engineering, ACT-independent Mdhs and their mutants from C. necator (Wu et al., 2016; Chen et al., 2018) and L. xylanilyticus (Lee et al., 2020; Le et al., 2021) have been reported and introduced into E. coli for methanol assimilation. As best candidate for synthetic methylotrophy, NAD⁺-dependent Mdh that can perform its function under both aerobic and anaerobic conditions (Zhang et al., 2017). Besides, it uses NAD⁺, which is ubiquitous and can provide electrons for metabolite products, as the cofactor. Therefore, it may be the best candidate for recombinant-based synthetic methylotrophs (Zhang et al., 2017).

In Gram-negative methylotrophs, the oxidation of methanol occurs in the periplasmic space by PQQ-dependent Mdh (Skovran et al., 2019). Pure PQQ-dependent Mdh was first described in 1967 (Anthony and Zatman 1967). To date, PQQ-dependent Mdh has been isolated and purified from several different strains of microorganisms including Pseudomonas sp. (Anthony and Zatman 1965; Anthony and Zatman 1967; Patel et al., 1972), Methylococcus capsulatus (Patel et al., 1972), Hyphomicrobium denitrificans (Nojiri et al., 2006), Methylorubrum extorquens (formerly Methylobacterium extorquens) (Anthony 2004; Liu et al., 2006; Nakagawa et al., 2012), Methyloversatilis universalis FAM5 (Kalyuzhnaya et al., 2008), Methylibium petroleiphilum (Kalyuzhnaya et al., 2008), Methylophaga aminisulfidivorans (Kim et al., 2012; Cao et al., 2018), Methylobacterium nodulans (Kuznetsova et al., 2012), Methylophilus sp. (Leopoldini et al., 2007; Li et al., 2011), Burkholderiales sp. (Kalyuzhnaya et al., 2008), Paracoccus denitrificans (Xia et al., 2003), M. radiotolerans (Hibi et al., 2011), Bradyrhizobium sp. (Fitriyanto et al., 2011), M. aquaticum (Masuda et al., 2018), Methylomicrobium buryatense (Deng et al., 2018), M. fumariolicum (Jahn et al., 2018), and Bradyrhizobium diazoefficiens (Wang et al., 2019). The PQQ-dependent Mdh contains a PQQ prosthetic group. The chemical structure of the PQQ prosthetic group has been confirmed by two independent research groups using a wide range of chemical and physical techniques, such as X-ray, UV/Vis absorption spectra, and HPLC (Anthony 1982). The role of the PQQ prosthetic group is capturing electrons from methanol oxidation and passing them to the cytochrome (Anthony 2004). The biggest disadvantage is the requirement of molecular oxygen for PQQ bio-synthesis (Velterop et al., 1995), while some desired intermediates as precursors of value-added products such as lactate must be produced under anaerobic conditions. Therefore, this limits the application of PQQ-dependent Mdhs.

In genomes of methylotrophs, PQQ-dependent Mdhs are generally encoded by MxaFI and XoxF. MxaFI consists of small (MxaI) and large (MxaF) subunits, encoding PQQ-dependent Mdh using calcium (Ca²⁺) as a cofactor (MxaFI-Mdh) (Anthony 2004). Another PQQ-dependent Mdh, which uses lanthanides (Ln³⁺) instead of Ca²⁺, is encoded by XoxF (XoxF-type Mdh) (Chistoserdova 2016; Skovran et al., 2019). XoxF-Mdh from M. extorquens AM1 is a representative of Ln³⁺-dependent Mdh that it was studied carefully to show the biochemical characterization. XoxF of M. extorquens AM1 showed better activity when La³⁺ or Ca²⁺ and La³⁺ were added together than when Ca²⁺ was used alone as part of the cofactor complex (Vu et al., 2016; Good et al., 2020). In addition, other elements of lanthanide (Ce³⁺, Nd³⁺, Pr³⁺, Sm³⁺, Eu³⁺, or Gd³⁺) were also found to be involved in the methanol oxidation activity (Pol et al., 2014). Lanthanides as important factor was suggested in regulatory and catalytic functions because the XoxF genes are required for transcription of the MxaFI (Vu et al., 2016).

Unlike NAD⁺-dependent and PQQ-dependent Mdhs, O₂-dependent AOX is obtained from eukaryotic methylotrophs and is located in the peroxisome of yeasts (Egli et al., 1980). First, formaldehyde and hydrogen peroxide (H₂O₂), which are highly toxic chemicals for cells, are created from methanol oxidation by O₂-dependent AOX. To protect the cells, dihydroxyacetone synthase (DAS) and catalase (CTA) work to transform them into non-toxic chemicals (Zhang et al., 2017). O₂-dependent AOX only function under aerobic conditions and thus, has limitations similar to those of PQQ-dependent Mdh. In addition, another important limitation AOX’s is that the electrons from methanol are not captured as useable energy by the cell, but wasted in the generation of peroxide.

The most important factor, which has a considerable effect on the activity of Mdhs, is cofactor binding. For NAD⁺-dependent Mdhs, a metal ion is involved in cofactor binding which may influence enzymatic activity (Hektor et al., 2002). Several metal ions have been examined for the effects on the methanol oxidation activity of Mdhs, such as Fe²⁺, Mn²⁺, Zn²⁺, Cu²⁺, Co²⁺, Ni²⁺, or Mg²⁺ ions (Sridhara et al., 1969; Arfman et al., 1991; Montella et al., 2005; Müller et al., 2015; Wu et al., 2016; Whitaker et al., 2017; Lee et al., 2020; Le et al., 2021). In general, the supplementation of Fe²⁺ or Mn²⁺ ions increase enzyme activity, and Mdh activity is inhibited by Cu²⁺, Co²⁺, or Zn²⁺ (Sridhara et al., 1969; Montella et al., 2005). In the case of Mdhs from Lysinibacillus xylanilyticus (Lxmdh), Mn²⁺ or Fe²⁺ reduce its activity, whereas and Zn²⁺, Cu²⁺, or Co²⁺ inhibit Lxmdh activity (Lee et al., 2020). For almost all NAD⁺-dependent Mdhs, Mg²⁺ increases the effect of enzyme activity (Arfman et al., 1989; Arfman et al., 1991; Müller et al., 2015; Whitaker et al., 2017; Lee et al., 2020; Le et al., 2021). For Mdhs from Cupriavidus necator (Cnmdh), Ni²⁺ is typically the chosen cofactor (Wu et al., 2016).

For PQQ-dependent Mdhs, Ca²⁺ plays a role in the active site (Anthony and Zatman 1967; Anthony 2004). The X-ray structure of Mdhs from M. extorquens, M. nodulans, Methylophilus sp, and P. denitrificans has been determined to have one molecule of PQQ and one Ca²⁺ ion in each large α-subunit, which is encoded by MxaF (Anthony and Williams 2003; Anthony 2004). Moreover, some types of Mdhs, which are encoded by XoxF, use Ln³⁺ instead of Ca²⁺, which is a part of cofactor complex for Mdhs encoded by MxaF (Egli et al., 1980; Skovran et al., 2019). Ln³⁺ was first suggested as a metal ion of the cofactor complex for PQQ-dependent Mdhs obtained from M. radiotolerans (Hibi et al., 2011) and Bradyrhizobium sp. (Fitriyanto et al., 2011) in 2011. Furthermore, Mdhs from M. extorquens AM1 (Nakagawa et al., 2012), M. aquaticum (Masuda et al., 2018), M. buryatense (Deng et al., 2018), M. fumariolicum (Jahn et al., 2018), and B. diazoefficiens (Wang et al., 2019) have been observed to be Ln³⁺-dependent Mdhs. Interestingly, the subunits of PQQ-dependent Mdh from M. aminisulfidivorans MP^T are coordinated by an Mg²⁺ ion instead of a Ca²⁺ ion or Ln³⁺ group (Cao et al., 2018). In addition, the activity of PQQ-dependent Mdhs under aerobic conditions with artificial electron acceptors in vitro requires the presence of an activator (e.g., ammonium salt) (Anthony 1982; Anthony and Williams 2003; Anthony 2004; Kuznetsova et al., 2012).

The other most important factors are the temperature and pH of the buffer in the enzyme assay. Almost all methanol dehydrogenases have high activity at high temperatures (∼55°C) and high pH (9–10). Mdhs from M. nodulans (Mnmdh) exhibits maximal activity at pH 9–10, and it increases linearly with increasing temperature from 20°C to 50°C (Kuznetsova et al., 2012). The optimum pH for Mdh from Pseudomonas sp. M27 (Patel et al., 1972), M. capsulatus (Patel et al., 1972), and M. extorquens AM1 (Liu et al., 2006) are also 9. Similarly, an assay involving NAD⁺-dependent Mdhs from thermotolerant methylotrophic Bacillus strains is performed at 45–50°C, using glycine/KOH buffer at pH 9.5 (Arfman et al., 1991; Krog et al., 2013). Lxmdh and its mutant or Cnmdh also function better in buffers with a pH of 9.5; however, Lxmdh and its mutant exhibit high activity at 55°C (Lee et al., 2020; Le et al., 2021), while the temperature for testing Cnmdh activity is 30°C (Wu et al., 2016). On the other hand, the conditions for the Mdhs from M. methanolicus (Bmmdh) and B. stearothermophilus (Bsmdh) reactions are similar, at pH 7.4 and 37°C (Müller et al., 2015; Whitaker et al., 2017). When examining the activity of Mdhs obtained from L. xylanilyticus or Burkholderiales using spectrometer experiments to detect the changes in absorbance, room temperature is preferred (Kalyuzhnaya et al., 2008; Le et al., 2021). Moreover, buffer systems with a pH of 8.8 are used for Burkholderiales Mdh assays (Kalyuzhnaya et al., 2008). On the whole, the Mdh assay requires the presence of an ion as the binding cofactor. This depends on the type and source of Mdh. For PQQ-dependent Mdhs, the activator for enzyme activity is required under aerobic conditions.

The molecular weight of most PQQ-dependent Mdhs has been identified as being between 112 and 158 kDa. The associated form of almost all PQQ-dependent Mdhs, which are Ca²⁺-dependent Mdhs, is a tetramer (α₂β₂). Therefore, it can be dissociated to α-subunits (56–76 kDa) and β-subunits (very small, ≤ 10 kDa) by a low pH or sodium dodecyl sulfate (SDS) (Anthony 1982), such as the Mdh from H. denitrificans (α: 65 kDa, β: 9 kDa) (Nojiri et al., 2006), M. extorquens (α: 62–65 kDa, β: 7.5–8.5 kDa) (Anthony 2004; Liu et al., 2006), M. aminisulfidivorans MP^T (α: 65–66 kDa, β: 7.5–7.6 kDa) (Kim et al., 2012; Cao et al., 2018), M. methylotrophus (α: 62 kDa, β: 8 kDa) (Leopoldini et al., 2007; Li et al., 2011), and M. radiotolerans (α: 60 kDa, β: 10 kDa) (Hibi et al., 2011). There are also some special cases with the heterodimer form (αβ), for example, Mdh from M. nodulans (α: 60 kDa, β: 6.5 kDa) (Kuznetsova et al., 2012). Besides, the associated form of La³⁺-dependent Mdhs is a homodimer (formed by two identical proteins), e. g, Mdhs from M. radiotolerans (120 kDa) (Hibi et al., 2011), Bradyrhizobium sp. (108 kDa) (Fitriyanto et al., 2011), M. extorquens AM1 (117 kDa) (Nakagawa et al., 2012), Methylacidiphilum fumariolicum SolV (63.6 kDa) (Jahn et al., 2018), and M. buryatense (Deng et al., 2018). On the other hand, the NAD⁺-dependent Mdh with a single subunit has a molecular weight of around 40 kDa. For instance, the molecular weight of NAD⁺-dependent Mdh from Bacillus sp. C1 (a thermotolerant methylotrophic Bacillus) is 43 kDa (Arfman et al., 1989; Arfman et al., 1991). Other B. methanolicus strains (MGA3 and PB1) show a similar molecular weight at 43 kDa (Müller et al., 2015; Witthoff et al., 2015; Price et al., 2016). Moreover, Cnmdh from C. necator N-1 (Wu et al., 2016) or Lxmdh from L. xylanilyticus (Lee et al., 2020) show respective molecular subunits at 40.7 or 42.8 kDa (Table 1). According to the previous report, NAD⁺-dependent Mdhs has decameric association structure (430 kDa) as native form (Vonck et al., 1991).

Although, MxaFI-Mdhs from M. extorquens AM1, with a high efficiency (k _cat/K _M) of methanol production, has been suggested as the best choice for engineering E. coli (Anthony and Williams 2003), it requires at least 11 gene products for its functional assembly (Chistoserdova et al., 2003). In addition, XoxF-Mdhs from M. extorquens AM1 would be required only three genes with a high catalytic efficiency (Keltjens et al., 2014), PQQ-dependent Mdhs are not suitable for synthetic methylotrophy using engineered E. coli. Because, PQQ as critical cofactor is critical limit that specially E. coli is not able to synthesize PQQ (Anthony 2004). In the case of O₂-dependent AOX, its product, H₂O₂, is also challenging because it is the highly toxic to most hosts. Therefore, only NAD⁺-dependent Mdh has been considered as the best candidate for synthetic methylotrophs (Zhang et al., 2017), which requires only one gene for functional production and can generate the reducing equivalent (NADH) to promote strain growth under both aerobic and anaerobic conditions. To successfully achieve methanol assimilation, the Mdh kinetics, including substrate affinity and catalytic activity, should be improved for methanol assimilation through directed evolution or rational approach based engineering. Various NAD⁺-dependent Mdhs from B. methanolicus (Vonck et al., 1991; De Vries et al., 1992; Hektor et al., 2002; Krog et al., 2013; Ochsner et al., 2014; Müller et al., 2015; Witthoff et al., 2015), C. necator (Wu et al., 2016), B. stearothermophilus (Whitaker et al., 2017), L. xylanilyticus (Lee et al., 2020) were reported in methanol conversion. Researchers are searching for NAD⁺-dependent Mdhs with higher activity and lower K _M from different microorganisms and improving their characteristics by a rational approach and directed evolution (Hektor et al., 2002; Ochsner et al., 2014; Roth et al., 2019; Lee et al., 2020; Le et al., 2021) (Table 2). Specially, the improvement of substrate affinity toward low concentration methanol is focused in the development of Mdh-driven synthetic methylotrophy because of the high toxicity of methanol for E. coli (Dyrda et al., 2019).

NAD⁺-dependent Mdhs from B. methanolicus that has been studied a lot (Vonck et al., 1991; De Vries et al., 1992; Hektor et al., 2002; Krog et al., 2013; Ochsner et al., 2014; Müller et al., 2015; Witthoff et al., 2015). They support cell growth and methanol uptake with high speed in native B. methanolicus. However, the catalytic activity of Mdhs from B. methanolicus in vitro and in vivo are limited because of the unclear mechanism of ACT (Hektor et al., 2002; Witthoff et al., 2015), even though ACT significantly improve the K _M value of Bmmdh (reduced from 1.8- to 14.0-fold) (Krog et al., 2013; Ochsner et al., 2014). Second, an ACT-independent Mdh from C. necator was developed and characterized for the kinetics and substrate specificity on 2016 (Wu et al., 2016). It showed the low affinity to methanol (132 mM for K _M) compared to that of Mdhs from B. methanolicus (170–360 mM for K _M) (Krog et al., 2013; Ochsner et al., 2014; Müller et al., 2015; Wu et al., 2016). Another study showed an Mdh from B. stearothermophilus, which shares 21–23% amino acid identity with the Mdh from B. methanolicus (Whitaker et al., 2017). The affinity of Mdh from B. stearothermophilus showed a lower value than that from Bmmdh and Cnmdh (20 mM for K _M), thus, it had superior performance in vivo than previously published Mdhs. In particular, Lee et al. found an Mdh from L. xylanilyticus, that had higher substrate specificity towards methanol than Bmmdh, Cnmdh and Bsmdh (Lee et al., 2020). In addition, it is also an ACT-independent Mdh with an impressively low affinity (3.23 mM for K _M).

To improve the activity of Mdhs, site-directed (Hektor et al., 2002; Ochsner et al., 2014), site-saturation (Wu et al., 2016) or random mutagenesis (Le et al., 2021) is used for creating Mdh mutants. In 2002, Hektor et al. used site-directed mutagenesis to confirm the role of various amino acid residues in the NAD(H) binding site in Mdh from B. methanolicus C1 (Hektor et al., 2002). All mutants are impaired in cofactor NAD(H) binding, though, some mutants (G95A, S97G, and S97T) retained Mdh activity. Finally, only the S97G mutant displayed as “fully activated” in Mdh reaction rates. Another study from Ochsner et al. investigated the effect of site-directed mutations in the predicted active site of Mdh from B. methanolicus MGA3 (Ochsner et al., 2014). The V_max of Bmmdh S98G increased two-fold compared with that of its wild-type (WT), yet its K _M value also increased in the absence of ACT. Even upon adding ACT, the catalytic efficiency of Bmmdh S98G was similar to that of WT (a doubling of V_max with a slight reduction in K _M). Meanwhile, Bmmdh2 S101G lost the activity on methanol. For Mdh from C. necator, the site-saturation mutagenesis on the Mdh2 A169 site was constructed (Wu et al., 2016). In the first round of screening, eight possible positive variants with over 50% activity improvement (based on the Nash reaction) were selected from 2000 screened variants, and, finally, CT1-2 was used as the template for another error-prone PCR library in the second round of screening. Afterward, CT4-1, the recombinant of three mutations (A169V, A31V and A26V), which showed a low K _M (21.6 mM) and an unchanged k _cat (0.2 s⁻¹) compared with WT Mdh2, was created by various rounds of high throughput screening (HTS). For studying the activity of Mdh from L. xylanilyticus, eight residues within 4.5 Å of the center of the docked substrate were selected to contribute toward site-directed mutagenesis (Lee et al., 2020). Finally, the mutations S101V (K _M = 10.35), T141S (K _M = 51.24) and A164F (K _M = 36.83) improved the enzyme’s specific activity towards methanol compared to that of the Lxmdh WT. Furthermore, a random mutant library of L. xylanilyticus Mdh was constructed and high throughput screened by an formaldehyde detectible biosensor (Le et al., 2021). As a result, several mutants were characterized by high catalytic efficiency and low K _M compared with Lxmdh WT and its published mutants. Thus, mutant Lxmdh E396V, which has the highest catalytic efficiency (79-fold that of WT catalytic efficiency) and an impressive K _M value (0.01 mM), was found. Moreover, the K _M value of another Lxmdh mutant, K318N, was also impressive (0.046 mM). Nevertheless, the recombinant of two mutations (E396V and K318N) had a higher K _M value compared with each mutant (0.233 mM).

Many alcohol dehydrogenases (ADHs), which can catalyze methanol oxidation, may be treated as Mdhs. Although, the catalytic efficiency of methanol oxidation by ADHs is low, it is another good candidate for synthetic methylotrophy. As an example, the AdhA from Corynebacterium glutamicum R has shown a low K _M value of methanol activity (97 mM) compared with Bmmdh and Cnmdh (Kotrbova-Kozak et al., 2007; Wu et al., 2016). A number of ADH enzymes has been tested for the methanol oxidation activity without kinetic values, such as ADHs from Lysinibacillus sphaericus, Lysinibacillus fusiformis, Bacillus coagulans and Desulfitobacterium hafniense (Müller et al., 2015).

Furthermore, critically, improving methanol oxidation rates by kinetically improved Mdh variants would only be enabled in cells where there is sufficiently fast of formaldehyde assimilation (Woolston et al., 2018). This is important for the development of Mdh-directed evolution approaches. This is covered in the synthetic methylotrophy section of this review.