Due to the high toxicity of formaldehyde, its metabolism requires sensitive and fine-tuned regulation systems. Particularly, in methylotrophic microorganisms, formaldehyde metabolism is often regulated through multilevel cascade systems. For example, in the methylotrophic P. denitrificans, the formaldehyde metabolism is controlled by a two-component system consisting of FlhR and FlhS [163,164]. The FlhRS system regulates the expression of enzymes that are either involved in production of formaldehyde (Mdh and methylamine dehydrogenase (Madh) or its consumption (GD-Faldh, Fgh) [40,163,164,165]. The FlhRS system is activated, and its expression is induced by the presence of formaldehyde and by the depletion of heterotrophic substrates [163,164]. This regulatory system consists of a signal (FlhS) and a response regulator (FlhR) that binds to DNA [164]. FlhS is a histidine kinase that, when activated through the binding of effector molecules, catalyzes the phosphorylation of FlhR [164]. As soon as FlhR is activated through phosphorylation, it binds to target promoters of C1 metabolism gene clusters, resulting in the expression of genes encoding Fgh and GD-Faldh [163,164]. The upregulation of the expression of these genes takes place during growth on methanol, while they are constitutively expressed at basal levels also during heterotrophic cultivation [164]. Consequently, GD-Faldh and Fgh are synthesized, and thus formaldehyde is converted to CO₂, which prevents the accumulation of its toxic concentrations [164]. This means that initially, only the formaldehyde-consuming enzymes are synthesized. When formaldehyde concentration increases, the activated FlhR binds to promoters of the operons mxa (encoding Mdh) and mau (encoding Madh), which, however, does not directly lead to their activation [163,164]. The expression of mxa and mau is induced by binding the additional regulator MxaX or MauR for expression of mxa and mau, respectively [164]. The regulatory systems derived from P. denitrificans were used to create synthetic regulators that can potentially be applied for dynamic gene expression control in synthetic methylotrophs. For example, the sensoring kinase domains of MxaY or FlhS derived from P. denitrificans were fused with the cytoplasmic catalytic domain of the osmosensor histidine kinase EnvZ from E. coli in order to create a chimeric sensor histidine kinase that responds to the presence of methanol in the environment [163,166]. A similar approach was used to create a methanol sensor by combining the sensing domain of MxcQ derived from M. organophilum XX or M. extorquens AM1, or M. extorquens AM1-derived MxbD with the transmitter domain of EnvZ from E. coli [167,168].

Besides methanol-sensing regulators, M. extorquens AM1 possesses a unique, recently discovered formaldehyde sensor enhanced formaldehyde growth protein A (EfgA), which contributes to formaldehyde detoxification not through enzymatic oxidation but relying on binding with this compound [169]. In response to the transient increase of intracellular formaldehyde concentration, EfgA leads to a rapid halt of protein translation and arrest of cell growth which could limit formaldehyde-induced protein damage [169]. Moreover, the changes in the translation are potentially linked to the global transcriptional response to formaldehyde stress mediated by EfgA [170]. Transcriptional response targets biosynthesis of free formaldehyde, contributes to increased formaldehyde consumption by Fae, mitigates proteotoxicity and genotoxicity, and is reversed when formaldehyde concentration decreases [170]. Expression of efgA is regulated by TtmR, a formaldehyde-responsive MarR family transcription factor, and both EfgA and TtmR are required for the optimal transition from multicarbon to C1 growth [171]. It was shown that heterologous expression of M. extorquens AM1-derived efgA in E. coli increases its formaldehyde resistance, indicating the potential application of EfgA in strategies to increase formaldehyde tolerance during the engineering of synthetic methylotrophy [169].

Methylotrophic growth activates formaldehyde assimilation and dissimilation pathways which should actively control intracellular formaldehyde concentrations. In the facultative methylotroph B. methanolicus MGA3 some of the genes involved in the RuMP formaldehyde assimilation cycle are upregulated during growth on methanol versus heterotrophic conditions. However, details of the expression regulation are not elucidated, and the regulator is not yet known. B. methanolicus MGA3 cells grown on methanol are more sensitive to formaldehyde than MGA3 cells grown on a nonmethylotrophic substrate [172]. The increased formaldehyde sensitivity during methylotrophic growth might be caused by the already high formaldehyde concentration in the cells grown in methanol and thus the saturation of formaldehyde assimilation and dissimilation pathways in this bacterium. For that reason, supplementation with external formaldehyde can lead to increased formaldehyde toxicity [172]. One of the regulators present in B. methanolicus MGA3 is HxlR, which controls the expression of chromosomal genes hps and phi, upregulated by formaldehyde [133]. It was shown that the introduction of additional copies of hps and phi through plasmid-based overexpression increases the tolerance of B. methanolicus MGA3 cells to high methanol concentrations confirming the central role of that operon in C1 metabolism [133].

The structure of the formaldehyde-responsive transcription factor HxlR was also studied in the nonmethylotroph B. subtilis. Similarly to TtmR, HxlR belongs to the MarR family of transcription factors, and it controls the expression of hxlAB in B. subtilis [86,173]. HxlR recognizes formaldehyde through a protein intra-helical cysteine-lysine cross-linking reaction at its N-terminal α1 helix, which in turn leads to a conformational change and transcriptional activation [173]. The resulting intrahelical methylene bridge is a protein modification with a conformational change that allosterically induces transcriptional activation of HxlR [173]. Another regulator responsible for controlling the expression of genes involved in formaldehyde metabolism in B. subtills is AdhR. AdhR regulates the expression of the BSH-dependent formaldehyde dissimilation pathway in B. subtilis and belongs to an NmlR clade within the family of MerR repressor-activators [19]. MerR regulators are sensitive to a wide range of molecules such as soft transition metal ions, the superoxide anion, and drug-like compounds, whereas the members of the NmlR clade respond to oxidative and carbonyl stressors [81,174,175,176]. Similar to other formaldehyde sensors, a cysteine residue is conserved within the NmlR clade; for example, Cys52 is conserved in AdhR from B. subtilis [81]. Replacement of this residue with alanine leads to the creation of a strain where adhA (adhC) is not transcribed in a formaldehyde rich environment [81].

Regulation of expression of the formaldehyde metabolic pathway in C. glutamicum is not well characterized. The expression pattern of the gene encoding NAD-linked MSH-dependent formaldehyde dehydrogenase is not known, and neither is its regulation [91]. Expression of ald encoding acetaldehyde dehydrogenase that catalyzes the oxidation of formaldehyde depends on the carbon source used for the cultivation of C. glutamicum. The activity of Ald increases about 10-fold when ethanol is a carbon source as compared to growth with glucose or mixtures of glucose with ethanol [91,177]. This process is regulated by RamA and RamB and putatively by GlxR [91,177]. Thus, due to differential gene expression, the importance of Ald and FadH might vary depending on the physiological conditions [91]. Methanol catabolism is subject to carbon catabolite repression in the presence of glucose and is dependent on the transcriptional regulator RamA, which was previously shown to be essential for the expression of adhA and ald [90].

A well-characterized system for the control of formaldehyde metabolism is present in heterotrophic E. coli. In this bacterium, yeiG encoding Fgh is constitutively expressed, while the expression of Fgh-encoding frmB, which belongs to the frmRAB operon, increases by 20- to 100-fold over basal levels in the presence of formaldehyde in the environment [98,99]. FrmR is a member of the CsoR/RcnR family of metal ion-sensing transcriptional repressors, which is responsible for controlling the frmRAB operon [178,179,180]. In the absence of formaldehyde, FrmR binds to the promoter of the frmRAB operon (P_frm), while in its presence FrmR changes its conformation, which leads to the dissociation of the P_frm-FrmR complex [180]. The change of protein conformation is caused by the formation of methylene bridges that link adjacent proline (Pro2) and cysteine (Cys35) residues in the FrmR tetramer [180]. The allosteric mechanism of FrmR is triggered directly by formaldehyde in vitro [180]. Sensitivity to formaldehyde requires a cysteine (Cys35 in FrmR) conserved in all DUF156 proteins [181].

As highlighted by the limited success in a full transfer of methylotrophy, and the importance of fine-tuning the C1 metabolic landscape, an important approach to synthetic methylotrophy is the implementation of dynamic formaldehyde regulation mechanisms. The formaldehyde-inducible promoter P_frm was engineered to obtain variants differing in their basal and induced expression levels [182]. A variant of the formaldehyde-responsive promoter characterized with higher basal and induced expression levels compared with P_frm was used for the control of mdh and hxlAB in a ΔfrmAΔpgi E. coli genetic background, which led to improved biomass yield in comparison to the strain where the native E. coli P_frm was used [182]. Furthermore, using the formaldehyde-inducible promoter P_frm to drive direct regulation of rpe and tkt genes involved in the regeneration of Ru5P led to significantly improved methanol assimilation into intracellular metabolites in E. coli [118]. Global gene regulation is an additional factor that should be considered in establishing synthetic methylotrophy. The use of the non-native substrate methanol for growth likely triggers the response characteristic for nutrient-limiting conditions in E. coli [105]. Such response is characterized by diverting resources away from active growth and division in favour of maintenance and stress resistance leading to inhibition of RNA synthesis [105]. This leads to decreased translation and conservation of amino acids concurrent with the upregulation of many amino acid biosynthetic genes [105]. It was shown that the activation of stringent response via overproduction of guanosine tetraphosphate (ppGpp) or enzymes involved in its biosynthesis (RNA polymerase-binding transcription factor DksA and the stress response sigma factor RpoS) enhances methanol utilization in synthetic methylotroph E. coli by enabling the biosynthesis of several limiting amino acids using carbon derived from methanol in comparison to the control strain where such amino acids cannot be synthesized [105].

Altogether, a comprehensive understanding of formaldehyde metabolism and its regulation in native methylotrophs is an invaluable asset in designing strategies for its introduction into nonmethylotrophic species. The hitherto research showcases the importance of finding a balance between oxidation of C1 substrates to formaldehyde, endogenous formaldehyde dissimilation, and introduction of synthetic formaldehyde assimilation pathways in order to properly regulate carbon flux towards assimilation and maintain formaldehyde below toxic levels. As exemplified in this review, formaldehyde dissimilation pathways and their regulation seem to be relatively conserved among different bacterial species regardless of their trophic lifestyle, which means that native pathways on nonmethylotrophs can potentially be used in the engineering efforts for synthetic methylotrophy.