During the methanol assimilation via the serine pathway, one glycine molecule reacts with formaldehyde (derived from methanol) to produce L-serine. This very step was utilized for the production of L-serine with glycine as a co-substrate. Using resting cells to prevent glycine toxicity and the freeze-thawing method for facilitating substrate permeability up to 54.5 g/L L-serine was produced (Sirirote et al., 1986). Violacein, a secondary metabolite derived from the shikimate pathway, has also been demonstrated for production from methanol. Overexpression of the violacein biosynthesis pathway genes encoded by the vio operon, consisting of five genes (vioA, vioB, vioC, vioD, and vioE) caused 11.7 mg/L of violacein production from M. extorquens AM1. Subsequently, through directed evolution of the host (for overcoming unidentified bottlenecks) and substrate co-utilization strategy (for increasing supply of reducing power equivalent), a final violacein production titer of 118 mg/L was achieved from methanol and acetate (Quynh Le et al., 2022). Even though the production titer was reportedly lower than that of established E. coli, C. glutamicum, or Yarrowia lipolytica strains (Fang et al., 2015; Sun et al., 2016; Tong et al., 2021), the estimated production cost in terms of substrate and supplement showed substantially lower values with the methylotrophic host.

Methanol dependent growth of M. extorquens AM1 is associated with a significant metabolic flux passing through the serine and EMC cycle, generating a steady supply of acetyl CoA. With this acetyl CoA as a precursor, 3-hydroxy propionic acid (3HP) production was attempted. 3HP, a precursor for acrylic acid, and 1,3-propanediol, which could be polymerized to form biodegradable polymers, was targeted for production in M. extorquens AM1 through the overexpression of malonyl-CoA reductase (mcr) gene of Chloroflexus aurantiacus DSM 635 encoding a bifunctional enzyme with alcohol dehydrogenase and aldehyde dehydrogenase activities. Using an adaptively evolved strain of M. extorquens AM1 for a faster growth rate (Van Dien and Lidstrom, 2002), a 3-HP titer of 69.8 mg/L was obtained (Yang et al., 2017). To further increase the production titer, increasing the enzymatic activity of Mcr (especially the aldehyde dehydrogenase counterpart of the bifunctional enzyme) along with a sufficient supply of the malonyl-CoA precursor (namely acetyl-CoA) was suggested (Liu et al., 2016). Accordingly, a synergistic methanol assimilation pathway designed by incorporating the RuMP cycle increased the acetyl CoA supply. Expressing B. methanolicus derived hps and phi genes for completing the RuMP cycle and pfk for flux distribution from F6P, the engineered strain showed an increased cell growth and methanol consumption rate. Using this newly engineered chassis of M. extorquens AM1, 3-HP production was increased up to 0.857 g/L in the fed-batch bioreactor condition (Yuan et al., 2021).

2-Hydroxyisobutyric acid (2-HIBA), a branched C4-hydroxycarboxylic acid, is a precursor of methyl methacrylate, the key intermediate for poly-methyl methacrylate, also known as an alternative glass. An EMC pathway intermediate, 3-hydroxybutyryl-CoA, was employed for the production of 2-HIBA. The coenzyme B12-dependent (R)-3-hydroxybutyryl-CoA mutases (RcmAB) of Bacillus massiliosenegalensis JC6 that catalyze the interconversion of (R)-3-hydroxybutyryl-CoA to (R)-2-hydroxyisobutyryl-CoA were overexpressed in M. extorquens AM1. By utilizing the poly-3-hydroxybutyrate (PHB) overflow metabolism, the engineered strain was able to produce 2-HIBA to about 2.1 g/L along with PHB as a byproduct, with a maximal combined yield (2-HIBA and PHB) of 0.11 g/g methanol (Rohde et al., 2017).

Although it was a low titer, itaconic acid production has also been demonstrated in M. extorquens AM1. Cis-aconitic acid an intermediate of the TCA cycle was decarboxylated with the overexpression of cis-aconitate decarboxylase (Cad) of (Aspergillus terreus) resulting in the production of 31.6 mg/L itaconic acid from methanol (Lim et al., 2019). Strengthening the TCA cycle along with its upstream supply chain was suggested as a potential approach for further increasing the production titer.

Mevalonate production was demonstrated in M. extorquens AM1 harboring an artificial operon that consisted of the HMG-CoA synthase (HmgS) gene from Blattella germanica, the HMG-CoA reductase (HmgR) gene from Trypanosoma cruzi, and the thiolase (PhaA) gene from Ralstonia eutropha. This engineered strain produced up to 2.2 g L−1 mevalonate with a yield and productivity of 28.4 mg/g and 7.16 mg/L/h under fed-batch fermentation conditions (Zhu et al., 2016). With further media optimization, the concentration, yield, and productivity of mevalonate were increased to 2.59 g/L, 48.90 mg/g, and 12.33 mg/L/h, respectively (Cui et al., 2017). Additionally, with the improvement of acetyl-CoA flux (7%) by using a biosensor-assisted transcriptional regulator (QscR) for increasing the NADPH generation and overexpression of fumC (encoding fumarase C), the highest yield of mevalonate (2.67 g/L) in engineered M. extorquens AM1 has been reported (Liang et al., 2017). Mevalonate being a precursor of isoprenoid compounds, these strains offers a wide expansion of product spectrum from methanol. Various bulk and value added isoprenoid production strategies established in primary industrial microbes (Navale et al., 2021) could be easily adapted in this host. Relatively, the sesquiterpenoid α-humulene has been successfully produced in M. extoquens AM1 by heterologous expression of α-humulene synthase (ZSS1) from Zingiber zerumbet and farnesyl pyrophosphate (FPP) synthase from Saccharomyces cerevisiae along with isoprenoid precursor supply from mevalonate pathway of Myxococcus xanthus (hmgs, hmgr, mvaK1, mvaK2, mvaD, and fni). By using the crtN (diapophytoene dehydrogenase) knock-out pigment-less strain of M. extorquens AM1 (Van Dien et al., 2003), a final product concentration of up to 1.65 g/L was obtained in two-phase fed-batch cultivation with dodecane as an extraction solvent for avoiding intracellular product accumulation (Sonntag et al., 2015). This production titer of α-humulene is already in competitive range with their production from E. coli (Harada et al., 2009). Taking together, all the different chemicals and production stratigies presented above M. extorquens AM1provides a reliable platform for the rational engineering of methylotrophic bacteria for industrial application.

D-Lactic acid, a monomer of the biodegradable polymer PLA (polylactic acid) with high mechanical properties and hydrolysis resistance (Tsuji, 2005), was targeted for production from methanol in P. pastoris. D-Lactate dehydrogenase (D-LDH) from Leuconostoc mesenteroides was integrated into the rDNA locus via a multicopy integrative plasmid and through post transformational gene amplification strategy, leading to engineered strain successfully producing D-lactate (3.48 g/L) under fermentation condition (Yamada et al., 2019). Because of the widespread application of TCA intermediates like fumaric acid, malic acid, and succinic acid in food, chemical, and pharmaceutical industries, and an aim to replace petroleum-derived commodity chemical maleic anhydride, the reductive tricarboxylic acid (TCA) pathway was overexpressed in P. pastoris. With the multicopy integration of the pyruvate carboxylase gene (pc) and the cytoplasmic malate dehydrogenase gene (mdh1) in the genome of P. pastoris, Zhang et al., obtained production titers of 0.76, 42.3, and 9.42 g/L for fumaric acid, malic acid, and succinic acid, respectively (Zhang et al., 2015) from a complex medium with methanol supplement. Recently, malic acid production with methanol as a sole carbon source was also attempted. Through an elaborate process involving: 1) expression of heterologous malic acid producing genes along with transporter, 2) knocking out competing pathway (succinic acid and ethanol production pathway), along with methanol dissimilation pathway, and 3) modifying XuMP cycle for optimum methanol assimilation, an engineered P. pastoris strain was designed for malic acid production. The final strain produced 2.79 g/L malic acid (Guo et al., 2021).

P. pastoris has been used to produce nootkatone, a highly demanded and prized aromatic sesquiterpenoid naturally found in grapefruit, pummelo, or Nootka cypress trees. Initially, a whole-cell biocatalytic system was created by coexpressing the Arabidopsis thaliana cytochrome P450 reductase (CPR) and the Hyoscyamus muticus premnaspirodiene oxygenase (HPO), which hydroxylated extracellularly added (+)-valencene to (+)-nootkatone. Followed by the introduction of valencene synthase from Callitropsis nootkatensis for production of (+)-valencene (Beekwilder et al., 2014). This strain, with the additional overexpression of an endogenous P. pastoris ADH and S. cerevisiae, truncated Hmg1p (tHMG1) successfully produced 208 mg/L (+)-nootkatone from methanol using dodecane bilayer in a fed-batch bioreactor (Wriessnegger et al., 2014).

In an attempt to develop a P. pastoris-based in vivo fungal polyketide production system, 6-methylsalicylic acid (6-MSA) production was demonstrated by expressing Aspergillus nidulans phosphopantetheinyl transferase (PPtase) gene npgA and A. terreus 6-methylsalicylic acid synthase (6-MSAS) gene atX driven by alcohol inducible AOX1 promoter. 6-MSAS, the first fungal polyketide synthase (PKS) gene to be cloned (Dimroth et al., 1976), is one of the best-characterized fungal PKSs. In an initial fed-batch culture, high cell density was achieved (using glycerol as feed), followed by the production of 6-MSA through the methanol induction phase. The strain was able to produce 6-MSA up to 2.2 g/L under methanol induction for 20 h until an intracellular toxic level accumulation of 6-MSA. Promoting higher cell density before methanol induction or developing a fermentation and bio-separation coupled process for releasing 6-MSA were suggested as practical solutions to further increase production levels (Gao et al., 2013).

Anti-hypercholesterolemia pharmaceutical lovastatin (a potent inhibitor of HMG-CoA reductase) and its precursor, monacolin J, have also been produced from P. pastoris. Biosynthetic pathway genes for these chemicals from A. terreus were strategically assembled to follow a pathway splitting and co-culture strategy. Avoiding the accumulation of pathway intermediates and relieving metabolic stress due to overexpression of complex pathways, a yield of 593.9 mg/L monacolin J and 250.8 mg/L lovastatin were achieved from methanol (Liu et al., 2018a). The lower level of lovastatin was presumably due to the pathway distribution at monacolin J and its partial permeability to the cellular membrane. The lovastatin production was further improved up to 419 mg/L after overexpression of TapA protein, a putative lovastatin efflux pump from the native lovastatin-producing strain A. terreus (Liu et al., 2018b).

Margatoxin (MgTx) is a toxin naturally found in scorpion venom. Due to its high affinity for blocking voltage-gated potassium (Kv) channels, this toxin has been widely used for studying the physiological function of Kv ion channels in various cell types, including immune cells. MgTx maintain the biologically active tertiary structure with three disulfide bonds (Johnson et al., 1994). The chemical synthesis of peptide toxins and production from recombinant E. coli require oxidative refolding for proper disulfide bond formation, resulting in low yield (Turkov et al., 1997; Jensen et al., 2009). With the capability to generate high disulfide-rich peptides, the P. pastoris expression system was able to produce 83 ± 3 mg/L of fully functional recombinant margatoxin (rMgTx) (Naseem et al., 2021).

Overall, P. pastoris represents a promising microbial cell factory for methanol-based bio manufacturing. Compared to the methylotrophic bacteria, the eukaryotic nature of this organism enables higher tolerance to methanol and extreme environmental conditions (pH or temperature), in addition to the possession of protein assembly, folding and post-translational modification systems.

Growth or cultivation of E. coli utilizing methanol as the sole carbon source has been of limited success; hence, most studies until now rely on a co-substrate feeding strategy (methanol along with a general substrate) for cell growth and biochemical production.

A formaldehyde dehydrogenase (frmA) knock-out strain of E. coli was developed for the efficient uptake of formaldehyde, overexpressing Mdh of B. stearothermophilus and RuMP pathway enzymes Hps and Phi from B. methanolicus. The E. coli strain successfully produced the valuable compound naringenin using methanol and yeast extract. The introduction of 4-coumaroyl CoA ligase of A. thaliana and chalcone synthase of Petunia hybrida brought out 3.5 mg/L of naringenin production, where 18% of its carbon was derived from methanol (Whitaker et al., 2017). This recombinant strain was further engineered for enhanced methanol assimilation by incorporating five non-oxidative PPP genes from B. methanolicus into the chromosome and deletion of pgi, rerouting the glycolytic carbon flow towards Ru5P through the oxidative PPP. Following the introduction of the 1-butanol production pathway (Kataoka et al., 2015) and the acetone production pathway (Mermelstein et al., 1993) into the resulting E. coli strain, successful production of acetone and 1-butanol up to 2.6 g/L and 1.8 g/L, respectively, using methanol and glucose as co-substrates was achieved (Bennett et al., 2018b). However, methanolic carbon incorporation was reported to be very low, about 3.6% in acetone and 0.7% in butanol. The improvement in product titer and the amount of methanol consumption were not well correlated, and metabolite levels, flux profiles, and/or other unknown cellular/metabolic responses were hypothesized for the observed effect. To further increase the methanol co-assimilation, a synthetic methanol auxotrophic strain of E. coli was created by blocking essential genes of the PPP, resulting in the co-assimilation of methanol and xylose. The methanol auxotrophic strain was then engineered for the production of ethanol and 1-butanol. An effective increase in methanol utilization was observed with a production titer of 4.6 g/L ethanol and 2.0 g/L 1-butanol with methanolic carbon incorporation of 43% and 71%, respectively (Chen et al., 2018).

Amino acid production is usually associated with high NADPH-demanding synthases (Bommareddy et al., 2014). Incorporating a methanol assimilation module could provide an extra supply of reducing equivalent NADPH. Correspondingly, methylotrophic E. coli engineered for lysine production was able to improve production twofold with heterologous expression of NADH kinase from S. cerevisiae (Wang et al., 2019a). With NADH identified as a potent kinetic inhibitor of Mdh (Woolston et al., 2018a), this particular step further drives the methanol metabolism in this strain.

C. glutamicum, a well-studied and preferred industrial workhorse, has also been targeted for methanol metabolism. By expressing Mdh from B. methanolicus, along with Hps and Phi, in a strain devoid of endogenous aldehyde dehydrogenase genes ald and fadH, a methylotrophic C. glutamicum was engineered, and cadaverine production was attempted. Using 13C methanol and either glucose or ribose as a co-substrate, 1.5 g/L of cadaverine with 5%–15% labeled carbon was produced from this strain (Leßmeier et al., 2015). In another study, a methanol-dependent C. glutamicum strain created via ribose 5-phosphate epimerase (rpe) knock-out, equipped with Mdh from B. methanolicus, and Hps and Phi from B. subtilis, has been tested for cadaverine production. Combining the methanol-dependent complementation of this metabolic cutoff strain with ALE (Adaptive Laboratory Evolution) for increasing methanol dependency, the final strain produced 163 mg/L cadaverine with 43% labeled carbon using 13C-methanol and ribose as carbon substrates (Hennig et al., 2020). Another methanol-dependent C. glutamicum, a ribose 5-phosphate isomerase (rpi) deficient mutant, with Mdh of B. stearothermophilus and RuMP enzymes (Hps and Phi) of B. methanolicus has been engineered for production of glutamate. In a minimal medium supplemented with methanol and xylose (4 g/L each), the strain produced 90 mg/ml glutamate (Tuyishime et al., 2018). Further increasing the methanol tolerance with ALE the production titer was improved up to 230 mg/L (Wang et al., 2020). As a native glutamate producer, these low production titers indicate poor methanol assimilation and metabolism; developing strategies for enhancing methanol tolerance and incorporation are the current challenges for this host.

In addition to the native methylotrophic yeast P. pastoris discussed above, attempts have been made to introduce methylotrophy in S. cerevisiae. Compared to the prokaryotic bacterial platforms, S. cerevisiae possesses great potential as a host for introducing methylotrophy due to its high methanol tolerance (Yasokawa et al., 2010). By integrating the methanol oxidation pathway of P. pastoris into the chromosome, a recombinant S. cerevisiae strain capable of growth on methanol as a sole carbon source was developed. The recombinant strain consumed up to 1 g/L methanol, producing 0.26 g/L pyruvate (Dai et al., 2017). Additionally, the native capacity of methanol utilization has also been realized in S. cerevisiae (Espinosa et al., 2020). For further advancement, it may be worthwhile to investigate how this capability works in conjunction with the optimization of the expression strength and balance of heterologous methylotrophic pathway enzymes.

In this pathway, the oxidation of formaldehyde to formate is achieved in a single step catalyzed by heterologous Faldh from P. putida, simplifying the four-step process in the native serine cycle. Additionally, the hydroxypyruvate reductase (Hpr) route was avoided, where native Hpr exhibit higher catalytic activity for the irreversible conversion of intermediate glyoxylate to glycolate rather than converting hydroxypyruvate to glycerate. Instead of employing serine as an amino group donor, glyoxylate is transaminated with alanine to generate glycine in a reaction catalyzed by S. cerevisiae alanine-glyoxylate transaminase (Agt). Glycine accepts MTHF to generate serine which is finally deaminated to pyruvate through serine dehydratase (Sdh) from Cupriavidus necator. PEP is regenerated by the action of endogenous phosphoenolpyruvate synthetase (Pps), thus avoiding the formation of the intermediate hydroxypyruvate (Figure 4). E. coli equipped with this pathway reported to produce 2.2 g/L acetate and 1.6 g/L ethanol with methanol and xylose as co-substrates. 13C-labeling results showed methanol assimilation into acetate and ethanol at 38% and 25%, respectively (Yu and Liao, 2018).

Another derivative of the serine cycle is designed to avoid the competition of pathway flux with the central metabolism, increasing thermodynamic favorability and avoiding energy-expensive carboxylation. In this cycle, the promiscuous activity of E. coli threonine aldolase (LtaE) catalyzes the direct condensation of glycine with formaldehyde to generate serine. Serine is directly converted to pyruvate, bypassing formaldehyde condensation to the CH2-THF route of the serine cycle, which follows the condensation of pyruvate with formaldehyde generating 4-hydroxy-2-oxobutanoate (HOB) via the promiscuous activity of E. coli 2-keto-3-deoxy-L-rhamnonate aldolase (RhmA). HOB is subsequently aminated to homoserine by HOB aminotransferase (Hat). Homoserine is later metabolized by homoserine kinase (Hsk) and threonine synthase (Ts) to produce threonine. Finally, the activity of LtaE cleaves threonine producing acetaldehyde and regenerating glycine (Figure 4). Acetaldehyde, thus produced, is further oxidized to acetyl-CoA (He et al., 2020). Using E. coli auxotrophic strains, the functionality of this cycle was demonstrated in vivo (He et al., 2020).

Methanol condensation cycle (MCC) is a RuMP pathway derived carbon conserved and ATP-independent synthetic biocatalytic pathway that uses enzymatic reactions of the non-oxidative glycolysis (NOG) to convert formaldehyde to acetyl-CoA and water (Bogorad et al., 2013; Bogorad et al., 2014). Similar to the RuMP cycle, formaldehyde is combined with Ru5P to form Hu6P, which is further isomerized to F6P. Unlike the RuMP cycle, where F6P is phosphorylated to FBP and later cleaved to GAP and DHAP, in the MCC, the phosphoketolase (Pkt) cleaves F6P to acetyl phosphate and erythrose 4-phosphate. The acetyl phosphate can be readily converted to acetyl-CoA by a phosphate acetyltransferase (Pta), and erythrose 4-phosphate is used to regenerate Ru5P (Figure 4). This pathway can increase the carbon yield by avoiding the decarboxylation of pyruvate for acetyl-CoA formation accompanying CO₂ release (Bogorad et al., 2014). Using this pathway, 610 mg L−1 ethanol and 170 mg L−1 butanol production were demonstrated in vitro.

Another novel C1 assimilation pathway based on phosphoketolase (Pkt) was developed through the in silico combinatorial algorithm called comb–flux balance analysis (Yang et al., 2019). In this multienzyme system, two formaldehyde molecules are first condensed to glycolaldehyde by an engineered benzoylformate decarboxylase (Gals) which behaves like an aldolase. Subsequently, glycoaldehyde condenses with glyceraldehyde-3-phosphate (G3P) to form Ru5P, which is isomerized to Xu5P. Finally, Pkt cleaves Xu5P generating G3P and producing the acetyl-CoA precursor acetylphosphate (Figure 4). In vitro demonstration of this pathway showed the production of 462.6 mg/L acetate after pathway optimization (Yang et al., 2019).

Similar to the GAA pathway and developed by the same group, the glycolaldehyde-allose 6-phosphate (GAPA) pathway condenses the formaldehyde-derived glycoaldehyde with E4P generating 2R, 3R-stereo allose 6-phosphate (A6P), catalyzed by the novel aldolase DeoC. A6P is then isomerized to d-allulose 6-phosphate (Au6P) by allose 6-phosphate isomerase/ribose 5-phosphate isomerase B (RpiB) and subsequently epimerized to F6P by D-allulose-6-phosphate 3-epimerase (AlsE). Finally, F6P is hydrolyzed by Pkt to produce acetylphosphate regenerating E4P (Figure 4). With this pathway, the in vitro carbon yield was further improved (94%) compared to the GAA (88%) (Mao et al., 2021).

The formolase pathway is a novel, computationally designed carbon fixation pathway. The de novo engineered enzyme formolase (FLS), catalyzing the carboligation of three molecules of formate to a DHA molecule (Siegel et al., 2015), was used to incorporate carbon into the central metabolism (Figure 4). Combining the expression of NAD-dependent Mdh from B. methanolicus with formolase and native dihydroxyacetone kinase (Dhak), a linear pathway for methanol assimilation was demonstrated for the first time in E. coli (Wang et al., 2017). Despite being unable to grow exclusively on methanol, the strain displayed greater methanolic carbon incorporation in the proteinogenic amino acids compared to the parental strain after adaptive evolution for improved methanol utilization.

The Reductive Glycine Pathway (rGlyP) is a synthetic pathway, which theoretically provides the most energy-efficient formate assimilation route. This pathway starts with the ligation of formate with H4F and further reduces to CH2-THF. Then glycine cleavage/synthase system (GCS) reacts CH2-THF with CO2, ammonia, and NADH, generating glycine. Following this, another CH2-THF condenses with glycine to form serine. The pathway starting from formate up to serine is fully reversible, and the direction of metabolic flux is determined by the concentration of substrates and products (Bar-Even et al., 2013). This pathway was successfully established in E. coli, creating strains able to grow on formate as the sole carbon source (Yishai et al., 2017; Kim et al., 2019). Additionally, with the introduction of Mdh from Pseudomonas putida, a short-term ALE evolved strain capable of utilizing methanol via this pathway has been reported (Kim et al., 2020). The engineered strain grew on methanol and CO2 with a doubling time of 54 ± 5.5 h and a biomass yield of 4.2 ± 0.17 gDCW/mole methanol. rGlyP has also been functionally demonstrated in S. cerevisiae (Gonzalez de la Cruz et al., 2019).

The synthetic acetyl-CoA (SACA) pathway is a linear C1 assimilation pathway where two formaldehyde molecules are condensed to glycolaldehyde catalyzed by a glycolaldehyde synthase (Gals). Subsequently, an Actinobacteria-derived Pkt with acetylphosphate synthase activity converts the glycolaldehyde to acetylphosphate. Finally, acetyl-CoA is generated via the action of Pta (Figure 4). Although theoretically one of the thermodynamically favorable pathways, the enzymes involved (Gals and Pkt) displayed very low substrate affinities in vivo. The in vitro demonstration of this pathway by 13C-labeled metabolites achieved a carbon yield of ∼50%; however, when introduced to E. coli, only 17% average carbon labeling was detected in PEP (Lu et al., 2019).

The 2-Hydroxyacyl-CoA lyase (HACL) pathway is another C1 assimilation pathway based on the activity of 2-hydroxyacyl-CoA-lyase (HACL). This enzyme can act reversibly and catalyze the ligation of carbonyl-containing molecules with formyl-CoA to produce C1-elongated 2-hydroxyacyl-CoAs (Cotton et al., 2020). In this pathway, formaldehyde is converted to formyl-CoA via an acyl-CoA reductase, and the HACL catalyzes the ligation of formaldehyde with formyl-CoA to generate glycolyl-CoA, which can be converted to glycolate (Figure 4). Engineered E. coli whole-cell biocatalyst ultimately produced 1.2 g/L of glycolate in 24h from formaldehyde (Chou et al., 2019).

Ketohydroxybutyrate (KHB) pathway is a pyruvate-based formaldehyde assimilatory pathway utilizing the aldol condensation activity of the natural enzyme 2-keto-4-hydroxyglutarate (KHG) aldolase. In this pathway, formaldehyde condenses with pyruvate to form HOB, which can be further decarboxylated via alpha-keto acid decarboxylase to 3-hydroxypropionaldehyde (3-HPA) and finally reduced to a value-added product 1, 3-propanediol (1, 3-PDO) using the enzyme 1, 3-PDO oxidoreductase. Engineered E. coli expressing this pathway successfully demonstrated the production of 32.7 mg/L 1, 3-propanediol from methanol with glucose supplement (Wang et al., 2019b).

All the aforementioned novel pathways have been designed to make effective use of endogenous cellular resources during methanol metabolism. Although functionally illustrated as proof of concept in vitro and/or in vivo for the assimilation of C1 substrates, these novel engineered pathways are still far from optimum. The difficulties of increasing enzyme efficiencies, efficient metabolic rerouting, and regulation of host metabolism are still hurdles to overcome for transforming synthetic methylotrophs into a methylotrophic platform organism.