Engineering the bioconversion of methane and methanol to fuels and chemicals in native and synthetic methylotrophs

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Highlights

  • Engineering aerobic and anaerobic methane activation for fuel and chemical production.

  • Developing synthetic methylotrophs for conversion of methanol to fuels and chemicals.

  • Engineering strategies to overcome the limitations of synthetic methanol utilization.

Methylotrophy describes the ability of organisms to utilize reduced one-carbon compounds, notably methane and methanol, as growth and energy sources. Abundant natural gas supplies, composed primarily of methane, have prompted interest in using these compounds, which are more reduced than sugars, as substrates to improve product titers and yields of bioprocesses. Engineering native methylotophs or developing synthetic methylotrophs are emerging fields to convert methane and methanol into fuels and chemicals under aerobic and anaerobic conditions. This review discusses recent progress made toward engineering native methanotrophs for aerobic and anaerobic methane utilization and synthetic methylotrophs for methanol utilization. Finally, strategies to overcome the limitations involved with synthetic methanol utilization, notably methanol dehydrogenase kinetics and ribulose 5-phosphate regeneration, are discussed.

Introduction

Abundant natural gas supplies have made methane and methanol promising substrates for biological production of fuels and chemicals [1••]. These one-carbon (C1) compounds are at least 50% more reduced than traditional sugars, for example, glucose, allowing for improved product titers and yields [2••]. Worldwide, the amount of recoverable natural gas is estimated to be 7.2 × 103 trillion ft3 [1••]. In the US alone, estimates approach 2 × 103 trillion ft3. At current energy usage rates, this is enough natural gas to supply the US for 100 years. Methane is also a potent greenhouse gas, having a warming potential 21 times that of CO2. As a result, along with the food versus fuel debate, biological gas-to-liquid (GTL) conversion technologies are promising alternatives for fuel and chemical production. This review discusses recent progress made toward understanding and engineering native methanotrophs and synthetic methylotrophs for production of fuels and chemicals. Advancements in aerobic and anaerobic methane utilization will first be discussed, followed by those made toward engineering synthetic methylotrophs for methanol utilization. Finally, difficulties with engineering synthetic methanol utilization and strategies to overcome them will be detailed.

Section snippets

Aerobic methane utilization to produce fuels and chemicals

The physiology and biochemistry of aerobic methanotrophs, which utilize methane as their sole carbon and energy source, have been extensively reviewed [3, 4]. The first step in methane assimilation is oxidation to methanol by methane monooxygenase (MMO) [5], followed by oxidation to formaldehyde by pyrroloquinoline quinone (PQQ)-containing methanol dehydrogenase (MDH) [3, 4]. Type I methanotrophs are gammaproteobacteria, which assimilate formaldehyde via the ribulose monophosphate (RuMP)

Anaerobic methane utilization to produce fuels and chemicals

Anaerobic oxidation of methane (AOM) is a significant biogeochemical process in marine and freshwater sediments and is important in methane release to the atmosphere (Figure 1) [29, 30]. Anaerobic methane oxidizing archaea, or ANaerobic MEthanotrophs (ANME), were first discovered obligately associated with bacterial partners that used the reducing equivalents generated during methane oxidation by ANME to reduce sulfate [30]. ANME belong to four phylogenetic clusters within Euryarchaeota:

Conversion of methane to methanol for use as a substrate for synthetic methylotrophs

Along with the aforementioned biological oxidation of methane to methanol, chemical conversion of methane to methanol is also possible. As compared to the biological oxidation of methane, chemical conversion is faster albeit suffers from low selectivity and high process demands, for example, elevated temperatures and pressures [2••]. As a result, the biological oxidation of methane is more ideal from an energetics perspective. However, as described above, several challenges remain before the

Enzyme and pathway considerations for synthetic methanol utilization

Methanol is first oxidized to formaldehyde via a methanol oxidoreductase (Figure 2), which include NAD-dependent and PQQ-dependent MDHs from bacteria and alcohol oxidases (AOXs) from yeast [2••]. NAD-dependent MDHs are ideal for synthetic methylotrophy since they function aerobically and anaerobically, are expressed from a single gene and conserve electrons in the form of NADH [2••, 52•].

Formaldehyde is next assimilated via the RuMP pathway, ribulose bisphosphate (RuBP) pathway or serine cycle [

Sourcing and engineering methanol dehydrogenases (MDHs) for improved kinetic properties

NAD-dependent MDHs generally exhibit higher affinity toward higher alcohols, for example, 1-butanol, and methanol oxidation is unfavorable under standard conditions, explaining why many native methylotrophs are thermophilic [2••]. A limited number of NAD-dependent MDHs have been characterized, notably those from B. methanolicus strains MGA3 and PB1, which each contain three distinctive MDHs with different kinetic properties [55]. In a recent study, we sourced an NAD-dependent MDH from the

Cell-free metabolic engineering to demonstrate and improve methanol utilization

Cell-free metabolic engineering has been used to demonstrate methanol conversion and improve MDH kinetic limitations. Bogorad et al. developed a methanol condensation cycle (MCC) by combining the RuMP pathway with nonoxidative glycolysis (NOG) for carbon-conserved, redox-balanced and ATP-independent higher alcohol production (Table 1) [58]. Importantly, sugar phosphates were required to prime MCC, suggesting the importance of sustained Ru5P levels for methanol utilization. Although methanol

Engineering Escherichia coli to assimilate methanol for in vivo growth and metabolite production

Muller et al. reported in vivo 13C-methanol assimilation in E. coli via incorporation of Mdh2, HPS and PHI from B. methanolicus (Table 1) [52]. The engineered E. coli exhibited up to 39.4% 13C-labeling in glycolytic and PPP intermediates. RuMP pathway cycling was demonstrated as higher-order mass isotopomers were observed. Although methanol assimilation was achieved, no growth on methanol was reported, suggesting limitations downstream of methanol oxidation.

Whitaker et al. reported

Other synthetic methylotrophs for in vivo methanol assimilation and metabolite production

Witthoff et al. demonstrated in vivo methanol assimilation in C. glutamicum using a similar strategy as those used in E. coli (Table 1) [60]. The engineered strain exhibited a methanol uptake rate of 1.7 mM h−1 and a 30% improvement in biomass titer in glucose minimal medium. Up to 25.7% 13C-labeling in M+1 mass isotopomers was observed in intracellular metabolites using a formaldehyde dissimilation deficient strain, constructed via deletion of acetaldehyde dehydrogenase (ald) and

Developing methanol and formaldehyde responsiveness in synthetic methylotrophs

One limitation of synthetic methylotrophy is the inability to regulate gene expression in response to methanol and/or formaldehyde, which leads to reduced gene expression and metabolic activity during growth on methanol [66]. Native methylotrophs regulate gene expression via methanol-responsive and/or formaldehyde-responsive promoters or systems [67, 68]. Upregulation of RuMP pathway and PPP genes in B. methanolicus during methylotrophic growth improves methanol tolerance and uptake rate [67].

Strategies to improve ribulose 5-phosphate (Ru5P) (re)generation

A critical limitation of synthetic methylotrophy is inefficient Ru5P regeneration. One strategy to improve Ru5P regeneration involves refactoring the expression of native PPP genes using native or engineered Pfrm promoters or a methanol-sensing system (Figure 3) [68, 69••], which would upregulate gene expression during growth on methanol, emulating native methylotrophs and providing sufficient flux for Ru5P regeneration. This strategy is readily applicable to other target genes as well that may

Exploring amino acid metabolism to improve synthetic methanol assimilation

Since yeast extract, which is primarily composed of amino acids, stimulates synthetic methylotrophy, the metabolism of all 20 amino acids and the regulatory networks in which they are involved were examined [72]. It was determined that co-utilization of threonine leads to improved methanol assimilation in a synthetic E. coli methylotroph, which resulted from activation of endogenous C1 metabolism via high flux from threonine to glycine to serine under threonine growth conditions (Figure 2) [72

Future perspectives and recommendations

Two key limitations were identified while attempting to engineer synthetic methylotrophs for methanol utilization. First, methanol oxidation is limited by MDH kinetics. Sourcing alternative or engineering current MDHs for improved kinetics can overcome this limitation. Second, methanol assimilation is limited by inefficient Ru5P regeneration. Several strategies can overcome this, including refactoring native PPP gene expression for methanol-responsiveness, incorporating a heterologous

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgement

Financial support from ARPA-E through contract no. DE-AR0000432 is gratefully acknowledged.

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