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Engineering the bioconversion of methane and

methanol to fuels and chemicals in native and synthetic methylotrophs

1,2 1,2 1

R Kyle Bennett , Lisa M Steinberg , Wilfred Chen and

1,2

Eleftherios T Papoutsakis

3 3

Methylotrophy describes the ability of organisms to utilize 2 10 trillion ft . At current energy usage rates, this is

reduced one-carbon compounds, notably methane and enough natural gas to supply the US for 100 years. Meth-

methanol, as growth and energy sources. Abundant natural gas ane is also a potent greenhouse gas, having a warming

supplies, composed primarily of methane, have prompted potential 21 times that of CO2. As a result, along with the

interest in using these compounds, which are more reduced food versus fuel debate, biological gas-to-liquid (GTL)

than sugars, as substrates to improve product titers and yields conversion technologies are promising alternatives for

of bioprocesses. Engineering native methylotophs or fuel and chemical production. This review discusses

developing synthetic methylotrophs are emerging fields to recent progress made toward understanding and

convert methane and methanol into fuels and chemicals under engineering native methanotrophs and synthetic methy-

aerobic and anaerobic conditions. This review discusses lotrophs for production of fuels and chemicals. Advance-

recent progress made toward engineering native ments in aerobic and anaerobic methane utilization will

methanotrophs for aerobic and anaerobic methane utilization first be discussed, followed by those made toward engi-

and synthetic methylotrophs for methanol utilization. Finally, neering synthetic methylotrophs for methanol utilization.

strategies to overcome the limitations involved with synthetic Finally, difficulties with engineering synthetic methanol

methanol utilization, notably methanol dehydrogenase kinetics utilization and strategies to overcome them will be

and ribulose 5-phosphate regeneration, are discussed. detailed.

Addresses

1 Aerobic methane utilization to produce fuels

Department of Chemical and Biomolecular Engineering, University of

and chemicals

Delaware, 150 Academy St., Newark, DE 19716, USA

2

The Delaware Biotechnology Institute, Molecular Biotechnology The physiology and biochemistry of aerobic methano-

Laboratory, University of Delaware, 15 Innovation Way, Newark, DE trophs, which utilize methane as their sole carbon and

19711, USA

energy source, have been extensively reviewed [3,4]. The

first step in methane assimilation is oxidation to methanol

Corresponding author: Papoutsakis, Eleftherios T ([email protected])

by methane monooxygenase (MMO) [5], followed by

oxidation to formaldehyde by pyrroloquinoline quinone

Current Opinion in Biotechnology 50

2018, :81–93 (PQQ)-containing methanol dehydrogenase (MDH)

This review comes from a themed issue on Energy biotechnology [3,4]. Type I methanotrophs are gammaproteobacteria,

which assimilate formaldehyde via the ribulose monopho-

Edited by Akihiko Kondo and Hal Alper

sphate (RuMP) pathway. Type II methanotrophs, which

assimilate formaldehyde via the serine cycle, are alpha-

proteobacteria [4]. A third group of aerobic methanotrophs,

https://doi.org/10.1016/j.copbio.2017.11.010 Type X, utilize the RuMP pathway for formaldehyde

0958-1669/ã 2017 Elsevier Ltd. All rights reserved. assimilation but express low levels of serine cycle enzymes

and grow at higher temperatures [3].

Two types of MMOs have been identified [5,6]. Nearly

all methanotrophs express a membrane-bound particulate

MMO (pMMO), and a few also express a soluble MMO

Introduction (sMMO). pMMO is an integral membrane hydroxylase

Abundant natural gas supplies have made methane and with three subunits arranged as an a3b3g3 trimer, encoded

methanol promising substrates for biological production by the pmoCAB operon, and contains two Cu-containing

of fuels and chemicals [1 ]. These one-carbon (C1) active sites in the N-termini and C-termini of pmoB

compounds are at least 50% more reduced than traditional [6]. sMMO contains three components: a hydroxylase,

sugars, for example, glucose, allowing for improved prod- encoded by mmoX, mmoY and mmoZ, a reductase,

uct titers and yields [2 ]. Worldwide, the amount of encoded by mmoC, and a regulatory protein, encoded

3

recoverable natural gas is estimated to be 7.2 10 tril- by mmoB [5,6]. The hydroxylase is an a2b2g2 dimer with

3

lion ft [1 ]. In the US alone, estimates approach a diiron active site in the alpha subunit [5]. pMMO has a

www.sciencedirect.com Current Opinion in Biotechnology 2018, 50:81–93

82 Energy biotechnology

narrow substrate specificity and oxidizes shorter alkanes into lactic and acetic acid with increased ATP and

up to five carbons [6] whereas sMMO has a broader decreased CO2 production [19]. Overexpression of phos-

substrate range that includes aromatic and heterocyclic phoketolase in M. buryatense led to a 2.6-fold improve-

compounds [6]. ment in biomass and lipid yield from methane [20 ].

In addition to oxygen, some methanotrophs use alternate Methylomonas sp. 16a is an interesting methanotroph due

electron acceptors for methane activation. A methane- to high-level production of C30 carotenoids [21], but the

oxidizing, nitrite-reducing enrichment culture from fresh- production of larger carotenoids remains challenging due

water sediment was dominated by one bacterial species to lack of genetic tools. Episomal gene expression for

[7], and metagenomic sequencing led to the construction synthesis of C40 carotenoids, astaxanthin and canthaxan-

1

of the full draft genome of a proposed new species, thin, resulted in yields of 2.4 g gDW [22]. Increased

Methylomirabilis oxyfera [8], which possesses a pMMO yields were obtained by optimizing chromosomal inte-

and an incomplete denitrification pathway. Methane is gration location [23] and co-expression of bacterial

oxidized with nitrite and a pathway was proposed in hemoglobins [24].

which two molecules of NO could be used to produce

N2 and O2 for methane oxidation [8]. Methane oxidation Efforts have also been made to engineer methanotrophs

coupled to nitrate reduction was described for Methylomonas for high-volume chemicals, for example, lactic and suc-

denitrificans under hypoxia [9]. cinic acids. Overexpression of lactate dehydrogenase

(LDH) from Lactobacillus helveticus in M. buryatense

There is increased interest in engineering methanotrophs improved lactate production by 70-fold over the wild-

for converting methane into fuels and chemicals. Improv- type strain, resulting in 0.8 g/L [25 ]. Expression of the

ing methane oxidation, either by MMO overexpression or succinate synthesis pathway in M. capsulatus Bath

enhanced activity via protein engineering, could increase resulted in 70 mg/L [26 ]. Trace-level production of

efficiency. However, MMO expression in heterologous 1,4-butanediol [27 ] and isobutanol [28 ] has also been

hosts has largely failed [10]. A number of genetic tools reported.

have been developed for methanotrophs [10], including

conjugation for introducing genetic material from E. coli. Although aerobic methane conversion to fuels and che-

Methylomicrobium buryatense 5G is emerging as a tractable micals has been demonstrated, only low yields were

host for metabolic engineering with advances including achieved at small scale. During the oxidation of methane

engineering of a strain capable of IncP-based vector repli- to methanol via MMO, two electrons are required to

cation for episomal gene expression [11], development of simultaneously reduce O2 to H2O. Recovery of these

selection/counter-selection markers for allelic exchange electrons is achieved in the subsequent step of methanol

[11] and transformation using electroporation [10]. oxidation to formaldehyde. Therefore, the result is the

redox-neutral conversion of methane to formaldehyde,

Currently, Methylosinus trichosporium is the preferred spe- which results in a 36% energy loss [1 ]. Since formalde-

cies for methanol production, which requires a co-sub- hyde possesses the same degree of reduction as traditional

strate and inhibition of MDH [12]. Another strategy for sugars, for example, glucose, product yields achieved

methanol production is co-feeding methane and ammonia from aerobic methane conversion are expected to be

to a nitrifying culture where the methanol produced by comparable to those of aerobic sugar metabolism. Fur-

action of ammonia monooxygenase cannot be used by the thermore, yields of reduced fuels and chemicals will be

nitrifiers [13]. A third strategy uses an engineered BM-3 limited under aerobic conditions as oxidative phosphor-

cytochrome P450 monooxygenase from Bacillus megaterium ylation competes for reducing equivalents in the form of

for methane oxidation [14]. NAD(P)H. Scale-up of aerobic methane conversion also

presents a challenge, as methane and oxygen gas transfer

One product from methane is polyhydroxybutyrate limitations result in poor kinetics. Although these chal-

(PHB), a biopolymer and plastic substitute [15,16]. lenges can be addressed by enhancing the volumetric

Methanotrophs synthesize intracellular PHB as a source mass transfer coefficient (kLa), either from increased gas

of reducing equivalents for growth under nutrient-limit- flow rate, agitation or improved reactor design, these

ing conditions albeit yields are modest and of low molec- improvements result in larger operating and capital

ular weight [15,16]. Methylobacterium organophilum CZ-2 expenses [1 ].

was reported to accumulate up to 57% PHB under nitro-

gen limitation [17]. Another product are storage lipids in Anaerobic methane utilization to produce

the form of triacylglycerides (TAGs), which can be con- fuels and chemicals

verted to biodiesel [18]. TAG accumulation is promoted Anaerobic oxidation of methane (AOM) is a significant

under oxygen-limiting, nitrogen-limiting or phosphate- biogeochemical process in marine and freshwater sedi-

limiting conditions [18]. In some methanotrophs, carbon ments and is important in methane release to the atmo-

flux can be routed through the phosphoketolase pathway sphere (Figure 1) [29,30]. Anaerobic methane oxidizing

Current Opinion in Biotechnology 2018, 50:81–93 www.sciencedirect.com

Bioconversion of methane and methanol Bennett et al. 83

Figure 1

+ Fe3+ 2 H

Fe2+ HdrDE MP MPH2 syntrophic Mhc partner MP 2 NADH CoM-S-S-CoB + 2H+ 2 NAD+ Mhc HS-CoM CH4 2 CoM-S-S-CoB pyruvate lactate Mcr H4MPT Hbd

MPH HS-CoB methyl-S-CoM 4 Fdox + 2 HS-CoA 2 Na+ Mtr Por + 4 Fdred + 2 CO2+ 2 H methyl-H4MPT HS-CoA F420 Mer ADP F420H2 ATP HS-CoM + Pi methylene-H4MPT F420 Mtd Cdh acetyl-CoA acetate F420H2 P Acd F H i MP 420 2 methenyl-H4MPT Mch Pta HS-CoA 2 H+ Fpo 2 Fdred Ack formyl-H4MPT acetyl-PO 2- acetate F 4 2 420 Ftr ADP ATP

MPH formyl-MFR + Pi Fox 2 HS-CoB Fmd + Fred 2 HS-CoM 2 CoM-S-S-CoB CO2 CoM-S-S-CoB HdrABC F HS-CoM red FrhB F420H2 Fred HdrABC F MvhD Fox MP ox HS-CoB MQ F420 Fox Rnf Fred Nar

+ MPH2 MQH 2 Na 2 2- ˜ NO2 MP MPH2 2- NO3

Current Opinion in Biotechnology

Proposed anaerobic methane oxidation pathways of archaeal methanotrophs and engineered methanotrophic Methanosarcina acetivorans C2A.

Enzymes are shown in bold within rectangles. Enzymes heterologously expressed in M. acetivorans C2A are shown in blue, and pathways utilized

by the engineered M. acetivorans strain to produce acetate and lactate are shown with blue arrows. Enzymes and pathways present only in

ANaerobic MEthanotrophs (ANME) are shown in green. Enzymes and cofactors are as follows: Acd, ADP-forming acetyl-CoA synthetase; Ack,

acetate kinase; Cdh, CO dehydrogenase; Fmd, formylmethylfuran dehydrogenase; Fpo, F420 dehydrogenase (note: this enzyme is replaced by the

homolog Fqo in some ANME); Frh, F420-reducing hydrogenase; Ftr, formylmethanofuran:H4MPT formyltransferase; H4MPT,

tetrahydromethanopterin; Hbd, 3-hydroxylbutyryl-CoA dehydrogenase; HdrABC, soluble heterodisulfide reductase; HdrDE, membrane-bound

heterodisulfide reductase; Mcr, methyl-coenzyme M reducase; Mch, methenyl-H4MPT cyclohydrolase; Mer, methenyl-H4MPT reductase; MF,

methanofuran; Mhc, multiheme cytochrome C; MP, methanophenazine; MQ, methanoquinone (note: some ANME possess MQ instead of MP as

lipophilic electron carrier); Mtd, methenyl-H4MPT; Mtr, methyl-H4MPT:coenzyme M methyltransferase; Mvh, F420-non-reducing hydrogenase; Pta,

phosphotransacetylase; Por, pyruvate ferredoxin oxidoreductase; Rnf, methanophenazine reductase.

www.sciencedirect.com Current Opinion in Biotechnology 2018, 50:81–93

84 Energy biotechnology

3+ 4+

archaea, or ANaerobic MEthanotrophs (ANME), were to particulate Fe and Mn oxides [40], likely mediated

first discovered obligately associated with bacterial part- by multiheme cytochrome c enzymes [39 ]. Direct elec-

ners that used the reducing equivalents generated during tron transfer between Methanosarcina barkeri [42] or Metha-

methane oxidation by ANME to reduce sulfate [30]. nosaeta harundinaceae [43] and Geobacter metallireducens

ANME belong to four phylogenetic clusters within Eur- during methanogenesis on ethanol was also demon-

yarchaeota: ANME-1, ANME-2, ANME-3 and GOM Arc strated. The direct electrical connections observed for

I (formerly ANME-2d) [30]. ANME-1 are related to ANME and methanogens suggest that bioreactors for

Methanosarcinales and Methanomicrobiales whereas others methane oxidation to fuels and chemicals could utilize

lie within Methanosarcinales [30]. ANME possess homo- electrochemistry instead of syntrophic partners to remove

5

logs of all methanogenesis enzymes (except for the N , reducing equivalents produced during methane 10

N -methylene-tetrahydromethanopterin reductase activation.

(Mer) in ANME-1), and metatranscriptomic studies of

consortia performing AOM demonstrated transcription of Currently, no ANME isolate exists, however, engineered

these enzymes, suggesting methane oxidation to CO2 strains can now be envisioned as a number of genetic tools

occurs through a reversal of methanogenesis [31,32]. have been developed for tractable strains including

The key enzyme involved in AOM is a homolog of the Methanococcus maripaludis [44] and Methanosarcina species

methyl-coenzyme M reductase (MCR), which catalyzes [45]. These include selection/counter-selection markers,

the formation of methane in methanogenic bacteria (Fig- transformation strategies and replicating vectors for

ure 1) [33 ]. MCRs from ANME and methanogens share episomal expression. Recently, Cas9-mediated genome

similarity although the MCR from ANME-1 has addi- editing of M. acetivorans was demonstrated utilizing

tional features, including a tetrapyrrole derivative of the native homology-dependent repair machinery [46]. The

nickel-containing F430 cofactor and cysteine-rich side first report of engineered methane oxidation in a metha-

chains [33 ]. nogen involved overexpression of ANME-1 MCR in

Methanosarcina acetivorans C2A [47 ]. High cell densities

10 1

Despite similarities in MCR structure and sharing (10 cells mL ) of the MCR-expressing strain con-

enzymes for AOM, sustained and efficient methane oxi- sumed 15% of supplied methane after 5 days and pro-

3+

dation in methanogens has been challenging. Trace duced 10 mM acetate coupled to the reduction Fe . Pro-

methane oxidation has been observed in several metha- duction of lactate using the same strain was also reported

1

nogens [34], and the MCR purified from Methanothermo- [48 ], and the yield of 0.59 g g methane was 10-fold

bacter marburgensis was found to convert methane and the greater than that reported for aerobic production [25 ].

heterodisulfide CoM-S-S-CoB into methyl-coenzyme M Additional work engineered an air-adapted strain of M.

(CH3-S-CoM) and coenzyme B (CoB) with rates consis- acetivorans [49] to express the ANME-1 MCR, and it was

tent to in vivo values (Figure 1) [35 ]. Additionally, in cultivated in the anode compartment of a microbial fuel

vitro methanol production from CH3-S-CoM was demon- cell along with Geobacter sulfurreducens and methane-accli-

strated using the Methanosarcina barkeri methanol:coen- mated anaerobic digester sludge to produce electricity

zyme M methyltransferase (MtaABC) [36]. MCRs from [50]. Although methane-fueled microbial fuel cells have

methanogens that catalyze trace methane oxidation share been previously proposed [51], the power density pre-

common features, including four of the five key post- sented in this work was over twice that obtained previ-

translational modifications in the active site [37]. ously [50]. It was postulated that the engineered

M. acetivorans strain converts methane to oxidized inter-

Reversal of methanogenesis to oxidize methane requires mediates, for example, acetate, which are consumed by

a suitable electron acceptor [30]. Removal of reducing G. sulfurreducens coupled to anode reduction to produce

equivalents by syntrophic partner organisms, for example, electricity. Interestingly, all three components of the

sulfate-reducing bacteria, is essential for AOM to be an consortium were found to be essential for methane

energy-yielding process [30]. Studies have demonstrated oxidation coupled to electricity production [50], suggest-

direct electron transfer between ANME and bacterial ing that anode reduction occurred through electron

partners [38,39 ]. AOM can also be coupled to the shuttles instead of direct contact of a biofilm with the

3+

reduction of nitrate [31], insoluble oxides of Fe and electrode.

4+

Mn [40] or soluble electron acceptors such as humic

acids, chelated ferric iron, 9,10-anthraquinone and 2,6- Limitations of AOM coupled to fuel and chemical pro-

disulfonate [41 ]. The archaeal partner in a consortium duction include slow growth and low energy yields of

with M. oxyfera, which demonstrated methane oxidation anaerobic methanotrophs, resulting in slow AOM rates

coupled to denitrification was sequenced, and a draft [30]. The MCR-expressing M. acetivorans strain also

genome was assembled with the uncultivated organism demonstrated slow growth [47 ] despite methane oxida-

3+

Methanoperedens nitroreducens [7], which contains narGH tion coupled to Fe reduction being 3.5-times more

1

for nitrate reduction. Later research reported that Metha- energetic (454 kJ mol [40]) than hydrogenotrophic

1

noperedens-like organisms can couple methane oxidation methanogenesis (131 kJ mol [44]) under standard

Current Opinion in Biotechnology 2018, 50:81–93 www.sciencedirect.com

Bioconversion of methane and methanol Bennett et al. 85

conditions. All known ANME are related to cytochrome- Enzyme and pathway considerations for

containing Methanosarcinales, which possess multiple synthetic methanol utilization

+ +

routes for energy capture through H and Na transloca- Methanol is first oxidized to formaldehyde via a methanol

tion, and all known ANME genomes encode multiheme oxidoreductase (Figure 2), which include NAD-depen-

cytochrome c enzymes [30–32]. However, the mode of dent and PQQ-dependent MDHs from bacteria and

energy capture during methane oxidation is unknown, alcohol oxidases (AOXs) from yeast [2 ]. NAD-depen-

limiting the ability to elucidate why observed growth dent MDHs are ideal for synthetic methylotrophy since

rates for methanogens are orders of magnitude greater they function aerobically and anaerobically, are expressed

than for ANME or engineered methanogens [30]. from a single gene and conserve electrons in the form of

NADH [2 ,52 ].

As with the aforementioned aerobic scenario, although

anaerobic methane conversion to fuels and chemicals has Formaldehyde is next assimilated via the RuMP pathway,

been demonstrated, only low yields were achieved at ribulose bisphosphate (RuBP) pathway or serine cycle

small scale. However, anaerobic methane conversion does [2 ]. Formaldehyde is a toxic intermediate that must be

not suffer from the same limitations that aerobic meth- consumed quickly, and an efficient assimilation pathway

ane conversion does. During anaerobic methane conver- in essential to overcome endogenous formaldehyde dis-

sion, reducing equivalents in the form of NAD(P)H may similation [53]. Strategies to eliminate dissimilation

be conserved for production of reduced fuels and che- involve gene deletions, for example, formaldehyde dehy-

micals, thus providing the opportunity for improved drogenase ( frmA) in Escherichia coli (Figure 2) [54 ].

product yields from methane. Additionally, gas transfer The RuMP pathway is more bioenergetically favorable

limitations are not as critical under anaerobic conditions, than either the serine cycle or RuBP pathway [2 ,52 ].

resulting in lower operating and capital expenses as The two enzymes of the RuMP pathway, hexulose

compared to the aerobic methane conversion scenario. phosphate synthase (HPS) and phosphohexulose isom-

As a result, anaerobic methane conversion is more ideal erase (PHI), fix formaldehyde with the pentose phos-

for large scale production of fuels and chemicals. How- phate pathway (PPP) intermediate ribulose 5-phosphate

ever, anaerobic methane metabolism is slow compared (Ru5P) to generate hexulose 6-phosphate (H6P), which

with other systems, resulting in low growth rates and is isomerized to fructose 6-phosphate (F6P) (Figure 2)

productivities. Therefore, future efforts should be [2 ]. Ru5P is a critical intermediate within the RuMP

devoted to improving the rate of anaerobic methane pathway, and its sustained regeneration is necessary to

metabolism for enhanced growth and productivity to sustain methanol assimilation. In principle, only three

realize scale-up of anaerobic methane conversion to heterologous enzymes (MDH, HPS and PHI) are

fuels and chemicals. required to achieve synthetic methylotrophy. However,

recent studies have shown this is not the case and

instead, have shed light on additional limitations,

Conversion of methane to methanol for use as including poor MDH kinetics and insufficient Ru5P

a substrate for synthetic methylotrophs regeneration.

Along with the aforementioned biological oxidation of

methane to methanol, chemical conversion of methane to Sourcing and engineering methanol

methanol is also possible. As compared to the biological dehydrogenases (MDHs) for improved kinetic

oxidation of methane, chemical conversion is faster albeit properties

suffers from low selectivity and high process demands, for NAD-dependent MDHs generally exhibit higher affinity

example, elevated temperatures and pressures [2 ]. As a toward higher alcohols, for example, 1-butanol, and meth-

result, the biological oxidation of methane is more ideal anol oxidation is unfavorable under standard conditions,

from an energetics perspective. However, as described explaining why many native methylotrophs are thermo-

above, several challenges remain before the biological philic [2 ]. A limited number of NAD-dependent MDHs

oxidation of methane can be realized at large scale. In have been characterized, notably those from B. methano-

any case, methanol can serve as an alternative C1 sub- licus strains MGA3 and PB1, which each contain three

strate for production of fuels and chemicals. Native distinctive MDHs with different kinetic properties [55].

methylotrophs are poor industrial hosts since many are In a recent study, we sourced an NAD-dependent MDH

obligate aerobes and have limited genetic tools, which from the Gram-positive bacterium Bacillus stearothermo-

are not as well-developed and extensive as those of philus [56], which exhibits better reported kinetics than

platform organisms [2 ]. Therefore, there is consider- those from B. methanolicus. This MDH was used to

able interest in developing synthetic methylotrophs for achieve growth of engineered E. coli on methanol using

the conversion of methanol to fuels and chemicals. In a small amount of yeast extract [54 ]. The importance of

following sections, we discuss recent advancements improved kinetics was demonstrated by realizing that

toward achieving synthetic methylotrophy in several Mdh2 from B. methanolicus could not achieve methylo-

platform organisms. trophic growth under the same conditions. In both

www.sciencedirect.com Current Opinion in Biotechnology 2018, 50:81–93

86 Energy biotechnology

Figure 2

Glucose Methanol

frmA mdh

CO2 Formaldehyde

CO2 G6P GL6P 6PG Ru5P R5P rpi pgi phi hps rpe F6P H6P pfk fbp X5P FBP tkt tkt E4P fba GAP DHAP DHAP GAP fba SBP S7P C1 Pool glpX

(+) (-) (+)

KDPG 3PG Ser Gly CO2

(-) (-)

Irp

Thr Pyr AcCoA Biomass, Biofuels & Biochemicals

Current Opinion in Biotechnology

Strategies to improve synthetic methanol utilization. Methanol assimilation occurs via methanol dehydrogenase (mdh), hexulose phosphate

synthase (hps) and phosphohexulose isomerase ( phi). Formaldehyde dissimilation is eliminated via deletion of formaldehyde dehydrogenase

( frmA). Glucose carbon flux is rerouted through the oxidative pentose phosphate pathway (PPP) for ribulose-5-phosphate (Ru5P) generation via

deletion of phosphoglucose isomerase ( pgi) (shown in green). Heterologous non-oxidative PPP enzymes from Bacillus methanolicus

(phosphofructokinase ( pfk), fructose-bisphosphate aldolase ( fba), transketolase (tkt), ribulose phosphate epimerase (rpe) and sedoheptulose

bisphosphate (glpX)) regenerate Ru5P from fructose-6-phosphate (F6P) (shown in blue). Regulation of endogenous one-carbon (C1) metabolism

via leucine-responsive regulatory protein (lrp) as an alternative route for methanol assimilation (shown in red). Remaining enzymes: fructose

bisphosphatase ( fbp), ribose phosphate isomerase (rpi). Remaining metabolites: glucose-6-phosphate (G6P), 6-phosphogluconolactone (GL6P),

6-phosphogluconate (6PG), ribose-5-phosphate (R5P), fructose bisphosphate (FBP), dihydroxyacetone phosphate (DHAP), glyceraldehyde 3-

phosphate (GAP), xylulose-5-phosphate (X5P), erythrose 4-phosphate (E4P), sedoheptulose bisphosphate (SBP), sedoheptulose-7-phosphate

(S7P), 3-phosphoglycerate (3PG), pyruvate (Pyr), ketodeoxyphosphogluconate (KDPG), serine (Ser), glycine (Gly), threonine (Thr), acetyl-CoA

(AcCoA).

instances, E. coli was engineered to assimilate formalde- Cell-free metabolic engineering to

hyde via the RuMP pathway from B. methanolicus. Wu demonstrate and improve methanol utilization

et al. characterized an NAD-dependent MDH from a Cell-free metabolic engineering has been used to dem-

Gram-negative, mesophilic, non-methylotrophic bacte- onstrate methanol conversion and improve MDH kinetic

rium, Cupriavidus necator (Table 1) [57 ]. Directed limitations. Bogorad et al. developed a methanol conden-

molecular evolution was performed to engineer an sation cycle (MCC) by combining the RuMP pathway

MDH variant having improved kinetic properties. Three with nonoxidative glycolysis (NOG) for carbon-con-

mutations were identified (A26V, A31V and A169V), that served, redox-balanced and ATP-independent higher

when combined, improved methanol affinity and catalytic alcohol production (Table 1) [58 ]. Importantly, sugar

efficiency. phosphates were required to prime MCC, suggesting

Current Opinion in Biotechnology 2018, 50:81–93 www.sciencedirect.com

Bioconversion of methane and methanol Bennett et al. 87

Table 1

Strategies and achievements made toward synthetic methanol utilization in recent literature.

Organism Strategy Achievements

Escherichia coli (in vitro) Developed a methanol condensation cycle (MCC) by MCC produced 13.3 mM ethanol from 33.5 mM

Scale: mL [58 ] combining RuMP and NOG pathways for carbon- methanol at 80% carbon yield

conserved, redox-balanced and ATP-independent MCC produced 2.3 mM 1-butanol from 21.1 mM

higher alcohol production from methanol and sugar methanol at 50% carbon yield

phosphates in a cell-free system

13

Escherichia coli (in vivo) Performed in silico modeling to demonstrate that Observed up to 39.4% C-labeling in glycolytic and

Scale: mL [52 ] NAD-dependent MDH and RuMP pathway are best PPP intermediates, specifically hexose 6-phosphates

suited for biomass formation from methanol in E. coli Observed RuMP pathway cycling as indicated by

Characterized multiple recombinant NAD-dependent higher-order mass isotopomers

MDH and RuMP pathway enzymes in E. coli to identify No growth on methanol was reported

best candidates

Escherichia coli (in vitro) Identified an activator-independent NAD-dependent Increased methanol affinity (Km) from 132 mM in wild-

[57 ] MDH from C. necator N-1, a Gram-negative, type version to 21.6 mM in mutant variant

mesophilic, non-methylotrophic bacterium Increased methanol catalytic efficiency (Kcat/Km)

1 1 1 1

Performed directed evolution to improve enzyme from 1.6 M s in wild-type version to 9.3 M s in

kinetics toward methanol mutant variant

Decreased 1-butanol affinity (Km) from 7.2 mM in

wild-type version to 120 mM in mutant variant

Decreased 1-butanol catalytic efficiency (Kcat/Km)

1 1 1 1

from 903 M s in wild-type version to 48 M s in

mutant variant

Escherichia coli (in vitro, Constructed a scaffoldless enzyme complex of B. Improved in vitro F6P production from methanol by

in vivo) methanolicus Mdh3 and M. gastri Hps-Phi fusion using 97-fold

Scale: mL [59 ] an SH3-ligand interaction pair to improve Increased in vivo methanol oxidation rate by 9-fold

formaldehyde channeling and total in vivo methanol consumption by 2.3-fold

Incorporated E. coli lactate dehydrogenase as an

‘NADH sink’ to improve methanol oxidation kinetics

and carbon flux to F6P

Escherichia coli (in vivo) Identified a superior NAD-dependent MDH from B. Methanol supplementation improved biomass titers

Scale: mL, L [54 ] stearothermophilus to use with the B. methanolicus by up to 50% during growth with a small amount of

RuMP pathway in a DfrmA genetic background yeast extract

1 13

Supplied small amounts of yeast extract (1 g L ) as a Observed up to 53% C-labeling in glycolytic, PPP,

co-substrate to stimulate growth on methanol TCA cycle and biomass components, specifically 3PG

Incorporated heterologous pathway for naringenin Observed RuMP pathway cycling as indicated by

production from methanol higher-order mass isotopomers

Improved naringenin production by 650% over the

empty vector control in methanol and yeast extract

13

Observed up to 4.7% C-labeling in naringenin and

18% of the total naringenin pool contained at least one

carbon label

Escherichia coli (in vivo) Characterized the native formaldehyde-responsive Developed Pfrm variants that improved formaldehyde

Scale: mL [69 ] promoter (Pfrm) of the formaldehyde dissimilation induced expression up to 13-fold and formaldehyde

operon ( frmRAB) in E. coli response up to 3.6-fold

Performed directed evolution and fluorescence- Achieved autonomous and dynamic regulation of

activated cell sorting (FACS) in combination with high- methylotrophic growth in E. coli via controlling MDH

throughput sequencing (Sort-seq) to generate a Pfrm and RuMP pathway gene expression with native and

library engineered Pfrm variants

Escherichia coli (in vivo) Developed a methanol-sensing E. coli strain by Developed a rapid in vivo methanol detection system

Scale: mL [68] incorporating the MxcQ/MxcE two-component system in E. coli based on a two-component system from a

from M. organophilum XX native methylotroph

Constructed a chimeric two-component system by Achieved a dynamic response range of gene

combining the sensing domain (MxcQ) of M. expression using a range of methanol concentrations

organophilum with the transmitter domain (EnvZ) of E. (from 0.01 to 8%)

coli to control the response regulator (OmpR) of E. coli Maximum gene expression of ca. 2.5-fold was

for activation of the ompC promoter achieved with 0.05% methanol

Corynebacterium Incorporated B. methanolicus MDH and B. subtilis Achieved a methanol consumption rate of

1

glutamicum (in vivo) RuMP pathway into C. glutamicum 1.7 mM h in a glucose minimal medium

Scale: mL, mL [60 ] Supplied methanol as a co-substrate in a glucose Methanol supplementation improved biomass titers

minimal medium by up to 30% during growth in glucose minimal medium

13

Observed up to 25.7% C-labeling in M+1 mass

isotopomers of intracellular metabolites, specifically

S7P

www.sciencedirect.com Current Opinion in Biotechnology 2018, 50:81–93

88 Energy biotechnology

Table 1 (Continued )

Organism Strategy Achievements

13

Corynebacterium Incorporated B. methanolicus MDH and B. subtilis Observed up to 25% C-labeling in glycolytic and

glutamicum (in vivo) RuMP pathway into C. glutamicum for methanol PPP intermediates, specifically F6P

Scale: mL, mL [62 ] assimilation Observed RuMP pathway cycling as indicated by

Supplied methanol as a co-substrate in a glucose or higher-order mass isotopomers

13

ribose minimal medium Observed up to 15.7% C-labeling in the non-native,

Incorporated methanol assimilation pathway into a C. secreted product cadaverine in ribose minimal

glutamicum strain capable of non-native cadaverine medium

production

Corynebacterium Performed directed evolution to improve methanol Achieved improved growth rates on glucose minimal

glutamicum (in vivo) tolerance of C. glutamicum during growth on glucose medium in the presence of up to 2M methanol

Scale: mL [63] minimal medium Identified two point mutations responsible for

Performed genome sequencing to identify mutations improving methanol tolerance (A165T mutation of O-

responsible for improved methanol tolerance acetylhomoserine sulfhydrolase MetY and Q342*

mutation leading to a shortened CoA transferase Cat)

1

Saccharomyces Incorporated the methanol assimilation pathway Achieved 1.04 g L methanol consumption,

1

cerevisiae (in vivo) (AOX, CTA, DAS and DAK) from P. pastoris into S. 0.26 g L pyruvate production and a 3.13% increase

Scale: mL [64 ] cerevisiae in biomass titer in methanol minimal medium

1 1

Supplied small amounts of yeast extract (1 g L ) as a Improved methanol consumption to 2.35 g L and

co-substrate to stimulate growth on methanol biomass titer by 11.7% during growth with a small

amount of yeast extract

the importance of sustained Ru5P levels for methanol B. methanolicus (Table 1) [52 ]. The engineered E. coli

13

utilization. Although methanol conversion was achieved, exhibited up to 39.4% C-labeling in glycolytic

productivity decreased after five hours, suggesting insta- and PPP intermediates. RuMP pathway cycling

bility of intermediates. It was hypothesized that produc- was demonstrated as higher-order mass isotopomers

tivity and product titers could be improved by achieving were observed. Although methanol assimilation was

higher fluxes via protein engineering and/or media opti- achieved, no growth on methanol was reported,

mization, and MDH was identified as a critical enzyme for suggesting limitations downstream of methanol

improvement. oxidation.

Price et al. constructed a scaffoldless enzyme complex Whitaker et al. reported methylotrophic growth of

composed of Mdh3 from B. methanolicus and an HPS-PHI engineered E. coli by sourcing a superior MDH from

fusion protein from Mycobacterium gastri (Table 1) [59 ]. B. stearothermophilus, which was expressed with the

This complex promoted efficient formaldehyde channel- RuMP pathway from B. methanolicus in a DfrmA back-

ing to improve carbon flux from methanol to F6P. An ground (Table 1) [54 ]. Methylotrophic growth was

‘NADH sink’ was also developed by incorporating the achieved with a small amount of yeast extract. Upon

LDH from E. coli, which catalyzes the NADH-dependent yeast-extract exhaustion, growth was sustained on

reduction of pyruvate to lactate, to scavenge the NADH methanol for ca. 64 h, during which time ca. 10 mM

1 1

from methanol oxidation, preventing formaldehyde methanol was consumed at a rate of 19 mg gDW h ,

reduction. The complex with LDH improved in vitro significantly less than that of native methylotrophs,

F6P production by 97-fold compared to unassembled further suggesting limitations downstream of methanol

enzymes, and improvements were also realized in vivo oxidation. Methanol supplementation improved bio-

as the complex increased methanol uptake rate by 9-fold mass titers by nearly 50% in bioreactors, during which

1

and total methanol consumption by 2.3-fold compared to time a biomass yield of 0.344 gDW g methanol was

unassembled enzymes. The discrepancy between in vitro achieved, comparable to that of native methylotrophs.

13

and in vivo improvements highlights the difficulties with Up to 53% C-labeling was observed in glycolytic, PPP

engineering complex biological systems. When transition- and tricarboxylic acid cycle intermediates, as well as

ing from cell-free to in vivo conditions, additional biological hydrolyzed biomass components. RuMP pathway

factors must be considered, for example, gene regulation, cycling was also demonstrated as higher-order mass

that may be assumed negligible during in vitro studies. isotopomers were observed. By incorporating the nar-

ingenin pathway into engineered E. coli, naringenin

Engineering Escherichia coli to assimilate production was improved 650% over the empty vector

13

methanol for in vivo growth and metabolite control in C-methanol and yeast extract. Up to 4.7%

13

production average C-labeling was observed in naringenin with

13

Muller et al. reported in vivo C-methanol assimilation 18% of the total naringenin pool containing at least one

in E. coli via incorporation of Mdh2, HPS and PHI from carbon label.

Current Opinion in Biotechnology 2018, 50:81–93 www.sciencedirect.com

Bioconversion of methane and methanol Bennett et al. 89

Other synthetic methylotrophs for in vivo in variants with improved promoter activity. Methylo-

methanol assimilation and metabolite trophic growth of engineered E. coli was achieved with a

production small amount of yeast extract when mdh, hps and phi were

Witthoff et al. demonstrated in vivo methanol assimila- autonomously and dynamically regulated using native

tion in C. glutamicum using a similar strategy as those and engineered Pfrm variants.

used in E. coli (Table 1) [60 ]. The engineered strain

1

exhibited a methanol uptake rate of 1.7 mM h and a Selvamani et al. developed a methanol-sensing E. coli

30% improvement in biomass titer in glucose minimal strain by combining the sensing domain (MxcQ) of

13

medium. Up to 25.7% C-labeling in M+1 mass iso- Methylobacterium organophilum with the transmitter

topomers was observed in intracellular metabolites using domain (EnvZ) of E. coli to control the response regulator

a formaldehyde dissimilation deficient strain, con- (OmpR) of E. coli for activation of the ompC promoter

structed via deletion of acetaldehyde dehydrogenase (Figure 3, Table 1) [68]. This resulted in a rapid in vivo

(ald) and mycothiol-dependent formaldehyde dehydro- methanol detection system that exhibited a dynamic

genase (adhE) [61]. Lebmeier et al. engineered C. glu- range of gene expression in response to a wide range

13

tamicum to convert C-methanol into the non-native, of methanol concentrations (0.01–8%). ompC gene expres-

secreted product cadaverine using a similar strategy sion was improved a maximum of ca. 2.5-fold during

13

(Table 1) [62 ]. Up to 15.7% C-labeling in cadaverine exposure to 0.05% methanol. Ganesh et al. reported a

was observed in ribose minimal medium. C. glutamicum similar strategy that combined the sensing domain

was also evolved for improved methanol tolerance (MxaY) of Paracoccus denitrificans with the transmitter

(Table 1) [63]. domain (EnvZ) of E. coli [70].

Dai et al. demonstrated in vivo methanol assimilation in Strategies to improve ribulose 5-phosphate

the non-methylotrophic yeast Saccharomyces cerevisiae by (Ru5P) (re)generation

integrating AOX, catalase (CAT), dihydroxyacetone A critical limitation of synthetic methylotrophy is ineffi-

synthase (DAS) and dihydroxyacetone kinase (DAK), cient Ru5P regeneration. One strategy to improve Ru5P

all from Pichia pastoris, into the chromosome (Table 1) regeneration involves refactoring the expression of native

[64 ]. DAS and DAK compose the xylulose monopho- PPP genes using native or engineered Pfrm promoters or a

sphate (XuMP) pathway for formaldehyde assimilation methanol-sensing system (Figure 3) [68,69 ], which

1

[65]. The engineered strain consumed 1.04 g L meth- would upregulate gene expression during growth on

1

anol, produced 0.26 g L pyruvate and exhibited a methanol, emulating native methylotrophs and providing

3.13% improvement in biomass titer in methanol mini- sufficient flux for Ru5P regeneration. This strategy is

mal medium. Yeast extract supplementation improved readily applicable to other target genes as well that

1

methanol consumption to 2.35 g L and biomass titer may be later identified as important for synthetic

by 11.7%, consistent with previous findings in E. coli methylotrophy.

[54].

Another strategy to improve Ru5P regeneration is via

Developing methanol and formaldehyde overexpression of heterologous PPP enzymes. B. metha-

responsiveness in synthetic methylotrophs nolicus contains plasmid homologs of chromosomal non-

One limitation of synthetic methylotrophy is the inabil- oxidative PPP enzymes that have evolved to favor the

ity to regulate gene expression in response to methanol production of Ru5P from F6P for methanol assimilation

and/or formaldehyde, which leads to reduced gene (Figure 2) [71]. Overexpression of heterologous, methy-

expression and metabolic activity during growth on lotrophic PPP enzymes improves the kinetics and favor-

methanol [66]. Native methylotrophs regulate gene ability of Ru5P regeneration for improved methanol

expression via methanol-responsive and/or formalde- utilization in E. coli [66]. Expression of these genes could

hyde-responsive promoters or systems [67,68]. Upregu- also be refactored for methanol-responsiveness and/or

lation of RuMP pathway and PPP genes in B. methano- formaldehyde-responsiveness.

licus during methylotrophic growth improves methanol

tolerance and uptake rate [67]. Regulating gene expres- Another strategy to improve Ru5P generation involves

sion in response to methanol is a critical component for deletion of phosphoglucose isomerase ( pgi). Glycolysis is

synthetic methylotrophy. the primary route for glucose catabolism in wild-type

E. coli, limiting carbon flux through the PPP [66]. Dele-

Rohlhill et al. demonstrated methanol-responsiveness in tion of pgi reroutes glucose carbon flux through the

E. coli by refactoring mdh, hps and phi expression with the oxidative PPP for sustained Ru5P generation and

native formaldehyde-responsive promoter (Pfrm) of the improves methanol utilization in E. coli (Figure 2) [66].

formaldehyde dissimilation operon ( frmRAB) in E. coli Furthermore, deletion of pgi acts to conserve methanol

(Figure 3, Table 1) [69 ]. Directed evolution and Sort- carbon since F6P cannot be metabolized via decarboxyl-

Seq were performed to construct a Pfrm library, resulting ation reactions in the oxidative PPP.

www.sciencedirect.com Current Opinion in Biotechnology 2018, 50:81–93

90 Energy biotechnology

Figure 3

Methanol Formaldehyde MDH

P P Pfrm

MxcQ EnvZ

mdhhps phi

OmpR Episomal Expression PompC

rpe tkt

PompC Chromosomal Expression

fba glpX pfk

Current Opinion in Biotechnology

Strategies to regulate gene expression in response to methanol and formaldehyde. When the methanol-sensing domain (MxcQ) of

Methylobacterium organophilum is fused with the transmitter domain (EnvZ) of E. coli, the response regulator (OmpR) of E. coli activates the

ompC promoter (PompC). Upon methanol oxidation via MDH, formaldehyde activates the formaldehyde-responsive promoter (Pfrm) of E. coli. Genes

listed serve as examples: methanol dehydrogenase (mdh), hexulose phosphate synthase (hps), phosphohexulose isomerase ( phi),

phosphofructokinase ( pfk), fructose-bisphosphate aldolase ( fba), transketolase (tkt), ribulose phosphate epimerase (rpe) and sedoheptulose

bisphosphate (glpX).

Exploring amino acid metabolism to improve would further enhance synthetic methylotrophy and a

synthetic methanol assimilation strain capable of growth solely on methanol.

Since yeast extract, which is primarily composed of amino

acids, stimulates synthetic methylotrophy, the metabo- Future perspectives and recommendations

lism of all 20 amino acids and the regulatory networks in Two key limitations were identified while attempting to

which they are involved were examined [72]. It was engineer synthetic methylotrophs for methanol utiliza-

determined that co-utilization of threonine leads to tion. First, methanol oxidation is limited by MDH kinet-

improved methanol assimilation in a synthetic E. coli ics. Sourcing alternative or engineering current MDHs

methylotroph, which resulted from activation of endoge- for improved kinetics can overcome this limitation. Sec-

nous C1 metabolism via high flux from threonine to ond, methanol assimilation is limited by inefficient

glycine to serine under threonine growth conditions Ru5P regeneration. Several strategies can overcome this,

(Figure 2) [72]. To verify the phenotype, a global regula- including refactoring native PPP gene expression for

tor that regulates this pathway was identified and exam- methanol-responsiveness, incorporating a heterologous

ined. This regulator, the leucine-responsive regulatory non-oxidative PPP with improved F6P to Ru5P kinetics

protein (Lrp), represses threonine dehydrogenase and or deleting pgi to reroute glucose flux entirely toward

serine hydroxymethyltransferase, which respectively cat- Ru5P.

alyze the conversion of threonine to glycine and glycine

to serine. For improved methanol assimilation, these Since growth on methanol as the sole carbon source has

pathways should be active. Therefore, the lrp gene was yet been achieved, future studies should explore various

deleted in a synthetic E. coli methylotroph, which cellular mechanisms, not just those limited to methanol

resulted in improved growth and methanol assimilation oxidation and Ru5P regeneration. For example, transcrip-

compared to the lrp-intact strain [72]. This study provides tomic approaches may identify gene(s) that are indirectly

the basis for exploring other regulatory networks that involved in methanol metabolism but could prove

Current Opinion in Biotechnology 2018, 50:81–93 www.sciencedirect.com

Bioconversion of methane and methanol Bennett et al. 91

12. Ge XM, Yang LC, Sheets JP, Yu ZT, Li YB: Biological conversion

essential for achieving growth on methanol. Another

of methane to liquid fuels: status and opportunities. Biotechnol

example is to evolve current methylotrophic phenotypes Adv 2014, 32:1460-1475.

for improved properties and identify the mutations

13. Taher E, Chandran K: High-rate, high-yield production of

responsible, especially if autonomous growth on metha- methanol by ammonia-oxidizing bacteria. Environ Sci Technol

2013, 47:3167-3173.

nol as the sole carbon source is achieved. Another

approach is to screen genomic or enriched metagenomic 14. Arnold F, Meinhold P, Peters MW, Fasan R, Chen MMY: Alkane

oxidation by modified hydroxylases. US Patent 2017/0183689 A1.

libraries from native methylotrophs under conditions that

Pasadena, CA, US: The California Institute of Technology; 2017.

assess the impact of multiple genes combinatorially

15. Karthikeyan OP, Chidambarampadmavathy K, Cires S,

[73,74]. Endogenous C1 metabolism could also be

Heimann K: Review of sustainable methane mitigation and

explored as an alternative route for methanol utilization biopolymer production. Crit Rev Environ Sci Technol 2015,

45:1579-1610.

(Figure 2) [72]. Since methanol likely causes carbon

16. Khosravi-Darani K, Mokhtari ZB, Amai T, Tanaka K: Microbial

starvation responses in non-methylotrophic organisms,

production of poly(hydroxybutyrate) from C1 carbon sources.

these mechanisms could be explored as well.

Appl Microbiol Biotechnol 2013, 97:1407-1424.

17. Zuniga C, Morales M, Le Borgne S, Revah S: Production of poly-

beta-hydroxybutyrate (PHB) by Methylobacterium

Acknowledgement organophilum isolated from a methanotrophic consortium in a

two-phase partition bioreactor. J Hazard Mater 2011, 190:876-

Financial support from ARPA-E through contract no. DE-AR0000432 is 882.

gratefully acknowledged.

18. Strong PJ, Xie S, Clarke WP: Methane as a resource: can the

methanotrophs add value? Environ Sci Technol 2015, 49:4001-

4018.

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