Available online at www.sciencedirect.com ScienceDirect 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 Â 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 Â 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- À1 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-