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Scaffoldless Engineered Enzyme Assembly for Enhanced Methanol Utilization

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Scaffoldless Engineered Enzyme Assembly for Enhanced Methanol Utilization Scaffoldless engineered enzyme assembly for enhanced methanol utilization J. Vincent Pricea, Long Chena, W. Brian Whitakera,b, Eleftherios Papoutsakisa,b, and Wilfred Chena,1 aDepartment of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716; and bThe Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711 Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved September 27, 2016 (received for review February 4, 2016) Methanol is an important feedstock derived from natural gas and organisms requires electrons in the form of NADH and culture can be chemically converted into commodity and specialty chem- conditions largely microaerobic or anaerobic (7, 10). Thus, or- icals at high pressure and temperature. Although biological ganisms that can grow anaerobically or microaerobically and NAD- conversion of methanol can proceed at ambient conditions, there dependent Mdhs are essential for effective conversion of methanol + is a dearth of engineered microorganisms that use methanol to to desirable metabolites (7). Although NAD(P) -dependent Mdhs produce metabolites. In nature, methanol dehydrogenase (Mdh), are the best candidates for engineering synthetic methylotrophs which converts methanol to formaldehyde, highly favors the reverse like Escherichia coli, these enzymes have poor predicted thermo- reaction. Thus, efficient coupling with the irreversible sequestration dynamic properties and are thus dependent on maintaining low of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and intracellular formaldehyde and NAD(P)H concentrations to 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An ensure the reaction continues to favor methanol oxidation (7). emerging strategy to promote efficient substrate channeling is to Formaldehyde, produced from the oxidation of methanol, is sub- spatially organize pathway enzymes in an engineered assembly to sequently assimilated via distinct methylotrophic pathways, among provide kinetic driving forces that promote carbon flux in a desirable which the ribulose monophosphate (RuMP) pathway is bio- direction. Here, we report a scaffoldless, self-assembly strategy to energetically the most efficient (7). The best characterized NAD + organize Mdh, Hps, and Phi into an engineered supramolecular (P) -dependent Mdhs were isolated from the thermotolerant SCIENCES enzyme complex using an SH3–ligand interaction pair, which en- Bacillus methanolicus MGA3 strain, which harbors two chromo- hances methanol conversion to fructose-6-phosphate (F6P). To in- somal mdh genes and one plasmid-borne mdh gene (6, 9). In par- APPLIED BIOLOGICAL crease methanol consumption, an “NADH Sink” was created using ticular, MGA3 Mdh3, a chromosomal mdh, is a decameric enzyme Escherichia coli lactate dehydrogenase as an NADH scavenger, and has been shown to possess broad substrate specificity when thereby preventing reversible formaldehyde reduction. Combina- tested with a range of primary alcohols (9, 11, 12). tion of the two strategies improved in vitro F6P production by 97- In the widespread methylotrophic RuMP pathway for form- fold compared with unassembled enzymes. The beneficial effect of aldehyde assimilation (Fig. 1A) (7, 13), D-arabino-3-hexulose supramolecular enzyme assembly was also realized in vivo as the 6-phosphate (6HP) is first produced by a condensation reac- ENGINEERING engineered enzyme assembly improved whole-cell methanol con- tion between formaldehyde and ribulose 5-phosphate (Ru5P) by sumption rate by ninefold. This approach will ultimately allow di- 3-hexulose-6-phosphate synthase (Hps) (6, 13, 14). This is fol- rect coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metab- lowed by the isomerization of heluxose 6-phosphate (H6P) by olites from methanol. 6-phospho-3-hexuloseisomerase (Phi) (6, 13, 14) into fructose 6-phosphate (F6P), which can either enter central metabolism methane | methylotophs | supramolcular | scaffold | substrate channeling Significance ossil fuels are recognized as a limited energy source given the Fworld’s growing population and energy demands (1–3). Bio- Methane, the major natural-gas component, can be converted fuels and bio-based chemicals have gained tremendous traction industrially to syngas (a mixture of CO2, CO, and H2), which is over the past decade as a means to produce both an alternative used to produce methanol, an important industrial precursor energy source as well as to replace bulk chemical production that for producing commodity and specialty chemicals. Unlike is heavily reliant upon petrochemicals. Methanol, which can be methanol’s chemical conversion, which requires high tempera- produced inexpensively from natural gas or renewably through ture and pressure, its biological conversion, proceeding through reduction of carbon dioxide by hydrogen (2, 4), can serve as a its oxidation to formaldehyde, takes place at ambient condi- feedstock to produce biofuels, amino acids, and polymers (5, 6). tions but suffers from relative low rates. As metabolite bio- Biological conversion of methanol to value-added chemicals may synthesis by industrial organisms utilizes NADH as an electron provide more favorable, economic operating conditions (i.e., donor, methanol oxidation must be carried out by NAD-dependent lower temperature and pressure) than more traditional catalytic methanol dehydrogenases, which have poor thermodynamic conversions. characteristics at mesophilic conditions. To solve this problem, Methylotrophic bacteria consist of a wide array of organisms we developed an engineering strategy for supramolecular en- zyme assembly to dramatically enhance the carbon flux from that can use one-carbon (C1) compounds, and notably methane methanol to the key intermediate fructose-6-phosphate. and methanol, as their sole source of carbon and energy (7). The ability to use methanol is initiated by its oxidation to formaldehyde Author contributions: J.V.P., E.P., and W.C. designed research; J.V.P., L.C., and W.B.W. by methanol dehydrogenase (Mdh) encoded by an mdh gene (8). performed research; J.V.P. contributed new reagents/analytic tools; J.V.P., W.B.W., E.P., There are two classes of Mdhs among methylotrophic bacteria: the and W.C. analyzed data; and J.V.P., W.B.W., E.P., and W.C. wrote the paper. well-studied pyrroloquinoline quinone (PQQ)-containing Mdhs, The authors declare no conflict of interest. which are found in the most ubiquitous, Gram-negative, strictly This article is a PNAS Direct Submission. + aerobic methylotophs, and the NAD(P) -dependent, cytoplasmic 1To whom correspondence should be addressed. Email: [email protected]. Mdhs commonly found in thermophilic Gram-positive bacteria This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (9). Generation of most useful metabolites produced by industrial 1073/pnas.1601797113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1601797113 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 promising approach to improve the efficiency of cascade reac- tions (19–22). This spatial organization provides more stable enzyme structures, facilitates substrate channeling between ac- tive sites, and prevents the buildup of toxic intermediates (23– 25). The use of driving forces such as ATP generation or NADH consumption as a kinetic control element to enable directional operation of highly reversible enzyme reactions has also been reported (26, 27). We hypothesize that engineered supramolec- ular enzyme assemblies can be similarly created as a kinetic trap to enable fast and efficient methanol utilization. Unlike other reports based on the use of a scaffold (24, 28), which tend to produce large and disordered multienzyme complexes, we take advantage of the decameric nature of Mdh3 from B. methanolicus to construct a scaffoldless supramolecular enzyme complex com- posed of Mdh3 and a previously validated Hps–Phi fusion from Mycobacterium gastri to enhance methanol conversion to F6P. Our ability to enhance F6P synthesis is ideally suited for the recently proposed methanol condensation cycle (29), which combines the enzymes from the RuMP pathway and the nonoxidative glycolysis pathway for the in vitro synthesis of platform chemicals from methanol. Results High Ratio of Hps to Mdh Enhances in Vitro Methanol Utilization. B. methanolicus MGA3 Mdh3 has a reported kcat/Km value roughly 1,000-fold lower for methanol oxidation than for formal- dehyde reduction (9), indicating that a significant kinetic barrier must be overcome to achieve efficient methanol utilization. In methylotrophs, the irreversible conversion of formaldehyde by Fig. 1. (A) A schematic of the Mdh–RuMP pathway for methanol assimila- Hps and Phi is the driving force behind shifting the pathway tion. (B) H6P formation with increasing ratios of Mdh3–sSH3lig:Hps or Mdh3– equilibrium toward F6P formation. In B. methanolicus,ithasbeen sSH3lig–SH3–Hps enzyme complexes. Purified Mdh–sSH3lig was mixed with suggested that the level of Hps/Phi can impact methanol con- equal molar ratios of purified Hps ranging from 1:1–1:10. H6P was assayed sumption and cell growth (18), and the stoichiometric effect of according to the previously established method by Arfman (39). Error bars Mdh to Hps:Phi on the methanol consumption pathway remains represent the SD of at least three replicate experiments. unexplored either in native or synthetic
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