Methanol Condensation Cycle
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Building carbon–carbon bonds using a biocatalytic methanol condensation cycle Igor W. Bogorada,b,1, Chang-Ting Chena,1, Matthew K. Theisena,b,1, Tung-Yun Wua,1, Alicia R. Schlenza, Albert T. Lama, and James C. Liaoa,b,c,2 Departments of aChemical and Biomolecular Engineering and bBioengineering, University of California, Los Angeles, CA 90095; and cUCLA–DOE Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095 Edited by Lonnie O. Ingram, University of Florida, Gainesville, FL, and approved October 2, 2014 (received for review July 16, 2014) Methanol is an important intermediate in the utilization of natural must be lost during the production of acetyl-CoA, the precursor gas for synthesizing other feedstock chemicals. Typically, chemical for n-alcohols. approaches for building C–C bonds from methanol require high Here we constructed an enzymatic cycle to achieve the cata- temperature and pressure. Biological conversion of methanol to lytic condensation of methanol to higher alcohols with complete longer carbon chain compounds is feasible; however, the natural carbon conservation and ATP independence. This pathway is biological pathways for methanol utilization involve carbon diox- modified from the combination of RuMP coupled with a non- ide loss or ATP expenditure. Here we demonstrated a biocatalytic native pathway, nonoxidative glycolysis (NOG) (12) shown in pathway, termed the methanol condensation cycle (MCC), by com- Fig. 1A. However, the combined pathway, which we call the bining the nonoxidative glycolysis with the ribulose monophos- methanol condensation cycle (MCC), can be simplified to be- phate pathway to convert methanol to higher-chain alcohols or come completely ATP independent. A thorough list of reactions other acetyl-CoA derivatives using enzymatic reactions in a carbon- is given in Table S1. The first step in MCC is the oxidation of conserved and ATP-independent system. We investigated the robust- methanol to formaldehyde. This reaction can be catalyzed by ness of MCC and identified operational regions. We confirmed that three classes of enzymes: alcohol oxidase (13), quinone-dependent 13 the pathway forms a catalytic cycle through C-carbon labeling. methanol dehydrogenase (14), and NAD-dependent methanol de- With a cell-free system, we demonstrated the conversion of methanol hydrogenase (15). Only the last class of enzymes provides the cor- to ethanol or n-butanol. The high carbon efficiency and low operating rect reducing equivalents, which can be used to drive the reductive temperature are attractive for transforming natural gas-derived portion of MCC for ethanol or n-butanol formation (Fig. S2). methanol to longer-chain liquid fuels and other chemical derivatives. The core portion of MCC is the biochemical condensation of two formaldehydes with a CoA to form acetyl-CoA and water methanol metabolism | metabolic engineering | cell-free synthesis | (Fig. 1 B and C). Similar to the initial steps in the RuMP pathway, bio-ethanol | bio-butanol formaldehyde combines with ribulose-5 phosphate (Ru5P) to produce hexulose-6-phosphate, which is isomerized to fructose- ethanol is industrially produced from synthetic gas-derived 6-phosphate (F6P). The formaldehyde assimilation is catalyzed Molefins and alkanes (1–7). These reactions typically involve by hexulose-6-phosphate synthase (Hps) and phosphohexulose high temperatures and pressures that require large capital in- isomerase (Phi), respectively. A similar conversion could be achieved vestment (8, 9). The condensation of methanol to higher-chain using dihydroxyacetone synthase (12) and fructose-6-phosphate alcohols such as ethanol or n-butanol is thermodynamically favor- aldolase (16) as shown in Fig. S2. Once fructose-6-phosphate able (ΔG°′ = −68 and −182 kJ/mol, respectively), but the direct condensation of methanol to higher-chain alcohols has been quite Significance challenging. Using the Guerbet reaction, methanol can upgrade n- short alcohols (such as propanol) to longer alcohols; however, With the recent discoveries of large reserves of natural gas, the methanol cannot self-couple (10). Metal acetylides can convert efficient utilization of one-carbon compounds for chemical syn- methanol to isobutanol, although this process was demonstrated thesis would reduce the raw material cost for the petroleum- to be noncatalytic (11). based chemical industry. Methanol is produced industrially from Nature has evolved several distinct ways to assimilate methanol methane and is a feedstock chemical for the synthesis of higher to form metabolites necessary for growth. In principle, metabo- carbon compounds. However, current chemical synthesis of lites resulting from these methylotrophic pathways can be used higher carbon compounds from methanol requires high temper- to form higher-chain alcohols, although inherent pathway limi- ature and pressure. Natural biological pathways for methanol tations prevent complete carbon conservation (Fig. S1). In the utilization are carbon and ATP inefficient. Here we constructed ribulose monophosphate pathway (RuMP), three formaldehydes a synthetic biocatalytic pathway that allows the efficient con- condense to pyruvate, which is decarboxylated to form acetyl-CoA version of methanol to higher-chain alcohols or other higher and CO2, reducing the carbon efficiency to 67%. The serine path- carbon compounds without carbon loss or ATP expenditure. way requires an external supply of ATP to drive otherwise unfa- The high carbon efficiency and favorable operating conditions vorable reactions. Similarly, oxidation of methanol to CO2 followed are attractive for industrial applications. by CO2 fixation using the Calvin–Benson–Bassham (CBB) cycle also requires additional ATP. To generate the required ATP input, Author contributions: I.W.B., C.-T.C., M.K.T., T.-Y.W., and J.C.L. designed research; I.W.B., extra carbon must be spent to drive oxidative phosphorylation. To C.-T.C., M.K.T., T.-Y.W., A.R.S., and A.T.L. performed research; I.W.B., C.-T.C., M.K.T., T.-Y.W., our knowledge, natural methylotrophs are not capable of using the and J.C.L. wrote the paper; and I.W.B. and J.C.L. invented the MCC pathway. reductive acetyl-CoA pathway, which can produce acetyl-CoA The authors declare no conflict of interest. without carbon loss or ATP requirement through carbon reas- This article is a PNAS Direct Submission. similation after complete oxidation of methanol. This route is Freely available online through the PNAS open access option. extremely oxygen sensitive and difficult to engineer due to the 1I.W.B., C.-T.C., M.K.T., and T.-Y.W. contributed equally to this work. complex cofactors involved, and achieving carbon conservation 2To whom correspondence should be addressed. Email: [email protected]. would require that CO2 produced in methanol oxidation is com- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pletely reassimilated. Thus, in all native pathways some carbon 1073/pnas.1413470111/-/DCSupplemental. 15928–15933 | PNAS | November 11, 2014 | vol. 111 | no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1413470111 Downloaded by guest on October 2, 2021 A B C 2 CH3OH Major MCC Mode with Xpk Minor MCC Mode with Fpk 2 CH O 2 CH O CO2 2 NADH 2 2 Rpe Rpe 2 Ru5P X5P 2 Ru5P X5P ATP 2 CH2O Hps Hps Rpi Rpi 2 H6P 2 H6P R5P R5P RuMP Phi Tkt Phi Tkt MCC 2 F6P G3P 2 F6P G3P G3P Fpk Tkt NOG E4P S7P E4P S7P Tal Tal X5P AcP AcCoA Xpk Pta 2 NADH AcP AcCoA AcCoA Higher Alcohols Pta Fig. 1. Conversion of methanol to higher n-alcohols. (A) The MCC is the combination of RuMP with NOG that bypasses ATP dependency. See Table S1 for details. (B) The major MCC mode uses the more active X5P-phosphoketolase (Xpk). (C) The minor MCC mode can achieve the same result with the less active F6P-phosphoketolase (Fpk). is formed, half of the molecules are saved for carbon rear- fructose-1,6-bisphosphate aldolase (Fba), and fructose-1, rangement to regenerate Ru5P, whereas the rest are cleaved 6-bisphosphatase (Fbp). irreversibly by phosphoketolase (F/Xpk). This promiscuous A unique feature of MCC is the conservation of phosphates in enzyme can cleave either F6P (Fpk) or X5P (Xpk) to acetyl- the cycle. The sum of all sugar phosphates remains constant phosphate and its corresponding sugar phosphate (17). Although throughout the catalytic cycle as long as no nonenzymatic degra- Xpk activity is higher, both Xpk and Fpk can be used to achieve dation reactions occur to deplete the sum. No new sugar phosphate the same net conversion. F/Xpk is able to conserve ATP by intermediates are generated or degraded by pathway reactions. phosphorylating the two carbon keto group cleaved from F6P or Other pathways like CBB and RuMP do not conserve phosphate groups and instead lose these high energy bonds by the action of X5P using inorganic phosphate. The produced acetyl-phosphate phosphatases. The final phase in MCC involves the reduction of can be readily converted to acetyl-CoA by the phosphate ace- acetyl-CoA to alcohols (Fig. S3). Ethanol can be directly produced tyltransferase (Pta). By avoiding pyruvate decarboxylation to from acetyl-CoA by an acylating aldehyde dehydrogenase and an form acetyl-CoA, no carbon is lost. The E4P produced then alcohol dehydrogenase. The biosynthesis of n-butanol can be ac- reacts with F6P through a series of reactions involving transaldolase complished by a pathway previously established (18) that involves (Tal), transketolase (Tkt), ribose-5 phosphate isomerase (Rpi), reactions similar to the one use by Clostridia. and ribulose 5-phosphate epimerase (Rpe), to regenerate two molecules of Ru5P to complete the cycle. MCC does not in- Results volve an essential RuMP enzyme (phosphofructokinase) and To demonstrate the feasibility of MCC, we first focused on avoids three NOG enzymes: triose phosphate isomerase (Tpi), the core portion from formaldehyde to acetate using purified ABCEMRA Analysis Simulated Acetate Isotope DistribuƟon In vitro ProducƟon of Acetate 4 4 60 60 1 61 3 61 3 62 62 Total 2 Total R,M 2 Y 0.5 (mM) c Acid c Acid (mM) c Acid Ɵ Ɵ 1 1 Ace Ace 0 0 0 0.1 1 10 0.5 5 50 500 5000 0.5 5 50 500 5000 Fold Change in Enzyme F/Xpk (RelaƟve Units) F/Xpk ConcentraƟon (mg/L) Fig.