In Vitro Reconstitution and Optimization of the Entire Pathway to

Jan 12, 2017 - Glucose and fatty acids play essential physiological roles in nearly all living organisms, and the pathway that converts glucose into f...
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In Vitro Reconstitution and Optimization of the Entire Pathway to Convert Glucose into Fatty Acid Zheng Liu,†,§ Yuchen Zhang,‡,§ Xiaoge Jia,‡ Mengzhu Hu,‡ Zixin Deng,‡ Yancheng Xu,*,† and Tiangang Liu*,‡ †

Department of Endocrinology, Zhongnan Hospital of Wuhan University, Wuhan 430071, China Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China

‡

S Supporting Information *

ABSTRACT: Glucose and fatty acids play essential physiological roles in nearly all living organisms, and the pathway that converts glucose into fatty acid is pivotal to the central metabolic network. We have successfully reconstituted a pathway that converts glucose to fatty acid in vitro using 30 purified proteins. Through systematic titration and optimization of the glycolytic pathway and pyruvate dehydrogenase, we increased the yield of free fatty acid from nondetectable to a level that exceeded 9% of the theoretical yield. We also reconstituted the entire pentose-phosphate pathway of Escherichia coli and established a pentose phosphate−glycolysis hybrid pathway, replacing GAPDH to enhance NADPH availability. Our efforts provide a useful platform for research involving these core biochemical transformations. KEYWORDS: glycolysis, pyruvate dehydrogenase, fatty acid biosynthesis, glucose, fatty acids, in vitro reconstitution

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ways, have been extensively investigated in model organisms.14−17 For example, concentrations of pathway intermediates, metabolic fluxes, and stress pathway dynamics have been analyzed in vivo via gene knockout.18 Despite these in vivo studies, the complexity of living systems hinders the complete understanding and recreation of these pathways.19 In order to gain a deeper understanding of these central metabolic pathways or specific enzymatic reactions, scientists have sought to excise complete enzymatic pathways from their native cellular environments.20 In studying the fermentation pathways of yeast using a yeast cell-free extract in 1897, Buchner et al. provided the earliest example of the concept of in vitro reconstitution of the glycolytic pathway.21 Welch and Scopes later reconstructed a glycolytic pathway using individually purified yeast enzymes in 1985.22 More recently, cell-free lysate-based glycolysis23,24 and synthetic glycolytic pathways have been manipulated in vitro for various purposes, such as enzyme prototyping and chemical manufacturing.25,26 In 1975, Reed et al. reconstituted the Escherichia coli PDC, one of the largest enzyme complexes across all known organisms, which catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA.27 Following this, Snoep et al. isolated and characterized the PDC of Enterococcus faecalis in 1993.28 In the fatty acid biosynthetic pathway, Yu et al. used purified protein components to reconstitute the E. coli FAS, which is

lucose and fatty acids are two of the most ubiquitous small molecules, appearing in nearly all life forms. Glucose is a crucial and common fuel used by almost all organisms, most of which process glucose using the glycolytic pathway (glycolysis or Embden−Meyerhof pathway). Fatty acids are crucial components of cell physiology as they act as structural components for membranes, energy storage molecules, precursors of hormones and intracellular messengers, and protein post-translational modifications moieties.1 In addition to these fundamental biological roles, glucose and fatty acids are also closely associated with the genesis and development of many diseases and metabolic disorders such as obesity, diabetes mellitus,2,3 and cardiovascular diseases.4,5 Most cancer cells exhibit increased aerobic glycolysis, known as the Warburg effect6,7 leading to interest in the development of novel glycolytic inhibitors for anticancer treatment.8 The essential role of fatty acids in membrane structure has focused attention on targeting the fatty acid biosynthetic pathway for development of novel antibacterial therapeutics.9,10 Moreover, due to the high energy density and chemical variability of fatty acids, attempts to engineer microbes to efficiently convert glucose into fatty acid derivatives have long been undertaken to produce renewable fuels.11−13 Owing to their ubiquity and importance, the central metabolic molecules and pathways involving glucose and fatty acid, such as glycolysis, fatty acid synthase (FAS), and pyruvate dehydrogenase complex (PDC), which acts as the essential interface between glycolysis and downstream metabolic path© 2017 American Chemical Society

Received: November 18, 2016 Published: January 12, 2017 701

DOI: 10.1021/acssynbio.6b00348 ACS Synth. Biol. 2017, 6, 701−709

Research Article

ACS Synthetic Biology

Figure 1. Schematic representation of the in vitro reconstitution system for the conversion of glucose to fatty acid. The complete pathway consists of three major parts: glycolytic pathway (red), pyruvate dehydrogenase complex (blue), and acetyl-CoA carboxlyase and fatty acid synthase (green).

(E1), AceF (E2) and Lpd(E3) of the PDC; AccA, AccB, AccC, and AccD of the ACC; FabA, FabB, FabD, FabF, FabG, FabH, FabI, FabZ, and ACP of FAS; and TesA, which is responsible for chain release (Figure S1). The feasibility of the reconstitution system and production of fatty acid from glucose were confirmed by mixing these purified components, cofactors, and 14C-labeled substrates in a testtube. Various substrates including 14C-labeled glucose, pyruvate, and acetylCoA were used in radioactive assays to validate the function of each part of the reconstitution system. Reactions with each substrates resulted in a single spot following radioactive thinlayer chromatography (TLC). The corresponding Rf values (approximately 0.37) were consistent with that of a 14C-labeled palmitic acid standard (Figure S2), indicating that fatty acid can be produced in this system from glucose and two other substrates. Notably, the rate of reaction was significantly decreased when 14C-labeled glucose or pyruvate were used individually as the substrate. These results indicate that the upstream glycolytic pathway and PDC may be the rate-limiting steps, providing a target for further optimization. Optimization and Titration of Components in the Glycolytic Pathway. In our initial reconstitution system, the production of fatty acid was relatively low and was only detectable in radioactive assays, as the amount produced was less than the GC−MS detection limit. In order to optimize the entire system step-by-step, we divided the pathway into its three major components: glycolysis, PDC, and FAS (Figure 1). Three enzymatic reactions have been reported to be ratelimiting steps and key regulatory points in glycolysis: the phosphorylation of glucose, catalyzed by glucokinase (Glk); the second phosphorylation of fructose-6-phosphate, catalyzed by two isozymes (PfkA and PfkB) of 6-phosphofructokinase in the preparatory phase; and the final substrate-level phosphorylation that forms pyruvate and ATP, catalyzed by two isozymes (PykF and PykA) of pyruvate kinase.33−36 In order to enhance the performance of glycolysis in our reconstitution system, we performed a titration experiment to examine the effect of the concentrations of enzymes that catalyze these reactions. Concentrations of glucokinase and 6-phosphofructokinase were varied from 0.1 to 10 μM, and that of pyruvate kinase was varied from 0.5 to 30 μM with a 1:1 mol-ratio for each isozyme (i.e., 1.0 μM 6-phosphofructokinase, 1.0 μM PfkA and

comprised of nine subunits [FabA, FabB, FabD, FabF, FabG, FabH, FabI, FabZ, and the acyl carrier protein (ACP)], along with a terminal thioesterase (TesA) responsible for chain release.29 Xiao et al. have extended the FAS in vitro system by including the four-subunit ATP-dependent acetyl-CoA carboxylase (ACC comprising AccA, AccB, AccC, and AccD).30 Despite these significant advancements, the in vitro reconstitution of the entire pathway to convert glucose to fatty acid has not yet been achieved. Here, for the first time, we successfully reconstituted in vitro the entire 19-step pathway that converts glucose to fatty acid. This consists of 30 enzymes from three major systems: the glycolytic pathway, the PDC, and the fatty acid biosynthetic pathway (Figure 1). Systematic optimization was performed to enhance the efficiency of the glycolytic pathway, and we investigated the contributions of two pairs of isozymes in this pathway. In vitro self-assembling and optimization of PDC from E. coli and E. faecalis were performed. In order to enhance NADPH availability, we replaced the NAD+-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP+-dependent GAPDH from Clostridium acetobutylicum. Furthermore, the entire pentose phosphate pathway (PPP) from E. coli was successfully reconstituted to create a hybrid pathway that can convert glucose to pyruvate using part of the glycolytic pathway to further enhance NADPH availability. On the basis of the information gleaned from our in vitro reconstitution system, we built and optimized a large enzymatic network in a testtube with high efficiency and stability. Our results may thus aid in the construction of large, complex in vitro systems, providing a platform for fundamental biochemical research.

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RESULTS AND DISCUSSION Establishment of an In vitro Reconstituted System of the E. coli Biosynthetic Pathway to Convert Glucose to Fatty Acid. We chose to derive these pathways from E. coli, which allows for each component to be overexpressed and purified and which contains one of the most well-described model pathways in biochemistry.15,16,31,32 To initiate our reconstitution of the pathway that converts glucose into fatty acid, we expressed and purified 30 recombinant proteins from E. coli: Glk, Pgi, PfkA/PfkB, FbaA, TpiA, GapA, Pgk, GpmA/ GmpM, Eno and PykA/PykF of the glycolytic pathway; AceE 702

DOI: 10.1021/acssynbio.6b00348 ACS Synth. Biol. 2017, 6, 701−709

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Figure 2. Titration of the rate-limiting enzymes in the glycolytic pathway: (A) glucokinase (Glk), (B) 6-phosphofructokinase (PfkA and PfkB), and (C) pyruvate kinase (PykF and PykA). Each assay mixture included 1.0 μM of all protein components except the one being titrated. Comparison of isozymes in the glycolytic pathway: (D) PfkA and PfkB, (E) PykF and PykA. Values represent averages (n = 3 for A, B, D and n = 2 for C, E), and error bars represent 1 s.d.

1.0 μM PfkB); concentrations of the other eight enzymes were fixed at 1.0 μM. When titrating the first step of glycolysis (the formation of glucose-6-phosphate, catalyzed by glucokinase), the initial rate of the reaction increased with increasing Glk in the range of 0.1 to 1.0 μM but then decreased from 1.0 to 10 μM (Figure 2A). This phenomenon indicates that the rates of ATP consumption and formation and of the formation of phosphorylation intermediates should be closely matched. Because initial input concentrations of glucose and ATP were fixed, an excess of glucokinase consumes ATP quickly to form excess amounts of the phosphorylation intermediates glucose-6-phosphate, fructose-6-phosphate, and 1,6-biphosphate. Inorganic phosphate is then sequestered in these intermediates, halting the subsequent ATP-synthesizing reactions. This result is consistent with findings from the reconstitution of the yeast glycolytic system.22 In the titrations of 6-phosphofructokinase and pyruvate kinase, increasing concentrations of PfkA/PfkB and PykF/PykA within a specific range yielded a 5−6-fold increase in the initial rate of reaction. Saturation occurred at protein concentrations above 2.5 μM for PfkA/PfkB (Figure 2B), while a concentration of PykF/PykA above 5 μM inhibited the initial rate of reaction (Figure 2C). On the basis of the results of titration studies, we determined that the optimal molar ratio of Glk:Pgi:PfkA:PfkB:FbaA:TpiA:GapA:Pgk:GpmA:GmpM:Eno:PykA:PykF is 1:1:2.5:2.5:1:1:1:1:1:5:5 in the glycolytic pathway. In addition to systematic titration of these enzymes, we investigated the contribution of different isozymes to the in vitro reconstituted glycolytic pathway by removing each isozyme individually (i.e., for 6-phosphofructokinase, 1.0 μM PfkA and 0 μM PfkB, followed by 1.0 μM PfkB and 0 μM PfkA). When testing the two isozymes of 6-phosphofructokinase, we found the initial rate of PfkB to be 3-fold faster than that of PfkA at an in vitro concentration of 1.0 μM (Figure 2D), despite the fact that previous biochemical research showed that the specific activity of PfkA in crude cell extract was much higher than that of PfkB.34 For pyruvate kinase, a comparison

assay was conducted in a 2-fold diluted system in which all enzyme concentrations were 0.5 μM. The initial rate of PykA was approximately 30% faster than that of PykF at 0.5 μM in vitro (Figure 2E), though previous biochemical research showed that PykA contributes little to overall pyruvate kinase activity in vivo.37,38 These differences between in vitro and in vivo enzyme activities may result from distinct activation and inhibition patterns34 as well as the differences in isozyme expression levels in vivo.39 Comparison of PDCs from E. coli and E. faecalis and Effects of Protein Ratios. After optimization of the glycolytic pathway, we turned to optimization of the second major component of the system, the PDC. The PDC exists in all known organisms. It catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA, a reaction that forms the interface between glycolysis and downstream metabolic pathways such as the tricarboxylicacid cycle (TCA or Krebs cycle) and the fatty acid biosynthetic pathway.15 In our reconstitution system, PDC produces acetyl-CoA, the building block of ACC and FAS, from the pyruvate created by glycolysis, forming one mole of NADH for each mole of acetyl-CoA. In the overall glucose-to-fatty acid pathway, the formation of one mole of acetyl-CoA yields two moles of NADH, while condensation of one molecule of malonyl-CoA consumes two molecules of NAD(P)H. In other words, there is an excess accumulation of NADH in our system during fatty acid formation. However, for many PDCs, including that of E. coli, the activity of dihydrolipoyl dehydrogenase (E3 subunit, Lpd) is strongly inhibited at high NADH/NAD+ ratios, thereby reducing activity of PDC.40,41 Therefore, in order to reduce the inhibition caused by excess accumulation of NADH in the in vitro reconstitution system, we titrated and optimized the E. faecalis PDC for high efficiency and insensitivity to a high NADH/NAD+ ratio.28,42 Unlike E. coli PDC, the PDC of E. faecalis is composed of four subunits: component E1α (PdhA), component E1β (PdhB), component E2 (dihydrolipoyl transacetylase, AceF), and component E3 (lipoamide dehydrogenase, Lpd). The E2 core 703

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Figure 3. Titration of the components forming the pyruvate dehydrogenase complex from E. faecalis: (A) pyruvate dehydrogenase subunit E1 (PdhAB), (B) subunit E2, dihydrolipoyl transacetylase (AceF), (C) subunit E3, dihydrolipoyl dehydrogenase (Lpd). The molar ratio of E1, E2, and E3 of the optimized PDC of the E. faecalis is 4:2:1. (D) Comparison of the initial rates of reaction of the E. coli and E. faecalis PDC with optimized ratios of the three subunits. Values represent averages (n = 3) and error bars represent 1 s.d.

of the E. faecalis PDC has an icosahedral symmetry that is similar to that of the eukaryotic PDC.27,28,43 In order to determine the optimal ratio for in vitro self-assembly of the E. faecalis PDC and E. coli PDCs, components of the E. faecalis PDC were systematically titrated from 1.0 to 4.0 μM (1.0 to 5.0 μM for E1) while the other enzymes were maintained at 1.0 μM (Figure 3A−C). Components of the E. coli PDC were subjected to the same procedure (data not shown). On the basis of the results of titration, we determined that the optimal E1:E2:E3 ratio for E. faecalis PDC was 4:2:1 and that of the E. coli PDC was 2:2:1 in vitro. Next, we compared the optimized PDCs from E. coli and E. faecalis in their optimal subunit ratios, revealing that the initial rate of reaction of the E. faecalis PDC is seven times greater than that of the E. coli PDC (Figure 3D). Optimization of Reducing Equivalents. Reducing equivalents, in this case NAD(P)H, are essential for the biosynthesis of many industrially valuable compounds, such as fatty acids,11 carotenoids,44 and polymers.26 When dealing with NADPH-dependent pathways, several strategies have been implemented to increase NADPH availability, such as overexpression of the endogenous pyridine nucleotide transhydrogenase (UdhA) to convert NADH to NADPH45 and replacement of an NAD+-dependent GAPDH with an NADP+dependent one.46,47 These strategies have been used to enhance the production of lycopene and poly(3-hydroxybutyrate). For pathways under anaerobic conditions, methods such as the engineering of cofactor-switched enzymes have been used to improve the production of isobutanol48 and fatty acid.49 In the fatty acid biosynthetic pathway, two reductases utilize NAD(P)H: FabG is active only with NADPH,50 whereas FabI exhibits activity with both NADH and NADPH.51 Previous work on the in vitro reconstitution of type II FAS indicates that NADH is not necessary for fatty acid biosynthesis.29 In our system, the production of one mole of myristic acid consumes 12 mol of NAD(P)H, but the original glycolytic pathway only

generates NADH. In order to facilitate the downstream NADPH-dependent fatty acid biosynthesis pathway, we used two distinct approaches to enhance NADPH availability. First, we replaced the NAD+-dependent GAPDH, which is encoded by the E. coli gapA gene, with an NADP+-dependent GAPDH, encoded by the gapC gene of C. acetobutylicum (Figure 4A). The initial rate of the reaction was determined by the rate of increase of A340 as a result of NAD(P)H formation. We also examined the effect of GapC concentration in our glycolytic system. Increasing the GapC concentration from 2.5 to 80 μM yielded a 10-fold increase in the initial rate of reaction, with saturation at protein concentrations higher than 60 μM (Figure 4B). The highest initial rate (217.06 ± 3.28 μM NADPH equivalents/min) was similar to that of the original glycolytic pathway with GapA. Second, we reconstituted the entire PPP using eight purified components: Zwf, Pgi, Gnd, Rpe, RpiA, TktA, TalA, and TalB (Figure S1). The PPP is one of the most essential sources of NADPH in living cells.38,52 The glyceraldehyde-3-phosphate and fructose-6-phosphate produced by the PPP can also be utilized by glycolysis. A hybrid glycolytic pathway combined with the PPP was built to convert glucose to pyruvate and form two types of reducing equivalents (Figure 4C). Step-wise Optimization of the Entire Pathway. On the basis of the findings of our titration studies, we estimated the optimal molar ratios of the components of the glycolysis and pyruvate dehydrogenase pathways. In the glycolytic pathway, the optimal molar ratio of Glk:Pgi:PfkA:PfkB:FbaA:TpiA:GapA:GpmA:Eno:PykA:PykF should be approximately 1:1:2.5:2.5:1:1:1:1:1:5:5. For the efficient and robust E. faecalis PDC, the optimal E1:E2:E3 ratio is 4:2:1. For the downstream ACC and FAS pathway, the enzyme ratio of AccA:AccB:AccC:AccD:FabA:FabB:FabD:FabF:FabG:FabH: FabI:FabZ:holoACP:TesA was fixed at 5:5:5:5:1:1:1:1:1:1:1:1:10:10, based on previous studies, balancing limitations of reaction volume and efficiency.29,30 704

DOI: 10.1021/acssynbio.6b00348 ACS Synth. Biol. 2017, 6, 701−709

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Figure 5. Step-wise optimization of the in vitro reconstitution system by adjustment of each part of the reaction and assessment of the performance of the PPP hybrid and GapC replacement pathways. Blue bars indicate the productivity and red bars indicate the titer of each reaction, as determined by GC−MS analysis. a = no NADPH added; + = addition of corresponding elements (for GapC + indicates that GapA in glycolysis was replaced with GapC). − = corresponding elements not added (for glycolysis, this notation refers to original glycolysis with all components at 1.0 μM). Values represent averages (n = 3) and error bars represent 1 s.d.

productivities of 33.23 ± 0.65 and 35.10 ± 0.64 μg/L/min, respectively, within 4 h. Although the GapC replacement pathway resulted in a similar fatty acid titer to that achieved with the optimized glycolytic enzyme concentrations and the E. faecalis PDC (20.94 ± 2.22 vs 30.04 ± 3.15 mg/L; second red column from left to right, Figure 5), the titer of the glycolysis/PPP hybrid pathway (9.92 ± 0.86 mg/L) was much lower. This may be because this reaction lacks a phosphoglucose isomerase (Pgi). Thus, the fructose-6-phosphate that is produced cannot be converted to glucose-6-phosphate and used in downstream pathways; instead, only glyceraldehyde-3phosphate can be utilized. Furthermore, we tested the cofactor recycling capacity of the GapC replacement pathway by removing the input of the reducing equivalents. In this case, fatty acid was produced from glucose at a mean productivity of 10.04 ± 0.57 μg/L/min within 4 h. This clearly indicates that the in vitro reconstitution system recycles its cofactors enough to support the entire pathway. The highest fatty acid titer achieved in this study, 30.04 ± 3.15 mg/L from 0.9 g/L glucose, which corresponds to 0.033 g fatty acid/g glucose consumed and represents 9.3% of the theoretical yield. Besides quantitative analysis, the fatty acid products of our system were also investigated via GC−MS (Figure S3). The GC−MS profile of the fatty acid showed that decanoic acid, lauric acid, myristic acid, and palmitic acid accumulated in our system. Lauric acid and myristic acid are the major types of fatty acid produced from glucose. It appeared that fatty acid chain length tended to be longer with an increasing rate of the reaction. Interestingly, virtually no unsaturated fatty acid was detected in our assays; only a tiny amount of the unsaturated fatty acid 9-hexadecenoic acid was detected in the pyruvateinitiated reaction (trace 6, Figure S3). On the basis of discrepancies between the fatty acids produced using different substrates (glucose, pyruvate, or acetyl-CoA), we hypothesize that an insufficient acyl-CoA building block pool hampers the system from synthesizing long-chain fatty acids. The pathway that converts glucose to fatty acid is relatively long, and

Figure 4. (A) Overview of the complete pathway when GapA is replaced by GapC. (B) Titration of GapC. In this case, 4.0 mM NADP + was added, while other cofactors and the substrate remained the same as in the original glycolysis assay. (C) Overview of the hybrid pentose phosphate−glycolytic pathway, showing the complete PPP and part of the glycolytic pathway. Black bubbles indicate glycolytic enzymes, red and blue bubbles indicate PPP enzymes. In this case, 4.0 mM NADP+ and 1.0 μM FeSO4 were added.

Our system provides a method of determining the contributions of specific enzymes or pathways, allowing us to optimize the entire pathway in a stepwise manner. First, we replaced the original glycolysis (all enzyme concentrations at 1.0 μM) with the optimized one, but fatty acid yield remained below the GC−MS detection limit (second column from left to right, Figure 5). Next, we replaced the E. coli PDC with that of E. faecalis while maintained the original glycolytic pathway. This resulted in successful detection of fatty acid by GC−MS with a mean productivity of 11.86 ± 0.86 μg/L/min within 4 h and a titer of 4.24 ± 0.16 mg/L at 12 h (third column from left to right, Figure 5). After individually replacing the glycolytic and PDC parts of the system, we combined these two replacements, resulting in a significantly improvement in the efficiency of the system, with a mean fatty acid productivity of 37.85 ± 3.15 μg/ L/min within 4 h and a titer of 30.04 ± 3.15 mg/L at 12 h (fourth column from left to right, Figure 5). These individual and combinatorial replacements indicate that both glycolysis and PDC play essential roles in the system; however, consistent with our speculations, NADH inhibition of the E. coli PDC appears to be the largest factor in fatty acid yield of this in vitro reconstituted pathway, almost completely blocking the reaction. We next investigated the glycolysis/PPP hybrid pathway and the GapC replacement pathway, resulting in mean fatty acid 705

DOI: 10.1021/acssynbio.6b00348 ACS Synth. Biol. 2017, 6, 701−709

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BL21(DE3) via transformation. Several single colonies were grown in LB medium with 50 μg/mL kanamycin at 30 °C until the OD600 reached 0.6−0.8. Cultures were then cooled to 18 °C and IPTG was added to a final concentration of 0.1 mM for induction. After further growth for 12−16 h, cells were harvested by centrifugation at 5000g and resuspended in 25− 35 mL buffer A (50 mM Tris−HCl, 300 mM NaCl, 4 mM βmercaptoethanol, pH 7.6). The cell suspension was lysed by homogenization at a variable pressure of 10 000−15 000 psig and centrifuged at 30 000g for 1 h. After filtration of the supernatant, 6% buffer B (500 mM imidazole in buffer A) was added to reduce nonspecific binding with the Ni-NTA column. The supernatant was applied to the Ni-NTA column (GE Healthcare, Marlborough, MA, USA) using buffer A + 6% buffer B for equilibration. Then, buffer A + 10−30% buffer B was used for washing and buffer A + 60−100% buffer B was used for elution (percentages were dependent on the protein being eluted). The eluent was applied to a HiTrapQ FF column (GE Healthcare) on a Biologic Duoflow chromatograph (BioRad, Hercules, CA, USA) for further purification. Freshly purified protein was concentrated using Amicon Ultra-15 centrifugal filter devices (Millipore, Darmstadt, Germany). Next, 2.5 mL concentrated protein was applied to a PD-10 column (GE Healthcare) equilibrated with storage buffer (100 mM phosphate buffer, 10% glycerol, pH 7.6) for buffer exchange. Protein concentrations were measured with a Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). Proteins were stored at −80 °C after flash freezing in liquid nitrogen. For E. coli and E. faecalis AceF proteins, plasmid pMH19, encoding the lipoate-protein ligase A (lplA) of E. coli, and plasmid pJX4, encoding lplA and lplA2 of E. faecalis, were cotransformed, and 5 μg/mL lipoic acid was added to the medium after induction to ensure complete conversion of the apoprotein into its holo form. All purified proteins use in this study are listed in Table S2. Reconstitution and Optimization of Fatty Acid Synthetic Components. In the initial reconstitution of the pathway that converts glucose to fatty acid, 1.0 μM of each protein component (except for 5 μM AccA/B/C/D and 10 μM TesA and holo-ACP), 1 mM ATP, 1 mM CoA, 1 mM thiamine pyrophosphate, 10 mM ADP, 10 mM NADPH, 10 mM NaHCO3, and 20 mM NAD+ were added to a reaction buffer containing 50 mM sodium phosphate buffer (pH 7.6), 10 mM potassium chloride, 1 mM Tris (2-carboxyethyl) phosphine (TCEP), 10 mM magnesium chloride, and 0.2 mM 2,3bisphosphoglyceric acid (2,3-BPG). Reactions were initiated by the addition of 5 mM glucose (10% D-[6-14C]Glucose; American Radiolabeled Chemicals, St. Louis, MO, USA) or 5 mM pyruvate (10% [1-14C] sodium pyruvate; American Radiolabeled Chemicals) and then incubated at 37 °C for 12 h. Radioactive TLC assays of fatty acid product were performed as previously described.29 In brief, the TLC silica gel 60 F254 aluminum plate (Merck, Darmstadt, Germany) was spotted with 20 μL hexane extract of fatty acid and was developed in hexane:ether:acetic acid in a ratio of 70:30:2 (v/v/v) as the mobile phase. The radioactivity of the developed plate was then detected on a Storm 825 scanner (GE Healthcare) using [1-14C] palmitic acid (American Radiolabeled Chemicals) as a standard. For kinetic assays of Glk, PfkA/B, and PykF/A, 1 μM of each protein component (except the one being titrated), 1 mM ATP, 5 mM ADP, and 5 mM NAD+ were added to a reaction buffer containing 50 mM sodium phosphate buffer (pH 7.6), 1 mM

downstream of FAS, it is of high efficiency. On the basis of these two factors, acyl-CoA building blocks may not accumulate in this system, as they have been shown to do in vivo53 or in a previous in vitro study using acetyl-CoA as a substrate.30 Furthermore, excess reducing equivalents of NAD(P)H are supplied with adequate enoyl-reductase FabI in our reconstitution system, resulting in virtually no accumulation of unsaturated fatty acids.

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CONCLUSIONS Here, we have successfully reconstituted an in vitro synthetic pathway for the conversion of glucose to fatty acid using 30 purified proteins. To our knowledge, this is the first time this metabolic pathway has been fully reconstituted with purified components. This platform can easily be used to assess the effect of various components by simply adding or omitting them from the testtube. In this study, we determined the feasibilities and efficiencies of a PPP/glycolysis hybrid pathway and a GapC replacement pathway. Both of these pathways convert glucose to fatty acid through a series of more than 20 enzymatic reactions. After the optimization of the glycolytic pathway, the system yielded a 6-fold increase in fatty acid production compared with unmodified glycolytic pathway. We also used an NADH-insensitive E. faecalis PDC to relieve cofactor inhibition between different pathways. Combining these optimizations together, the overall carbon conversion efficiency was estimated to be 9.3% of the theoretical efficiency. In summary, our system allows for the monitoring of inhibition and regulation in multiple central metabolic pathways at once, in contrast to assessments of individual metabolic pathways, which have been presented in previous in vitro reconstitution studies, such as those involving the fatty acid biosynthetic pathway29,30,54 or the terpenoid biosynthetic pathway.55 Looking forward, we expect to extend this system to larger metabolic networks. Such research will complement existing in vivo studies by maintaining both the flexibility of in vitro systems and the complexity of in vivo metabolic networks, enabling us to gain a deeper understanding of living systems from a unique point of view.

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METHODS Plasmid Construction. Genomic DNA of E. coli BL21(DE3) was extracted with Blood & Cell Culture DNA Mini kit (QIAGEN, Hilden, Germany). Genes from E. coli were individually amplified from genomic DNA by PCR and cloned into pET28a(+) (Novagen, Darmstadt, Germany) for subsequent protein overexpression and purification. Glyceraldehyde-3-phosphate dehydrogenase (GapC) from C. acetobutylicum and pyruvate dehydrogenase component E1α and E1β (PdhA and PdhB), dihydrolipoyltransacetylase E2 (AceF), dihydrolipoyl dehydrogenase E3 (Lpd), and lipoate-protein ligase A and A2 (LplA and LplA2) from E. faecalis were codon optimized and synthesized by Genewiz (Suzhou, China) and cloned into pET28a(+). Plasmids and synthesized DNA sequences are summarized in SI text. Protein Purification. All components of the glycolytic pathway, PDC, ACC, FAS, and the PPP were individually overexpressed and purified from recombinant strains of E. coli BL21(DE3) or BAP156 harboring the relevant plasmids. FAS and ACC proteins were purified as previously reported.29,30 To purify individual proteins of the glycolytic pathway, PDC, and PPP, the corresponding plasmids were introduced into 706

DOI: 10.1021/acssynbio.6b00348 ACS Synth. Biol. 2017, 6, 701−709

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potassium chloride, 1 mM TCEP, 1 mM magnesium chloride, and 0.2 mM 2,3-BPG. Reactions were initiated by adding 5 mM glucose, and incubated at 37 °C. In the cases of Glk and PfkA/ PfkB, upon adding glucose to initiate the reactions, all contents were quickly mixed, and the increase in absorbance at 340 nm due to NADH formation was measured in a microplate reader. In the case of PykF/PykA, a pyruvate assay kit (Biovision, Milpitas, CA, USA) was used to measure the pyruvate formed. For the in vitro self-assembly of PDC, the three purified components were thawed and mixed at the desired concentration on ice to form the activated complex, as reported previously.31,43 After a 1 h incubation on ice, substrates and cofactors were added to initiate the reaction. Overall activity of PDC was measured by monitoring the reduction of NAD+ at 340 nm.57 All reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Analysis of Fatty Acids Produced from Glucose in the Reconstituted System. The reaction mixture was prepared and incubated at 37 °C for 12 h as described above, except that uniformly labeled 13C-glucose (all six carbons labeled, SigmaAldrich) was used as the sole carbon source to remove any possible effect of contamination. In this series of experiments, “1” in enzyme ratio equals to 1.0 μM corresponding enzyme (i.e., Pgi:PfkA = 1:2.5, 1.0 μM Pgi and 2.5 μM PfkA). Substrate and cofactor concentrations are consistent with the initial reconstitution system, enzyme concentrations are modified in different assays based on titration information. For GapC replacement experiment, GapC concentration is 60.0 μM. In PPP/glycolysis hybrid pathway, concentrations of all PPP components are fixed at 1.0 μM. After reacting for 4 or 12 h, 5% acetic acid (v/v) was added to quench the reaction. The 13 C-labeled fatty acid product was extracted, derivatized to fatty acid methyl esters, and quantitatively analyzed by GC−MS using pentadecanoic acid as an internal standard according to a previously published method.58 Briefly, each 200 μL reaction mixture was mixed with 1 μL of 10 mg/mL pentadecenoic acid, and extracted twice with an equal volume of n-hexane. The upper organic phase was collected and rotavapored to near dryness. Then, fatty acids were esterified by being redissolved in 200 μL 5% (v/v) H2SO4 in methanol and incubated at 90 °C for 2 h. After the reaction, 500 μL 0.9% (w/v) NaCl was added to each sample, and then the fatty acid methyl esters were extracted with 350 μL hexane for GC−MS analysis. The GC− MS analysis of fatty acid esters dissolved in hexane phase was performed and quantified using a TRACE Ultra gas chromatograph connected to a TSQ Quantum XLS triple quadrupole mass spectrometer (Thermo Scientific). The GC capillary column used was a 30 m long TR-WAXMS with a diameter of 0.25 mm and a film thickness of 0.25 μm. Injections were performed in a split mode with a split ratio of 20:1. Helium was used as the carrier gas with a flow rate of 1 mL/min. The inject inlet and ion source temperatures were 220 and 240 °C, respectively. The temperature sequence was programmed as follows: 100 °C as an initial temperature for 5 min, then a 6 °C/min ramp to 240 °C, followed by a hold at 240 °C for 10 min. For identification of fatty acid esters, the fatty acid methyl ester C8−C22 mix standard (Sigma-Aldrich) was used. For quantification, fatty acid concentrations were determined by comparing the peak area of an individual fatty acid species with that of the internal standard using the Xcalibur software. Total fatty acid titer were calculated as the sum of all kinds of fatty acids.

Research Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.6b00348. Figures S1−S3; Tables S1−S2; Appendix S1 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +86-27-68755086. Fax: +8627-68755086. *E-mail: [email protected]. Tel: +86-27-67813107. Fax: +86-27-67813107. ORCID

Tiangang Liu: 0000-0001-8087-0345 Author Contributions §

Z.L. and Y.Z. contributed equally to this work. T.L. and Y.X. designed and analyzed experiments. Y.Z., Z.L., X.J. and M.H. conducted experiments. Y.Z., Z.L., Y.X., Z.D., and T.L. wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology of China 973 Grants (2012CB721000 and 2011CBA00800) and 863 Grants (2012AA02A701), the National Natural Science Foundation of China (31222002 and 81370872), as well as the Project supported by Science and Technology Department of Hubei Province and by J1 Biotech Co. Ltd. (2014091610010595). The authors thank Ms. Hui Tao for discussions of this manuscript.

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REFERENCES

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DOI: 10.1021/acssynbio.6b00348 ACS Synth. Biol. 2017, 6, 701−709