Development of a Synthetic Pathway to Convert Glucose to Hydrogen Using Cell Free Extracts

international journal of hydrogen energy 40 (2015) 9113e9124

Available online at www.sciencedirect.com ScienceDirect

journal homepage: www.elsevier.com/locate/he

Development of a synthetic pathway to convert glucose to hydrogen using cell free extracts

* ** Franklin Lu a, , Phillip R. Smith a,1, Kunal Mehta b,2, James R. Swartz a,b, a Department of Chemical Engineering, Stanford University, USA b Department of Bioengineering, Stanford University, USA article info abstract

Article history: Sustainable production of biochemicals from cellulosic biomass is an attractive alternative Received 26 February 2015 to chemical production from fossil fuels. We describe the further development of a three þ Received in revised form protein synthetic pathway to convert NADPH and H to hydrogen using a ferredoxin- þ 16 May 2015 NADP reductase, a ferredoxin, and the [FeFe] hydrogenase from Clostridium pasteurianum 1 1 Accepted 18 May 2015 at a rate greater than 14 mmol H2 L hr using natural enzymes. We also demonstrate the Available online 13 June 2015 feasibility of coupling this pathway to a cell-free extract to convert glucose to hydrogen in a potentially cost effective manner. Both of these accomplishments serve as the basis for Keywords: further engineering to optimize both the yield and productivity of a low-cost cell-free Biohydrogen process for the production of highly reduced biochemicals. Hydrogenase Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights Synthetic enzyme pathway reserved. þ Ferredoxin-NADP -reductase Ferredoxin Cell-free metabolic engineering

fossil fuels and requires less energy input would be attractive Introduction as a sustainable method to ensure future hydrogen supply. One such method could be the use of cellulosic biomass to Hydrogen is an important commodity chemical with more produce hydrogen using metabolically engineered organisms. than 50 million metric tons produced worldwide each year The DOE estimates that a billion tons of biomass feedstock with a value of approximately $100 billion [1,2]. The majority could be available in various forms for processing into bio- of this hydrogen is produced through high temperature steam chemicals [4]. In particular, a biomass to hydrogen process reformation of natural gas and is used directly in petroleum could be advantageous by converting locally grown agricul- reﬁning, the production of ammonia for fertilizer, or for other tural residuals to hydrogen and then immediately converting industrial processes such as metallurgical treatment [3].An that hydrogen into ammonia for fertilizer. This fertilizer could alternative to steam reformation that does not depend on then be delivered back to the agricultural areas from which

* Corresponding author. 060A Shriram Center, 443 Via Ortega, Stanford, CA 94305-5025, USA. Tel.: þ1 812 243 5863; fax: þ1 650 725 0555. ** Corresponding author. 093 Shriram Center, 443 Via Ortega, Stanford, CA 94305-5025, USA. Tel.: þ1 650 723 5398; fax: þ1 650 725 0555. E-mail addresses: [email protected] (F. Lu), [email protected] (P.R. Smith), [email protected] (K. Mehta), jswartz@stanford.edu (J.R. Swartz). 1 35 Highland Drive, Yardley, PA 19067, USA. Tel.: þ1 650 861 7567; fax: þ1 650 725 0555. 2 060A Shriram Center, 443 Via Ortega, Stanford, CA 94305-5025, USA. Tel.: þ1 949 636 4345; fax: þ1 650 725 0555. http://dx.doi.org/10.1016/j.ijhydene.2015.05.121 0360-3199/Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. 9114 international journal of hydrogen energy 40 (2015) 9113e9124

the feedstock originated, thereby minimizing transportation enzymes, including the hydrogenase which was increased 5.6 for both feedstock and product. fold [13]. These rates are encouraging however, as they Enzymatic depolymerization of cellulose and hemicellu- demonstrate the potential for in vitro bioconversion pro- lose from pretreated agricultural residuals such as corn stover ductivities similar to those of bioethanol plants While in vitro [5] releases the constituent sugars, which consist primarily of approaches can reach significantly higher yields compared to glucose and xylose as well as smaller amounts of other five- in vivo production, the processes described above have their carbon sugars. Engineered organisms could then convert own challenges in scale up due to the cost of producing and these sugars into biochemicals through sustainable processes purifying 13e15 enzymes. Selection of thermostable enzymes [6] operating at low temperature and pressure relative to that can be purified through simple heat treatment methods is existing petrochemical processes. one option to isolate multiple proteins without requiring Previous attempts to produce hydrogen with engineered costly resin based chromatography methods [14]. However, organisms have focused on engineering cells to produce finding the right combination of heat stable, highly active hydrogen in vivo. Wells et al. inserted a [NiFe] hydrogenase enzymes, that can all be expressed in a small number of host and associated maturation factors into an Escherichia coli BL21 cell lines still has its own challenges and currently still re- m 1 cell line and produced hydrogen at a rate of 0.7 mole H2 L quires several separate purification methods [15]. hr 1 [7]. Subdubi et al. used a similar approach, expressing the Our proposed process seeks to combine the benefits of low HydA hydrogenase from Clostridium butyricum in E. coli to cost in vivo enzyme production with high yield in vitro produce hydrogen at a rate of 0.625 mmol L 1 hr 1 [8]. Yoshida hydrogen production. We seek to accomplish this by engi- et al. produced hydrogen in E. coli using native formate neering an E. coli cell extract in which a three protein synthetic hydrogen lyase and pyruvate formate lysae by knocking out pathway is coupled to the pentose phosphate pathway. The competing pathways. By artificially concentrating their cell use of unpurified E. coli extracts eliminates the costly pro- culture to 94.3 g dry cell weight/L, or approximately an OD610 cessing required to separate and purify individual compo- of 230, they were able to produce hydrogen at a rate of nents of the pathway as well as avoids potential challenges 793 mmol L 1 hr 1 [9]. However, all these approaches were with heterologous expression of a completely synthetic limited in overall yield of glucose to hydrogen: the theoretical pathway, while in vitro conversion potentially eliminates the maximum conversion in vivo is only 2 mol of hydrogen per loss of energy to other metabolic pathways thereby improving mole of glucose using the formate-dependent pathway in the overall yield. facultative anaerobes such as E. coli and 4 mol of hydrogen per We seek to complete this pathway with a [FeFe] hydroge- mole of glucose in certain obligate anaerobes [9]. In practice, nase from Clostridium pasteurianum, CpI. While use of CpI re- yields of only 1.82e3.2 mol of hydrogen per mole of glucose quires both ferredoxin NADPH reductase (FNR) and ferredoxin have been reported [7e9] due to the need to maintain carbon (Fd), CpI has higher reported turnover rates than the hydrog- and energy flow to other metabolic pathways as well. These enases [16] previously used at the temperatures required for yields are prohibitively low for bioprocesses that will be using E. coli extracts. In addition, CpI has been expressed dominated primarily by feedstock cost. heterologously in E. coli at high yields and only requires three Alternatively, other researchers have produced hydrogen proteins for maturation, as opposed 9 required to heterolo- in vitro using purified enzymes. Woodward et al. [10] first gously express the [NiFe] hydrogenase in addition to the four demonstrated this possibility by combining the enzymes of different subunits that must be coexpressed [17,18]. So while it the pentose phosphate pathway with the [NiFe] hydrogenase may be possible to use other hydrogenases such as those from from Pyrococcus furiosus. This enzyme can directly convert Pyrococcus furiosus or other [NiFe] hydrogenases, in order to NADPH reduced by the pentose phosphate pathway to achieve the simplest process of generating an E. coli cell hydrogen. Woodward achieved a yield of 11.6 mol of H2 per extract without need for purification and have the highest mole of glucose, over 90% of theoretical conversion. However, potential rate of hydrogen production, we selected CpI as our this was produced at a very low overall rate. In another hydrogenase. example, Zhang et al. achieved a yield of 8.4 mol of hydrogen Our pathway is shown in Fig. 1 and is composed of 14 proper mole of glucose at a significantly higher rate of teins from the glycolytic and pentose phosphate pathways as þ 0.75 mmol L 1 hr 1 using the same system of purified enzymes well as our synthetic pathway of Ferredoxin NADP reductase, with the Pyrococcus furiosus hydrogenase [11]. In addition, Ferredoxin, and Hydrogenase. Glucose is first phosphorylated Zhang et al. later demonstrated that this in vitro pathway to glucose-6-phosphate, which is then oxidized through the could also convert xylose to hydrogen after xylose isomerase pentose phosphate pathway to reduce two molecules of þ and xylulokinase were included to generate xylose-5- NADP with the release of a single molecule of CO2. The phosphate, which could then enter the pentose phosphate resulting 5-carbon sugar is then recycled through the non- pathway [12]. From this in vitro cocktail, Zhang and coworkers oxidative pentose phosphate pathway. Six 5-carbon sugar achieved 95% conversion of xylose to hydrogen at a rate of units are converted back into five 6-carbon glucose-6- 1.95 mmol L 1 hr 1. The ability to incorporate these five car- phosphate molecules, which can then reenter through the bon sugars is particularly important in maximizing the yield oxidative pentose phosphate pathway. The complete break- of biochemicals from cellulosic biomass as a significant down of a single glucose unit requires six cycles through this þ portion of the feedstock is composed of five carbon sugars. pathway, thereby reducing a total of 12 NADP molecules. þ Further work by the Zhang group was also able to establish Our three-protein synthetic pathway (Ferredoxin-NADP rates as high as 157 mmol H2/L/hr using 100 mM Glucose 6 reductase, Ferredoxin, and Hydrogenase) potentially can uti- Phosphate and increased concentrations of their purified lize all of the resultant NADPH molecules to produce hydrogen international journal of hydrogen energy 40 (2015) 9113e9124 9115

e Fig. 1 Proposed pathway from glucose to H2. The pathway is composed of three main sections, glycolysis, the pentose phosphate pathway (PPP), and our synthetic pathway. 14 proteins are required. Each molecule of glucose is phosphorylated þ and then oxidized to reduce 2 molecules of NADP to NADPH. The resulting five carbon ribulose units must be recycled through the non-oxidative branch of the pentose phosphate pathway and gluconeogenesis. The equivalent of one molecule of glucose is completely processed after six glucose-6-phosphate molecules are oxidized to six molecules of ribulose-5- þ phosphate with the reduction of twelve molecules of NADP to NADPH and release of six molecules of CO2. The six molecules of the five carbon ribulose units are recycled to regenerate five molecules of six carbon glucose-6-phosphate. All glycolysis and pentose phosphate enzymes are native to E. coli with the exception of 1) S. cerevisiae Hexokinase purchased from Sigma. The E. coli proteins are 2) Glucose-6-Phosphate Dehydrogenase 3) 6-Phosphogluconate Dehydrogenase 4) Ribulose-5-Phosphate Isomerase 5) Ribose-5-Phosphate Epimerase 6) Transketolase 7) Transaldolase 8) Triosephosphate Isomerase 9) Fructose Bisphosphate Aldolase 10) Fructose-1,6-Bisphosphatase and 11) Phosphoglucose Isomerase. The þ heterologous proteins are 12) Ferredoxin NADP Reductase 13) Ferredoxin and 14) [FeFe] Hydrogenase.

þ and NADP for recycle when the extract is modified to avoid for protein engineering to further improve hydrogen produc- significant sinks for reducing equivalents. This suggests a tion rates. In addition, we also demonstrate the functionality theoretical yield of 12 mol H2 per mole of glucose. The recycling of using a cell extract to supply reducing equivalents from of carbon intermediates will produce the ATP required for the glucose. initial phosphorylation step when the 3 carbon phosphorylated intermediates (glyceraldehyde 3 phosphate and dihy- droxyacetone phosphate) are converted to glucose six Materials and methods phosphate. Since the majority of our pathway is composed of native E. DNA assembly coli enzymes that have evolved to function together, we decided to focus first on selecting the optimal heterologous Table 1 lists the proteins analyzed in this work. E. coli ferre- components of the added synthetic pathway. Consequently, doxin, E. coli FNR and Synechocystis PCC 6803 ferredoxin were þ we screened six ferredoxin-NADP reductases (E. coli, Ana- cloned directly from genomic DNA and were produced as baena variablis, Spinach Leaf (Spinacia oleracea), Pea Root (Pisum described previously [19,20]. The remaining genes were sativum), Rice Root (Oryza sativa), Corn Root (Zea Mays)) and assembled from sixty base pair oligonucleotides that were five ferredoxins (E. coli, Anabaena variablis, Synechocystis PCC designed using DNAworks [21] to encode the desired amino 6803, Zea mays, C. pasteurianum) by first assessing production acid sequence and used the preferred E. coli codons. See Table in E. coli. Those proteins that could be expressed in their active A.1 and A.2 for full amino acid and DNA sequences. The 50 form at sufficient titers were analyzed in three assays to and 30 sequences were modified to include restriction sites determine compatibility with other components in the and an additional five base pairs at each end to allow for pathway. This screen enabled roughly a 3-fold improvement efficient restriction enzyme digestion and insertion into in H2 production rate relative to our previous work [17]. cloning vectors. Oligonucleotides were ordered from IDT DNA Although these new rates still fall short of our goals and the Technologies (Coralville, IA). Overlap extension PCR with published rates with the [NiFe] hydrogenase from pyrococcus Phusion DNA polymerase (NEB, Ipswich, MA) was used to furiosus, we also see that the CpI hydrogenase is capable of assemble the full-length genes according to the methodology producing hydrogen much more rapidly then we can currently of Kodumal et al. [22]. Final DNA products were purified with achieve. This work also identified the best natural candidates the Qiagen PCR cleanup kit and stored in 10 mM Tris pH 8 at 9116

Table 1 e Protein produced in this work with culture and purification conditions. All genes were either amplified from genomic DNA or assembled using sixty base pair oligos using PCR overlap extension. Genes were inserted into the pET21b vector for transformation into BL21 E. coli cells. The transformed cells were cultured and the product purified as indicated above. þ Ferredoxins (Fds) Ferredoxin NADP reductases (FNRs) nentoa ora fhdoe nry4 21)9113 (2015) 40 energy hydrogen of journal international Protein Organism E. Coli Anabaena Synechocystis Zea Mays Clostridium E. Coli Pisum sativum Zea Mays Spinacia oleracea Anabaena Oryza sativa attributes variabilis PCC 6803 (Corn Root) Pasteurianum (Pea Root) (Corn Root) (Spinach Leaf) variabilis (Rice Root) Abbreviation EcFd AnFd SynFd ZmFd CpFd EcFNR PrFNR ZmFNR SpFNR AnFNR RrFNR Type 1 2Fee2S 1 2Fee2S 1 2Fee2S 1 2Fee2S 2 4Fee4S Bacterial Plant Non- Plant Non- Plant Bacterial Plant Non- photosynthetic photosynthetic Photosynthetic Photosynthetic photosynthetic # of AA 111 99 97 152 56 248 317 315 315 304 315 MW 12.331 10,830 10,363 16,123 5,630 27,751 35,620 35,310 35,470 34,130 35,310 Culture Temperature 30C 37C 30C 30C 30C 30C 30C 30C 30C 30C 30C conditions OD at Induction 5 5.5 3.5 3 0.5 2 2.5 2.5 3 3 2.5 [IPTG] 250 mM 500 mM 500 mM 500 mM 500 mM 500 mM 500 mM 500 mM 500 mM 500 mM 500 mM 2 mM Fe/Cys Yes Yes Yes Yes Yes No No No No No No 25 mM Fumarate No No No No Yes No No No No No No Post Induction Aerobic Aerobic Aerobic Aerobic Anaerobic Aerobic Aerobic Aerobic Aerobic Aerobic Aerobic Atmosphere Induction to 4.5 h 4 h 5 h 16 h 12 h 5 h 18 h 18 h 6 h 6 h 18 h Harvest Time Purification 1st Purification DEAE DEAE DEAE DEAE DEAE DEAE Unable to Unable to Unable to DEAE DEAE conditions Step produce produce produce Elution Linear Linear Step 150, Linear Step 75, Linear sufficient sufficient sufficient Linear Linear Conditions 150e500 mM 150e500 mM 1000 mM 0e1000 mM 150, 250, 20e300 mM quantities quantities quantities 0e500 mM 0e500 mM NaCl NaCl NaCl NaCl 400 mM NaCL for purification for purification for purification NaCl NaCl NaCl 2nd Purification N/A N/A Butyl Ammonium SP Sp Sepharose Sp Sepharose Step Sepharose Sulfate Sepharose Elution Linear 40% Linear Linear Linear Conditions 50%e0% Ammonium 25e500 mM 25e500 mM 25e500 mM e

Ammonium Sulfate NaCl NaCl NaCl 9124 Sulfate 3rd Puriﬁcation N/A N/A Ammonium N/A Step Sulfate Conditions 50% Ammonium Sulfate international journal of hydrogen energy 40 (2015) 9113e9124 9117

4 C until use. The linear PCR dsDNA product encoding the purification products were performed as required using Ami- protein was digested with SalI and NdeI (NEB, Boston, MA), con stirred-cell concentrators (Millipore Corp, Bedford, MA). and ligated into the pET21b expression vector (Novagen, San For the ferredoxins, ammonium sulfate precipitation and Diego, CA). The ligation product was transformed into DH5a hydrophobic interaction chromatography was used to further cells (Invitrogen, Carlsbad, CA) that were prepared using clarify elutions from the initial anion exchange step as indi- Zymo Research z-competent kit (Zymo Research, Irvine, CA) cated in Table 1. All proteins were eluted with salt gradients and transformed by heat shock at 42 C. The resulting pET21b and purified pools were identified by SDS PAGE analysis. Final vector was purified using a QIAprep Spin Miniprep Kit (Qia- chromatography products were concentrated and buffer gen, Valencia, CA), sequenced for verification, and retrans- exchanged into 25 mM Tris, pH 8.0. Protein concentrations formed into chemically competent BL21 (DE3) cells were determined spectrophotometrically where extinction (Invitrogen). For ferredoxins, expression vectors were trans- coefficients were available, by using a DNA binding dye (Qubit, formed into chemically competent BL21 (DE3) DiscR cells to Invitrogen, Carlsbad, CA) or with a Lowry protein assay kit allow for improved assembly of FeeS clusters. Transformed (Biorad, Hercules, CA). Final stocks were formulated in 5% strains were grown on LB media and maintained at 80 Cas glycerol or 20% sucrose, flash frozen in liquid nitrogen, and frozen stocks in 25% glycerol. See Table A.2 for DNA stored at 80 C. Sequences. Hydrogen production assay using dithionite In vivo expression and purification 2 Dithionite (S2O4 ) is a powerful reductant and is commonly Table 1 lists culture and purification conditions for each pro- used to reduce ironesulfur cluster-containing proteins [23]. tein produced in this work. CpI hydrogenase was prepared as In this assay, dithionite is used to reduce ferredoxin, which described by Kuchenreuther et al. [18]. Overexpression of each then passes electrons to the CpI hydrogenase. Reactions of the FNRs and ferredoxins was accomplished as follows with were conducted in 8.4 mL (Wheaton, Millville, NJ) anaerobic exceptions noted below. See Table 1 for specifics on temper- serum vials that were crimp-sealed with a septum and an ature, induction, and specific purification conditions. Frozen aluminum cap. As the atmosphere in the anaerobic glove- glycerol stocks of E. coli BL-21 (DE3) transformed with the box contained 2% hydrogen, the headspace of the vials was pET21b expression vector containing our gene of interest were purged with oxygen-free nitrogen for 5 min before initiation used to inoculate starter cultures grown in temperature of the reaction. The experiments were initiated by addition controlled incubators with an orbital shaking platform at of dithionite to a final concentration of 15 mM. The 280 rpm. Terrific Broth (TB) media with 4 mL/L of glycerol was dithionite solution was prepared anaerobically, sealed in a used as basal growth media. Cultures in late exponential glass serum vial with a septum and aluminum crimp-cap, phase were induced with IPTG to initiate protein expression. removed from the glovebox, and purged for 5 min with a For ferredoxin production, 2 mM ferric ammonium citrate was nitrogen stream. Additions were made via a glass syringe. supplemented to basal media and 2 mM cysteine was added at Experiments were conducted with the following composi- induction to aid in FeS cluster synthesis. For CpFd, cultures tion: 100 mM Tris pH 8, 100 mM Ferredoxin, 20 nM Hydrog- were incubated in an anaerobic glovebox (Coy Laboratories, enase, and 15 mM Dithionite (added to initiate the reaction). m Grass Lake, MI) with a 98% N2/2% H2 atmosphere. After in- The total volume for each experiment was 200 L. Reactions duction, all subsequent steps were performed anaerobically. were conducted at 37 C with stirring. The concentration of In addition, for the CpFd anaerobic culture phase, 25 mM hydrogen in the headspace of the vial was measured by fumarate was added to serve as a terminal electron acceptor. removing 200 mL of headspace gas with a gastight syringe After an induction period of 4e18 h (See Table 1), cultures were (Hamilton) and injecting the sample into a 6890 Gas Chro- harvested by centrifugation and resuspended in 1.5 mL of lysis matograph (Agilent Technologies, Palo Alto, CA) equipped buffer per gram of wet cell paste. The lysis buffer contained with a thermal-conductivity detector (TCD) and a Shincar- rLysozyme (5000 units/gram cell paste) (Novagen), Benzonase bon column (Restek Corp, Bellefonte, PA). The GC inlet was DNase (125 units/gram cell paste), 1 bugbuster, and one maintained at 40 C, the oven at 50 C, and the TCD at dissolved EDTA-free protease inhibitor tablet (Roche Molecu- 160 C. The nitrogen carrier gas flowrate was 20 mL/min. lar Biochemicals, Nutley, NJ) per 50 mL of buffer. The cell The reference gas flow through the TCD was also main- suspension was lysed in a continuous homogenizer (Avestin tained at 20 mL/min. Headspace hydrogen concentrations Emulsiflex-C50, Ottawa, Ontario, Canada) by a single pass at were determined from peak areas by comparing to calibra- 20,000 psig. Lysate was incubated at 25 C for 30 min for DNA tion curves generated using standards with known and peptidoglycan degradation and further cell lysis. After hydrogen concentrations. No other gaseous species were incubation, lysates were centrifuged and diluted in equili- detected by the GC analysis. The headspace was sampled bration buffer. Ion exchange chromatography was then per- over time and the measured concentrations were fit by formed to isolate the desired protein. A DEAE Sepharose Fast linear regression to determine the initial rate of reaction. 1 Flow column was used for all purifications, with an additional The volumetric hydrogen production rate (mmol H2 L cation exchange SP Sepharose column used to further purify hr 1) was determined by dividing the rate of hydrogen 1 the FNRs. The equilibration buffer was 25 mM Tris pH 8 for production (mmol H2 hr ) by the liquid reaction volume. DEAE and 25 mM Bis-Tris pH 5.5 for SP Sepharose with the salt The turnover number (TON) of the hydrogenase was deter- concentrations indicated in Table 1 used for loading and mined by dividing the molar hydrogen production rate by elution. Buffer exchanges and concentration of intermediate the molar concentration of the hydrogenase. 9118 international journal of hydrogen energy 40 (2015) 9113e9124

Cytochrome c reduction assay Results The cytochrome c assay was conducted as described in Smith et al., 2011 [20]. Briefly, reaction mixtures were prepared The eleven proteins selected for this work are listed in Table 1. anaerobically and incubated at 25 C in a VERSAmax Tunable Each gene was constructed individually from overlapping ol- Microplate Reader (Molecular Devices Corp., Sunnyvale, CA). igonucleotides with the exception of EcFNR, EcFd, and SynFd, Horse heart cytochrome c (SigmaeAldrich) was used as the which were amplified from genomic DNA. Each remaining terminal electron acceptor and NADPH (SigmaeAldrich) as the gene was codon optimized and expressed in E. coli using a lac electron source. The assay was monitored at 550 nm. The re- inducible promoter system. To ensure that we sampled the action mixture consisted of 100 mM Tris pH 7, 50 mM Cyto- full range of available FNRs, we tested members from each of chrome c,30mM Fd, and 10 nM FNR, and was initiated with the three major FNR families identified through comparative 500 mM of NAPDH. Spectrophotometric analysis of the absor- genomic studies by Karplus et al.: cyanobacterial, plant root, bance peaks for the ironesulfur clusters was used to confirm and plant leaf [24]. Within these groups, the functions were oxidation of ferredoxin before the reaction was initiated. The further subdivided between the non-photosynthetic root en- rate of reaction was determined via the BeereLambert law zymes, which physiologically function to consume NADPH using the extinction coefficient of cytochrome c and reduce nitrogenase and sulfases to facilitate nutrient (19,600 M 1 cm 1). The TON was calculate by dividing the uptake in the roots [25], and the photosynthetic leaf enzymes, observed molar rate of reaction by the molar concentration of which primarily transfer electrons from ferredoxin to NADPH FNR. as a product of photosynthesis [26]. We also evaluated native E. coli enzymes as an expression control, since our cell free Hydrogen production using NADPH extract is E. coli derived and these proteins would avoid any possible limitations caused by heterologous expression. Hydrogen production experiments were conducted as In selecting ferredoxins, we hypothesized that ferredoxins described by Smith et al. [20]. Briefly, experiments were con- would pair optimally with FNRs from the same species and ducted using 100 mL reaction volumes contained in 250 mL wells consequently we expected the highest cytochrome c rates installed inside of 8.4 mL glass vials. All reaction mixtures were from these pairs. However, we also require the ferrodoxin to prepared inside an anaerobic glovebox with a 2% hydrogen, couple with our CpI [FeFe] hydrogenase from C. pasteurianum. 98% nitrogen atmosphere. The initial reaction mixture con- Consequently, we also evaluated the clostridial ferredoxin sisted of 25 mM Tris pH 7, 100 mMFNR,80mM oxidized Fd, 2 mM (CpFd) to further evaluate which interactions are possibly CpI hydrogenase, 15 mM glucose-6-phosphate (G6P), 5 units of limiting the overall flux of our synthetic pathway. The FNRs G6P dehydrogenase (G6PD), and was initiated with 7 mM that we tested in detail were EcFNR, AnFNR (cyanobacteria), NADPH. The G6P and G6PD were added to regenerate NADPH and RrFNR (rice root). We also investigated several other FNRs from spinach leaf, corn root, and pea root, but were unable to during the reaction. The vials were then sealed, purged with N2, and warmed as described above. After temperature equilibra- express sufficient amounts of these enzymes in E. coli to test in tion, NADPH was added to initiate the reaction. The headspace our synthetic pathway. AnFNR and ZmFNR were reported to hydrogen concentration was determined as described previ- have turnover number (TONs) of greater than 200 per second, 1 which is significantly higher then the reported values for our ously. The volumetric hydrogen production rate (mmol H2 L hr 1) was determined by dividing the rate of hydrogen pro- previously tested EcFNR [20,27]. RrFNR has not previously 1 been produced heterologously and evaluated to our knowl- duction (mmol H2 hr ) by the liquid reaction volume. The turnover number (TON) of the hydrogenase or FNR was deter- edge, but we expected this to have a similar TON as ZmFNR mined by dividing the molar hydrogen production rate by the due to sequence similarity. The ferredoxins that we tested molar concentration of the specified enzyme. included the native redox partners for EcFNR (EcFd) and AnFNR (cyanobacterial), and a similar plant root ferrodoxin, Hydrogen production from glucose using cell extract ZmFd (corn root). We also tested the previously expressed SynFd (cyanobacterial) and the native redox partner for our hydrogenase from C. pasteurianum, CpFd. To test hydrogen production from glucose, the following re- þ agents were combined to a final volume of 200 mL: 50 mM We tested the ferredoxin-NADP reductases (FNR) and þ AnFNR, 80 mM CpFd, 1 mM CpI hydrogenase, 1 mM NADP , ferredoxins (Fd) in three separate assays. The first method þ 30 mM Glucose, 30 mM PEP, 10 mM Mg2 , 0.025 units/mL produced hydrogen from ferredoxin and CpI using dithionite Hexokinase, 55% (v/v) KC6 extract [29], and 20 mM sodium as the electron donor. Dithionite is a strong reducing agent phosphate buffer, pH 7. Control reactions were prepared with that reduces ferredoxin rapidly so that at limiting hydroge- noted deviations from this recipe. Hydrogen production ex- nase concentrations the rate of hydrogen production is periments were conducted as described in Fig. 2. Vials were determined primarily by the interaction of reduced ferredoxin with the CpI hydrogenase. Ferredoxin, hydrogenase, and sealed, purged with N2, and warmed as described above. Re- actions were initiated by addition of the glucose. Glucose dithionite were combined in a buffered solution inside a stock solutions were prepared anaerobically and purged with sealed anaerobic vial, and the rate of H2 evolution was oxygen-free nitrogen prior to initiation of the experiment. measured by analyzing headspace samples using a gas chro- Measurement of the headspace hydrogen concentration and matograph to assess hydrogen accumulation (Fig. 2). calculation of initial hydrogen production rates and TONs The second method analyzed the interaction of each FNR were performed as described previously. and ferredoxin using a colorimetric assay with NADPH as the international journal of hydrogen energy 40 (2015) 9113e9124 9119

Fig. 2 e Procedure for preparing the hydrogen production reaction mixture and measuring hydrogen production rates. 1) Inside an anaerobic glovebox, all reagents except the electron source are added to the cell extract (including puriﬁed FNR, Fd, and CpI) within an inner reaction well inside a glass vial. Wells were cut from standard 96 well plates (Fisher) and ﬁxed with an adhesive inside the glass vial. After reagent additions, vials were sealed with rubber stoppers and crimp caps (Wheaton).

For glucose to H2 experiments, additional reagents needed for H2 production from glucose were added including hexokinase and PEP to phosphorylate the glucose. Pyruvate kinase is provided by the cell extract. See materials and methods for additional details. 2) The vial is sealed and removed from the glovebox 3) The vial is purged with nitrogen for 5 min to replace the glovebox atmosphere initially in the vial (2% hydrogen 98% nitrogen) with 100% nitrogen. 4) The vial is warmed to 37 C for 5 min, repurged for 5 min to remove any hydrogen evolved from reduced species initially in the cell extract and placed back in the water bath 5) After thermal equilibrium, NADPH or glucose is added by syringe to initiate the reaction 6) The vial headspace is sampled periodically and injected into a GC to measure the headspace hydrogen concentration.

electron source and cytochrome c as the terminal electron Dithionite based hydrogen production acceptor. NADPH reduces the ﬂavin moiety in FNR, which in turn reduces ferredoxin. Cytochrome c is then reduced by Our ﬁrst analysis evaluated the hydrogen production rate of ferredoxin, which results in a change in absorbance at 550 nM. CpI hydrogenase when combined with each ferredoxin. Cytochrome c has a positive redox potential (þ0.26 mV [28]) and Dithionite is a strong reducing agent (0.66 V) that can directly the reduced ferredoxins have negative potentials (~ 340 mV to reduce ferredoxin, which in turn provides reducing equiva- 420 mV, [29,30]) so there is a strong driving force for electrons lents to the CpI hydrogenase. We used an excess of ferredoxin to be transferred from ferredoxin to cytochrome c. and a limiting concentration of hydrogenase (20 nM) to Finally, the last method evaluated our entire synthetic determine the maximum rate of hydrogen production with pathway with FNR, ferredoxin, and hydrogenase mixed each ferredoxin. This provides a relative turnover number for together in a sealed anaerobic vial in which NADPH was added the hydrogenase coupling with each ferredoxin. Direct and hydrogen gas accumulation measured by gas chroma- reduction of CpI by dithionite does occur, but at a much slower tography (Fig. 2). This method is similar to the dithionite- rate then mediated by the ferredoxins. This background rate based assay except that the electron source is NADPH, FNR has been subtracted from the turnover numbers reported in is present in addition to Fd and CpI, and the concentration of Fig. 3. There is no measurable hydrogen accumulation from reduced versus oxidized ferredoxin is controlled by the rela- dithionite and ferredoxin alone in the time period of our tive rates of reduction by FNR and oxidation by CpI. This analysis. method resembles our eventual pathway and should replicate The best ferredoxin was the ferredoxin from C. pasteur- the case when NADPH is generated rapidly by our extract. In ianum (CpFd) which enabled a hydrogenase turnover number this assay NADPH is added at high concentration (7 mM) to of ~1700 sec 1 (Fig. 3). EcFd had the lowest rate of hydrogen provide a driving force of reducing equivalents for FNR to production; near background level, while ZmFd, AnFd, and reduce ferredoxin. Using these three methods together we SynFd supported increasing turnover numbers. These results compared FNR-ferredoxin and ferredoxin-hydrogenase in- were consistent with our hypothesis that CpI has evolved for teractions to help determine the optimal components for optimal reactivity with its native electron donor, CpFd. How- future evolution. ever, there is still dramatic variation among the other 9120 international journal of hydrogen energy 40 (2015) 9113e9124

Our best ferredoxin from the dithionite screen, CpFd, did not produce the best cytochrome c reduction rate (Fig. 4), most likely due to the different binding interactions between the ferredoxins and FNRs from different species. The highest cytochrome c reduction rates were observed with AnFNR and a cyanobacterial ferredoxin. Surprisingly, the Fd from Syn- echocystis coupled with Anabaena FNR produced more rapid electron transfer rates then the matching ferredoxin from Anaebaena. Consequently, it seems possible that even the ferredoxin from the same species as the FNR may not have evolved for maximal activity but rather to fine-tune oxidation/ reduction rates to balance the needs of the organism. Alter- natively, the interaction between the ferredoxin and our final electron acceptor, horse heart cytochrome c, may also be influencing our observations. The background rates of cyto- Fig. 3 e Turnover number of CpI hydrogenase with the chrome C reduction observed from NADPH alone, ferredoxin electrons supplied from various ferredoxins with alone, ferredoxin and FNR (with no NADPH) were all less then dithionite as the electron source. Hydrogen production 6 mAU/min, which is less than 1% of the maximum rates from dithionite and CpI alone is subtracted. No measurable measured with AnFNR and SynFd. hydrogen is observed from dithionite and ferredoxin alone. The turnover numbers were calculated as the rate of Hydrogen production using NADPH hydrogen production (measured by gas chromatography) divided by the hydrogenase concentration. The We next tested the synthetic electron transfer pathway concentrations of each reagent were: 100 mM Ferredoxin, (including FNR, ferredoxin, and CpI hydrogenase) with NADPH 20 nM Hydrogenase, and 15 mM Dithionite. Error bars are as the electron donor. In this assay a high concentration of standard deviation of three replicates. Ec ¼ E. coli, NADPH drives reduction of ferredoxin by FNR. The reduced Syn ¼ Synechocystis PCC 6803,Cp¼ Clostridium ferredoxin then transfers electrons to CpI hydrogenase, which Pasteurianum, and Zm ¼ Zea Mays e Corn Root. produces hydrogen that accumulates in the headspace of an anaerobic vial. We then determine hydrogen production rates by sampling the headspace and measuring the accumulation with a gas chromatograph (Fig. 2). ferredoxins despite significant structural and sequence similarity amongst, for example, the photosynthetic ferredoxins. In addition to evolved binding surfaces, CpFd has a structural difference in both size and the presence of an additional FeS cluster that give this ferredoxin unique properties. One possibility is that the small size of CpFd (56 amino acids) may improve mass transfer by increasing diffusivity. Another possible reason for improved electron transfer could be the presence of two FeS clusters within CpFd, allowing the CpFd to be a two-electron carrier, whereas the other ferredoxins have a single FeS cluster. Consequently, a single binding event could result in the transfer of both electrons from a fully reduced flavin cofactor in the FNR. NMR studies indicate that intermolecular electron transfer between the two clusters is extremely fast (5 10 6 sec 1) [31]. The hydrogenase also requires two electrons for every molecule of H2 formed. Fig. 4 e Turnover number of FNRs as indicated by the rate Consequently, such a two-electron carrier would potentially of Cytochrome c reduction divided by the concentration of transfer those electrons from FNR to the hydrogenase in fewer the FNR. The reaction mixtures consisted of 50 mM binding steps than would be possible with ferredoxin that Cytochrome c,30mM Fd, and 10 nM FNR. Reactions were carries a single electron. initiated by the addition of 500 mM NADPH. Error bars are standard deviation of three replicates. (¡) Control is Cytochrome c activity background measured with AnFNR and SynFd without NADPH added. Other (¡) control combinations of FNR and Next, we evaluated combinations of FNRs and ferredoxins in a Fd without NADPH, Fd alone, and NADPH only were colorimetric assay using NADPH as the electron donor and comparable and less than 6 mAU/min (less then 1% of cytochrome c as the final electron acceptor. In these assays, maximum rates measured). Ec ¼ E. coli, FNR was present at a limiting concentration with an excess of Syn ¼ Synechocystis PCC 6803,Cp¼ Clostridium ferredoxin. The FNR turnover number was measured by the Pasteurianum,An¼ Anabaena variablis,Rr¼ Oryza sativa e change in absorbance at 550 nm. Rice Root, and Zm ¼ Zea Mays e Corn Root. international journal of hydrogen energy 40 (2015) 9113e9124 9121

The observed rates in our NADPH to hydrogen studies evaluate the propensity for ferredoxins to both accept electrons from an FNR and to deliver them to our CpI hydrogenase. The best hydrogen production rate of 14.8 mmol L 1 hr 1 was provided by rice root FNR and CpFd (Fig. 5A). The best pair from the cytochrome c tests, AnFNR and SynFd, supported a lower hydrogen production rate of 10 mmol L 1 hr 1, which is most likely because of the much better coupling of CpFd to CpI. The highest production rate obtained (using RrFNR, CpFd, and CpI hydrogenase) is more then 15-fold higher than our in vitro rates when we started this work [10]. While all these comparisons of different protein combinations were conducted at fixed concentrations, changes in e E. coli concentration of single synthetic pathway components (FNR, Fig. 6 Volumetric H2 productivity using unpurified Fd, CpI) did not result in further gains in hydrogen production cell extract to provide reducing equivalents from glucose rate. Interestingly, increasing the FNR concentrations only compared to hydrogen produced from exogenous NADPH decreased the calculated FNR turnover number, indicating a without cell extract. Error bars are standard deviation of less efficient use of the enzyme (Fig. 5B). While initial obser- three replicates. vations suggested that the FNR is not limiting; additional CpI hydrogenase or ferredoxin also did not increase hydrogen production rates (unpublished results). The turnover of the CpI hydrogenase in the NADPH to H2 assay is significantly in our lab [32]. 30 mM Glucose was added as the energy source þ lower then that measured in the dithionite assay, indicating along with 1 mM NADP to allow for rapid generation of that this protein is not being fully used either. At this point the NADPH. In addition, a yeast hexokinase (Sigma Aldrich) was limiting factor is not apparent. added to the extract, as less then 1 mmol L 1 hr 1 of hydrogen was produced without hexokinase (Fig. 6). While E. coli does Hydrogen production using glucose and cell extract have a native glucose kinase, the primary means of glucose phosphorylation is by a membrane-bound PEP-dependent In order to determine the feasibility of using an unpurified E. transporter complex [33], which most likely cannot function þ coli extract to reduce NADP , experiments were conducted to in the extract because the cell lysis produces inverted mem- evaluate hydrogen production from glucose using a cell-free brane vesicles which prevent access to both sides of the extract. Eventually, all proteins required in our pathway membrane [34]. E. coli also has a cytoplasmic glucose kinase, would be present in an unpurified cell extract to minimize the but it is down regulated during growth on glucose and is likely cost; but in these initial experiments, purified FNR, ferredoxin, present at low concentration, if at all, in the KC6 extract. In the and CpI were added to a cell extract containing pentose eventual cell extract, an effective glucose kinase will be phosphate pathway enzymes. The cell extract used for these expressed to avoid the requirement for this exogenous experiments was made from a strain of E. coli called KC6, enzyme. Since ATP is required to drive the conversion of which was originally developed for cell-free protein synthesis glucose to glucose-6-phosphate, 30 mM phosphoenolpyruvate

e Fig. 5 A: Volumetric productivity of H2 from NADPH as measured by gas chromatography. Initial rates were measured anaerobically with various FNR and Fd combinations providing reducing equivalents to CpI hydrogenase. The reaction mixture consisted of 100 mM FNR, 80 mM Fd, and 2 mM Hydrogenase. 7 mM NADPH was added to initiate the reaction. Error bars are standard deviation of three replicates. Ec ¼ E. coli, Syn ¼ Synechocystis PCC 6803,Cp¼ Clostridium Pasteurianum, An ¼ Anabaena variablis,Rr¼ Oryza sativa e Rice Root, and Zm ¼ Zea Mays e Corn Root. Fig. 5B: Turnover numbers of Rice

Root FNR and CpI Hydrogenase when NADPH drives H2 production. Turnover numbers are calculated by dividing the hydrogen production rate by the concentration of either FNR (black) or CpI hydrogenase (grey). Concentrations of FNR were varied as indicated on x-axis. [CpFd] ¼ 80 mM, [CpI] ¼ 2 mM. Error bars are standard deviation of three replicates when indicated. 9122 international journal of hydrogen energy 40 (2015) 9113e9124

was added to supply the native pyruvate kinase with a sub- hydrogen production with an approximately three-fold in- strate from which to generate ATP. However, this addition crease in hydrogen production rate from NADPH observed could likely be decreased significantly in the final reaction when using a plant root FNR relative to native E. coli FNR. system, as the conversion of fructose-1,6-bisphosphate to Interestingly, this FNR has evolved to naturally transfer elec- fructose-6-phosphate in gluconeogenesis generates ATP as trons from NADPH to reduce ferredoxin, as opposed to the ribulose-6-phosphate is recycled. Although the majority of photosynthetic FNRs that primarily generate NADPH with carbon recycles as fructose-6-phosphate (see Fig. 1) two three electrons from photosynthesis. Even when selecting revers- carbon phosphorylated molecules are made (G3P and DHAP) ible pathway enzymes, it may be important to select enzymes for every cycle and one ATP is thereby generated. Conse- from tissues that have evolved to favor the desired reaction quently, ATP should be available to phosphorylate the glucose direction. entering the pathway. We have also shown that an unpurified E. coli extract can With this protocol, glucose was successfully converted to convert glucose to NADPH for use by our synthetic pathway to hydrogen at a rate comparable to the rate measured when produce hydrogen at rates comparable to our synthetic NADPH was added directly to purified FNR, Fd, and Hydroge- pathway alone. In this situation, a large bolus of glucose was nase (Fig. 6). These results indicate the pentose phosphate added and we analyzed only the initial rate of hydrogen pro- pathway in a cell extract can produce NADPH at sufficient duction. It is likely that a large amount of the feedstock was rates to maximize H2 production through our current syn- used inefficiently to maintain this rate. Eventually, we plan to thetic pathway. produce our entire pathway in an engineered cell line and will These results also suggest a number of potential im- therefore fine tune expression levels of the pentose phosphate provements for our process: overexpressing hexokinase and pathway enzymes to increase both the rate of hydrogen pro- establishing methods for producing ATP will eliminate the duction and the conversion yield of glucose to hydrogen. need to add two components. Inactivating competing path- Nonetheless, this data demonstrates effective coupling to ways will help direct carbon flux in the extract to the recycling unpurified pentose phosphate pathway enzymes. We are now portion of the pentose phosphate pathway and gluconeo- working to reduce loss of reducing equivalents to side path- genesis. In addition, another method to produce ATP will be ways. In addition to maximizing the yield by eliminating side evaluated, the activation of oxidative phosphorylation which reactions, maintaining the stability of these extracts will also has been previously described in cell extracts [34]. Despite the be critical for the feasibility of large-scale biomass conversion. breakup of the cell membrane, fragments of the E. coli inner In our initial work we see hydrogen production activity of cell membranes reform into membrane vesicles. Many of these extracts for several days until our initial feed is exhausted, but vesicles are inverted but others that are orientated properly we have not tested hydrogen production beyond this duration. can continue to generate ATP from a proton gradient when If there are particularly labile parts of the pathway these reducing equivalents and O2 are provided. Only a small frac- proteins can be engineered for stability and modified in the tion of reducing equivalents obtained from glucose would be genome of our parent strain. required to produce the needed ATP. A closer look at the FNR activity at high Fd and CpI concentrations reveals that as the FNR concentration is increased, its turnover number still substantially decreases (Fig. 5B), even Discussion though these measured turnover rates are significantly lower than the turnover rate of the enzyme measured by the þ We evaluated a series of ferredoxin-NADP reductases and ferredoxin-mediated reduction of cytochrome c (~50 s 1). ferredoxins for use in a synthetic pathway to produce While this initially appears to indicate that the FNR is no hydrogen from glucose. Six FNRs and five ferredoxins were longer limiting at these conditions, when we analyze the cloned and expressed in E. coli, although several FNRs did not turnover of the hydrogenase in these same cases we find that express well enough to purify and evaluate in vitro. These the CpI hydrogenase turnover number is significantly lower poorly expressed enzymes were all plant enzymes (spinach (~3e5s 1) than its capacity: the dithionite-driven ferredoxin- leaf, pea root, and corn root FNRs) and may require post- mediated rate of ~1700 s 1. Consequently, it appears that translational modifications or other factors for proper as- neither enzyme is being used effectively and that there are sembly and folding that are missing in E. coli. By measuring limitations in this NADPH to H2 pathway independent of hydrogen production from dithionite, we clearly saw that protein concentrations. At this point, we do not understand effective interaction of the ferredoxin with the CpI hydroge- the reason for this limitation. It's possible that the competition nase is important in increasing the overall reaction rate. CpFd of FNR and CpI for ferredoxin may require a strict balance may confer significant advantages both because of its smaller between oxidized and reduced forms of ferredoxin, with the size and its two ironesulfur clusters that enable it to carry two addition of too much of either enzyme resulting in an imbal- electrons as well as having evolved to interact more effec- ance that offsets any potential gains in rate. Through further tively with it's native redox partner, CpI. Dithionite-based protein engineering we hope to gain further understanding of hydrogen production is more than four fold better with CpFd these limitations and discover how we can effectively modify than that mediated by the next-best ferredoxin. A 1000 range enzymes to improve electron transfer rates. We are encour- of hydrogen production was observed with different ferre- aged since to achieve our target of 400 kJ L 1 h 1 volumetric doxins indicating that the ferredoxin and hydrogenase inter- energy productivity, only 10% of the cytochrome c-measured action can have dramatic effects on hydrogen production rate. FNR turnover (5 s 1) is required at an FNR concentration of Improvements in FNR TONs also contributed to improved 100 mM (~3.6 g/L) (assuming a hydrogen fuel value of 120 MJ/kg international journal of hydrogen energy 40 (2015) 9113e9124 9123

H2). This compels us to search for an FNR mutant that can allows it to carry two electrons at once. The highest FNR TON come closer to its intrinsic turnover capability in the hydrogen in the cytochrome c assay was observed with the pairing of production pathway. AnFNR and SynFd, even better than when AnFNR was coupled While in this work purified enzymes of the synthetic with its natural partner, AnFd. This may indicate that organ- pathway were separately added to an E. coli extract, these isms have evolved for limited rates of ferredoxin reduction or proteins could easily be co-expressed in a mutant strain to could reflect limitation in interactions between certain ferre- produce a single extract that could be lysed and used directly doxins and cytochrome c. Finally, the conversion of glucose to for hydrogen production. However, the expression of these NADPH in a cell-free extract supplemented with purified FNR, proteins would need to be well controlled to produce a cell ferredoxin, and CpI hydrogenase generated hydrogen at a rate extract with high hydrogen production capability. More likely, similar to that of the synthetic pathway alone. Consequently, two separate strains could be made, one to optimally produce this work supports the feasibility of using unpurified extracts the FNR and pentose phosphate pathway enzymes and a for cell-free synthetic biology. In addition, this new pathway second to anaerobically produce the hydrogenase and ferre- enables the use of natural electron supply sources, such as doxin. The two extracts would then be blended to make a final glucose and NADPH, to enable screening for improved FNR extract mixture for hydrogen production. and hydrogenase function. This approach could be further extended by also co- expressing cellulases that could directly degrade pretreated biomass feedstock, producing a consolidated extract capable Acknowledgments of directly converting biomass to hydrogen in a single reaction mixture [35]. While many in vivo processes are challenged by We gratefully acknowledge the assistance of Dr. Alyssa Bing- byproducts released during the pre-treatment stages, such as ham and Jamin Koo in the overexpression and purification of acetic acid or furfural [36], our approach could be more flexible CpI [FeFe] hydrogenase. We thank Dr.’s Cem Albayrak and since viable cells are not present during hydrogen production, William Yang for providing the KC6 extract used in the glucose and the only requirement is that the enzymatic pathway we to hydrogen experiments and Global Climate and Energy desire is relatively stable. Project for funding. One ongoing challenge is the oxygen sensitivity of our [FeFe] hydrogenase, which is inactivated in aerobic environ- ments. Consequently, all work with hydrogen production was done anaerobically. Future processing and scale up of an Appendix A. Supplementary data anaerobic process could increase process complexity. There- Supplementary data related to this article can be found at fore, engineering the [FeFe] hydrogenase for oxygen tolerance http://dx.doi.org/10.1016/j.ijhydene.2015.05.121. will be helpful in developing a simpler large-scale process. While other [NiFe] hydrogenases exist that are tolerant to oxygen, these all catalyze much lower rates of H2 production references [37]. We therefore are working to discover O2 tolerant mutants of CpI. Using our cell-free lysate, we also have a unique opportu- [1] Armaroli N, Balzani V. The hydrogen issue. ChemSusChem nity to perform cell-free metabolic engineering and selectively 2011;4(1):21e36. knock out competing pathways that would be impossible to [2] Balat H, Kırtay E. Hydrogen from biomass e present scenario do in vivo [38]. Furthermore, while improvements in both rates and future prospects. Int J Hydrogen Energy and yields will be required for commercial feasibility, identi- 2010;35(14):7416e26. fying improved components of the synthetic pathway and [3] Ramachandran R, Menon R. An overview of industrial uses of hydrogen. Int J Hydrogen Energy 1998;23(7):593e8. demonstrating the feasibility of using an unpurified extract [4] U.S. Department of Energy. U.S. billion-ton update: biomass provide strong encouragement for us to further engineer our supply for a bioenergy and bioproducts industry. R.D. Perlack process. and B.J. Stokes (Leads), ORNL/TM-2011/224. Oak Ridge, TN: Oak Ridge National Laboratory; 2011. p. 227. [5] Saxena RC, Adhikari DK, Goyal HB. Biomass-based energy Conclusions fuel through biochemical routes: a review. Renew Sustain Energy Rev 2009;13(1):167e78. þ [6] Li H, Liao J. Engineering a cyanobacterium as the catalyst for We have evaluated six ferredoxin-NADP reductases and five the photosynthetic conversion of CO to 1,2-propanediol. 2013 ferredoxins for use in a synthetic pathway to produce Jan 12. p. 4. hydrogen from cellulosic biomass. Our highest rate of [7] Wells MA, Mercer J, Mott RA, Pereira-Medrano AG, Burja AM, hydrogen production came with a combination of Oryza sativa Radianingtyas H, et al. Engineering a non-native hydrogen (rice root) FNR, C. pasteurianum ferredoxin and C. pasteurianum production pathway into Escherichia coli via a cyanobacterial e [FeFe] hydrogenase. Significantly higher hydrogenase turn- [NiFe] hydrogenase. Metab Eng 2011;13(4):445 53. over was observed when CpI was combined with CpFd relative [8] Subudhi S, Lal B. Fermentative hydrogen production in recombinant Escherichia coli harboring a [FeFe]-hydrogenase to other ferredoxins. In addition to being the native electron gene isolated from Clostridium butyricum. Int J Hydrogen donor to CpI, CpFd is smaller than the photosynthetic ferre- Energy 2011;36(21):14024e30. doxins, which may reduce diffusion time between FNR and [9] Yoshida A, Nishimura T, Kawaguchi H, Inui M, Yukawa H. CpI, and also has an additional ironesulfur cluster, which Enhanced hydrogen production from glucose using ldh- and 9124 international journal of hydrogen energy 40 (2015) 9113e9124

frd-inactivated Escherichia coli strains. Appl Microbiol [25] Aliverti A, Faber R, Finnerty C, Ferioli C, Pandini V, Negri A, Biotechnol 2006;73(1):67e72. et al. Biochemical and crystallographic characterization of [10] Woodward J, Orr M, Cordray K, Greenbaum E. Biotechnology: ferredoxin-NADPþ reductase from nonphotosynthetic enzymatic production of biohydrogen. Nature tissues. Bochemistry 2001;40:14501e8. 2000;405:1014e5. [26] Carrillo N, Ceccarelli EA. Open questions in ferredoxin- [11] Zhang YHP, Evans BR, Mielenz JR, Hopkins RC, Adams MWW. NADPþ reductase catalytic mechanism. Eur J Biochem High-yield hydrogen production from starch and water by a 2003;270(9):1900e15. synthetic enzymatic pathway. PLoS One 2007;2:e456. [27] Martı´nez-Ju´ lvez M, Hermoso J, Hurley JK, Mayoral T, Sanz- [12] Wang Y, Huang W, Sathitsuksanoh N, Zhu Z, Zhang YHP. Aparicio J, Tollin G, et al. Role of arg100 and arg264 from High-yield production of dihydrogen from xylose by using a Anabaena PCC 7119 ferredoxin-NADPþ reductase for optimal synthetic enzyme cascade in a cell-free system. Angew NADPþ binding and electron transfer. Biochemistry Chem Int Ed 2013;52(17):4587e90. 1998;37(51):17680e91. [13] Rollin JA, Ye X, Martin del Campo J, Adams MWW, Zhang YH. [28] Margalit Rimona, Schejter Abel. Cytochrome c: a Novel hydrogen detection apparatus along with bioreactor thermodynamic study of the relationships among oxidation systems. Adv Biochem Eng Biotechnol 2014 Jul 15. http:// state, ion-binding and structural parameters. Eur J Biochem dx.doi.org/10.1007/10_2014_274. 1973;32:492e9. [14] Ninh PH, Honda K, Sakai T, Okana K, Ohtake H. Assembly [29] Prince Roger C, Adams Michael WW. Oxidation-reduction and multiple gene expression of thermophilic enzymes in properties of the two Fe4S4 clusters in Clostridium Escherichia coli for in vitro metabolic engineering. Biotechnol pasteurianum ferredoxin. J Biol Chem 1987;262(11):5125e8. Bioeng 2015;112:189e96. [30] Onda Yayoi, Matsumura Tomohiro, Kimata-Ariga Yoko, [15] Myung S, Rollin J, You C, Sun F, Chandrayan S, Adams MW, Sakakibara Hitoshi, Sugiyama Tatsuo, Hase Toshiharu. et al. In vitro metabolic engineering of hydrogen production Differential interaction of maize root Ferredoxin:NADP at theoretical yield from sucrose. Metab Eng 2014;24:70e7. oxidoreductase with photosynthetic and non- [16] Madden C, Vaughn MD, Diez-Perez I, Brown KA, King PW, photosynthetic ferredoxin isoproteins. Plant Physiol July Gust D, et al. Catalytic turnover of [FeFe]-hydrogenase based 2000;123:1037e45. on single-molecule imaging. J Am Chem Soc [31] Kyritsis P, Huber JG, Quinkal I, Gaillard J, Moulis JM. 2012;134:1577e82. Intramolecular electron transfer between [4fe-4s] clusters [17] Sun J, Hopkins RC, Jenney FE, McTernan PM, Adams MWW. studied by proton magnetic resonance spectroscopy. Heterologous expression and maturation of an NADP- Biochemistry 1997;36(25):7839e46. dependent [NiFe]-hydrogenase: a key enzyme in biofuel [32] Calhoun KA, Swartz JR. Total amino acid stabilization during production. PLoS One 2010;5:e10526. cell-free protein synthesis reactions. J Biotechnol [18] Kuchenreuther JM, Grady-Smith CS, Bingham AS, George SJ, 2006;123(2):193e203. Cramer SP, Swartz JR. High-yield expression of heterologous [33] Kundig W, Roseman S. Sugar transport II characterization of [FeFe] hydrogenases in Escherichia coli. PloS One constitutive membrane-bound enzymes II of the Esherichia 2010;5(11):e15491. coli phosphotransferase system. J Biol Chem [19] Boyer ME, Wang C-W, Swartz JR. Simultaneous expression 1971;246:1407e18. and maturation of the iron-sulfur protein ferredoxin in a [34] Jewett M, Calhoun K, Voloshin A, Wuu J, Swartz J. An cell-free system. Biotechnol Bioeng 2006;94:128e38. integrated cell-free metabolic platform for protein [20] Smith PR, Bingham AS, Swartz JR. Generation of hydrogen production and synthetic biology. Mol Syst Biol 2008;4:220. from NADPH using an [FeFe] hydrogenase. Int J Hydrogen [35] Olson D, McBride J, Shaw AJ, Lynd L. Recent progress in Energy 2012;37:2977e83. consolidated bioprocessing. Curr Opin Biotechnol [21] Hoover DM, Lubkowski J. DNAWorks: an automated method 2012;23(3):396e405. for designing oligonucleotides for PCR-based gene synthesis. [36] Pienkos P, Zhang M. Role of pretreatment and conditioning Nucleic Acids Res 2002;30:e43. processes on toxicity of lignocellulosic biomass [22] Kodumal SJ, Patel KG, Reid R, Menzella HG, Welch M, hydrolysates. Cellulose August 2009;16(4):743e62. Santi DV. Total synthesis of long DNA sequences: synthesis [37] Lenz O, Ludwig M, Schubert T, Burstel I, Ganskow S, Goris T,

of a contiguous 32-kb polyketide synthase gene cluster. Proc et al. H2 conversion in the presence of O2 as performed by Natl Acad Sci U S A 2004;101(44):15573e8. the membrane-bound [NiFe] hydrogenase of Ralstonia [23] Lambeth DO, Palmer G. The kinetics and mechanism of eutropha. ChemPhysChem 2010;11:1107e19. reduction of electron transfer proteins and other compounds [38] Guterl J-K, Garbe D, Carsten J, Stefﬂer F, Sommer B, Reiße S, of biological interest by dithionite. J Biol Chem et al. Cell-free metabolic engineering: production of 1973;248(17):6095e103. chemicals by minimized reaction cascades. ChemSusChem [24] Karplus A, Faber R. Structural aspects of plant 2012;5:2165e72. http://dx.doi.org/10.1002/cssc.201200365. ferrodoxin:NADPþ oxidoreductase. Photosyn Res 2004;81:303e15. 本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

学霸图书馆（www.xuebalib.com）是一个“整合众多图书馆数据库资源，

提供一站式文献检索和下载服务”的24 小时在线不限IP 图书馆。图书馆致力于便利、促进学习与科研，提供最强文献下载服务。

图书馆导航：

图书馆首页文献云下载图书馆入口外文数据库大全疑难文献辅助工具