Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to NRP
INNOVATIVE BIOMANUFACTURING -- CASCADE ENZYME FACTORIES FEATURING HIGH PRODUCT YIELDS AND LOW PROCESSING COSTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0229173
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jun 1, 2012
Project End Date
May 31, 2017
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Biological Systems Engineering
Non Technical Summary
Thanks to our recent breakthroughs - the production of 12 mol of hydrogen per mol of hexose and water as C6H10O5 (l) + 7 H2O (l)  12 H2 (g) + 6 CO2 (g) mediated by cascade enzymes, we suggest the use of sugars as a renewable energy source for hydrogen production and as a high-density hydrogen carrier with a gravimetric density of 8.33 mass H2 %. Furthermore, high-purity hydrogen is produced to permit direct coupling of the hydrogen-generating enzymatic reactions to the PEM fuel cells. The aims of this project are to break the last two obstacles of this novel sugar-to-hydrogen technology: (i) slow hydrogen generation rates and (ii) high biocatalyst costs. We plan to accelerate enzymatic hydrogen-generation rates to ~8 g H2/L/h by elevating reaction temperature, increasing total enzyme loadings, elevating substrate concentration, optimizing rate-limiting step enzyme ratio, and creating enzyme complexes (metabolons) for potential substrate channeling. Also, we will decrease biocatalyst costs by replacing all mesophilic enzymes with recombinant thermoenzymes produced by E. coli, immobilizing enzymes or their complexes for prolonged lifetime, as well as engineering three redox enzymes that can utilize low-cost, stable biomimetic coenzymes.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
51140992020100%
Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
2020 - Engineering;
Goals / Objectives
The goals of this project are to break the last two obstacles of cascade enzyme factories -- high biocatalyst costs involving enzyme stability and co-factor degradation and to increase reactions rates for more potential applications. The specific objectives are (i) to decrease biocatalyst costs by replacing all mesophilic enzymes with stable recombinant thermoenzymes, (ii) to engineer three redox enzymes that can utilize low-cost stable biomimetic cofactors, and (iii) to increase reaction rates mediated by synthetic enzyme pathways.
Project Methods
This project comprises three tasks as described in the following sections. Task 1. Decrease biocatalyst costs by systematic efforts Subtask 1.1. Clone, express, and purify four recombinant hyperthermophilic enzymes Subtask 1.2. Develop enzyme complexes by one-step enzyme complex immobilization Subtask 1.3. Produce low-cost P. furiosus hydrogenase SH1 Task 2. Engineer three oxidoreductases that can use low-cost biomimetic coenzymes by rational design and directed evolution Subtask 2.1. Engineer coenzyme specificity of a hyperthermophilic Thermotoga maritima 6-phosphogluconate dehydrogenase (Tm6PGDH) Subtask 3.2. Engineer coenzyme specificity of glucose-6-phosphate dehydrogenase and hydrogenase SH1 Task 3. Increase enzymatic hydrogen generation rates by ~100-1,000 fold Subtask 3.1. Increase reaction rates by ~10 fold Subtask 3.2. Milestone 2 -- Increase reaction rates by ~100-1000 fold

Progress 06/01/12 to 05/31/17

Outputs
Target Audience: Nothing Reported Changes/Problems:The PD is no longer employed atVirginia Tech, no information to report. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? N/A

Publications


    Progress 10/01/15 to 09/30/16

    Outputs
    Target Audience:Scientific community, biofuels, hydrogen, sustainable agriculture, biotechnology industry, government agencies, policy makers, and food-energy-water nexus. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In the past year, the PI, his group and his collaborators, published 15 journal articles and one book chapter pertaining to this topic. Two Ph.D. students, two postdocs and two visiting scholars received training. How have the results been disseminated to communities of interest?We published papers and gave invited talks in conferences and companies. What do you plan to do during the next reporting period to accomplish the goals?With the help of DOE EERE funding, we will further increase the hydrogen generation rate and decrease its production costs through our fundamental research. I will provide scientific advice to the Chinese company to help themestablish the first plant based on this novel biomanufacturing platform in 2017. I hope that the USA communities will realize great potentials of cascade enzyme factories for the biomanufacturing of clean energy, new food, biochemicals, etc.

    Impacts
    What was accomplished under these goals? We achieved the fastest biohydrogen production from sugars, being 310 mmole H2/L/h via the biomimetic electron transport chain and revised enzymatic pathway. In addition to theoretical yield of hydrogen production from carbohydrates (i.e., 1 mole of glucose plus water can generate 12 moles of hydrogen), we have succeeded in addressing all three biomanufacturing criteria - yield, titer and rate. Carbohydrate-based hydrogen could provide affordable green hydrogen for the hydrogen economy. Also, carbohydrate may be a new high-density hydrogen carrier. We designed a biomimetic in vitro enzymatic pathway for starch hydrolysis for cost-efficient generation of glucose 1-phosphate without costly ATP. As a result, this alternative method of starch hydrolysis could have an important role in biomanufacturing of a lot of products, such as hydrogen, bioelectricity, fructose, stevia, inositol, to name a few. We established a novel high-throughput screening method for coenzyme engineering. We succeeded in changing NADP-preferred glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from NADP to NAD. The engineered enzymes have the equivalent performance on NAD and NADP. Along with Chinese scientists, we published a perspective review about the history and perspective of biomanufacturing. In the viewpoint of history, cascade enzyme factories or advanced biotransformation catalyzed by in vitro synthetic enzymatic biosystems would be one of the most important disruptive technologies to revolutionize the agriculture and renewable energy systems. In collaboration with Chinese scientists and companies, we determined that the production cost of recombinant enzymes produced in E. coli could be decreased to $30-50 per kg of dry enzyme. Beyond the proof-of-concept experiments pertaining to enzymatic conversion of cellulose to starch (PNAS 2013), we have developed several new methods to decrease production costs of artificial amylose (linear starch). The introduction of starch branching enzyme enables us to produce amylopectin, another important component of natural starch. Via mice feeding tests, our international collaborators found out that artificial amylose (linear starch) has a much slower glycemic index than natural starch and artificial amylopectin. Such data suggest that we are able to tailor the properties of artificial starch to meet different food needs, and healthy artificial starch could decrease possibilities of obesity and diabetes. We believe that our cascade enzyme factory technology would address the key problem in the food-energy-water nexus and we will eventually feed the world with biomass-derived starch.

    Publications

    • Type: Book Chapters Status: Published Year Published: 2016 Citation: You C, Zhang Y-HP*. 2016. Ex vivo enzymatic conversion of non-food cellulose biomass to starch. In Springer Book -- Green Chemistry and Sustainable Technology (Quality Living Through Chemurgy and Green Chemistry). Editor: Peter C.K. Lau. Springer. ISBN 3662537028. (Invited book chapter).
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhong C, Wei P*, Zhang Y-HP*. 2016. A kinetic model of one-pot rapid biotransformation of cellobiose from sucrose catalyzed by three thermophilic enzymes. Chemical Engineering Science. Epub, DOI: 10.1016/j.ces.2016.1011.104
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhu ZG, Zhang Y-HP*. 2016. In vitro metabolic engineering of bioelectricity generation for the complete oxidation of glucose. Metabolic Engineering. Epub. DOI: 10.1016/j.ymben.2016.11.002
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhang Y-HP*, Sun JB, Ma YH. 2016. Biomanufacturing: history and perspective. Journal of Industrial Microbiology & Biotechnology. Epub. DOI: 10.1007/s10295-016-1863-2.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: You C*, Zhang Y-HP. 2016. Biomanufacturing by in vitro biosystems containing cascade enzymes. Process Biochemistry. Epub, DOI: 10.1016/j.procbio.2016.09.025.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Chen H, Zhu ZG, Huang Rui, Zhang Y-HP*. 2016. Coenzyme engineering of a hyperthermophilic 6-phosphogluconate dehydrogenase from NADP+ to NAD+ with its application to biobatteries. Scientific Reports. 6:36311.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Kim E-J, Wu C-H, Adams MWW, Zhang Y-HP*. 2016. Exceptionally high rates of biohydrogen production by biomimetic in vitro synthetic enzymatic pathways. Chemistry  A European Journal. 22:16047-16051.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Kim J-E, Huang R, Chen H, You C, Zhang Y-HP*. 2016. Facile insertion of a large-size random gene mutagenesis library into Escherichia coli plasmid without restriction enzymes. Biotechnology Journal. 11:1142-1150.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Collins J, Huston S, Zhang T, Sun FF, Zhang Y-HP, Fu JL*. 2016. A hidden transhydrogenase activity of an FMN-bound diaphorase under anaerobic conditions. PLoS One. 11(5): e0154865.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhang L, Yuan J, Xu Y*, Zhang Y-HP*, Qian XH*. 2016. New artificial fluoro-cofactor of hydride transfer with novel evaluation method of fluorescence sensing for redox biocatalysis. Chemical Communications. 52: 6471-6474.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Li YJ, Huang B, Wu H*, Li ZM*, Ye Q, Zhang Y-HP. 2016. Production of succinate from acetate metabolically engineered by Escherichia coli. ACS Synthetic Biology. 5(11), 1299-1307.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Moustafa HMA, Zaghloul TI, Zhang Y-HP*. 2016. A simple enzymatic assay for determining activities of phosphopentomutase from a hyperthermophilic bacterium Thermotoga maritima. Analytical Biochemistry. 501: 75-81.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Kim J-E, Zhang Y-HP*. 2016. Biosynthesis of D-xylulose 5-phosphate from D-xylose and polyphosphate through a minimized two-enzyme cascade. Biotechnology and Bioengineering. 113, 275-282.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Rollin JA, Ye XH, Martin del Campo JS, Adams MWW, Zhang Y-HP*. 2016. Novel hydrogen detection apparatus along with bioreactor Systems. Advances in Biochemical Engineering-Biotechnology. 152: 35-51.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Huang R, Chen H, Zhong C, Kim J-E, Zhang Y-HP*. 2016. High-throughput screening of coenzyme preference change of thermophilic 6-phosphogluconate dehydrogenase from NADP+ to NAD+. Scientific Reports 6:32644.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Moustafa HMW, Kim E-J, Zhu ZG, Wu C-H, Zaghloul TI, Adams MWW, Zhang Y-HP*. 2016. Water splitting for high-yield hydrogen production energized by biomass xylooligosaccharides catalyzed by an enzyme cocktail. ChemCatChem. 8: 28982902.


    Progress 10/01/14 to 09/30/15

    Outputs
    Target Audience:Scientific community, biofuels, hydrogen, sustainable agriculture, biotechnology industry, and government agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two Ph.D. students, three postdocs and two visiting students receivedtraining. How have the results been disseminated to communities of interest?We published numerous scientific journal papers and book chapters. Biomass hydrogen paper was highlighted by a lot of news media, such as EurekAlert - Discovery by Virginia Tech may be breakthrough for hydrogen cars (Apr 6, 2015) MIT Technology Review - A new source of hydrogen for fuel cell vehicles (Apr 20, 2015) Chemical Engineering Progress (AIChE) - Metabolic Engineering Delivers Low-Cost Hydrogen (May 2015) The Independent (UK) - Breakthrough in hydrogen-powered cars may spell end for petrol stations (Apr 6, 2015) International Business Times (UK) - Hydrogen fuel made from corn husks could be renewable energy breakthrough for cars (Apr 7, 2015) R&D Magazine - Discovery may be breakthrough for hydrogen cars 20 minutes (France) -- L'hydrogène : bientôt du carburant propre bon marché (Apr 6, 2015) Spiegel Online (German) -- Erneuerbare Energie: Dreimal mehr Wasserstoff aus Mais (Apr 7, 2015) Tech Times - Corn husks, cobs, and stalks may fuel green cars of the future (Apr 7, 2015) What do you plan to do during the next reporting period to accomplish the goals?Data collection and analysis; preparation of peer-reviewed research publications and abstracts/presentations.

    Impacts
    What was accomplished under these goals? Based on the proof-of-concept experiments pertaining to enzymatic conversion of cellulose to starch (PNAS 2013), we have developed several new methods to decrease production costs: (1) We can utilize commercial cellulase by selective removal of beta-glucosidase, (2) We can decrease cellulase use via substrate channeling among the cellulose-cellulose-microbe complex, (3) We identify high-end application of synthetic starch. We believe that our technology has no problem in its commercialization. As a result, we will feed the world by using nonfoodbiomass eventually. Sustainable agriculture will occur by switching the cultivation of annual crops to perennial plants. We achieved high-yield hydrogen production from biomass sugars and increased reaction rates by nearly 20-fold by using mathematical modeling. This key breakthrough has been published in PNAS (2015). A lot of media covers this accomplishment, such as MIT Technology Review, Chemical Engineering Progress (official journal of AIChE). We discovered the most thermostable isoamylase, which is suitable for simultaneous starch gelatinization and enzymatic starch hydrolysis. We have invented the best (far less costly) technologies to produce tagatose from starch instead of lactose, produce inositol from starch, and produce arabinose from xylose. As a result, we will revolutionize sweetener industries by providing affordable functional sweeteners: (1) replace fructose by its epimer -- low calorie functional tagatose; and (2) neutralize sucrose by using arabinose.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Rollin JA, Martin del Campo JS, Myung S, Sun FF, You C, Bakovic AE, Castro RL, Chandrayan S, Wu C-H, Adams MWW, Senger R, Zhang Y-HP*. 2015. High-yield hydrogen production from biomass by in vitro metabolic engineering: Mixed sugars coutilization and kinetic modelling. Proceedings of the National Academy of Sciences of the United States of America. 112: 4964-4969.
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Cheng K, Zhang F, Sun F-F, Zhu, Z-G, Chen H-G*, Zhang Y-HP*. 2015. Doubling power output of starch biobattery treated by the most thermostable isoamylase from an archaeon Sulfolobus tokodaii. Scientific Reports. 5:13184.
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhu ZG, Zhang Y-HP*. 2015. Use of non-immobilized enzymes and mediators achieved high power densities in closed biobatteries. Energy Science and Engineering. 3: 490-497.
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Chen HG, Zhang Y-HP*. 2015. New biorefineries and sustainable agriculture and: increased food, biofuels, and ecosystem security. Renewable & Sustainable Energy Reviews 47: 117-132.
    • Type: Book Chapters Status: Published Year Published: 2015 Citation: Zhu ZG, Zhang Y-HP*. 2015. Chapter 4: Chemical biotechnology of in vitro biosystems for biomanufacturing. In the book of Chemically Promoted Biotechnology and Bioengineering. Royal Society of Chemistry, UK (Invited book chapter) (pp98-121). Print ISBN: 978-1-84973-810-1. DOI: 10.1039/9781782620129-00098
    • Type: Book Chapters Status: Published Year Published: 2015 Citation: JE Kim, Zhang Y-HP*. 2015. Chapter 9. Use of carbohydrates for hydrogen storage. In Compendium to Hydrogen Energy - Volume 2: Hydrogen storage, transmission, transportation and infrastructure. Edited by: Ram B. Gupta  Series editors: Angelo Basile & T. Nejat Veziroglu (Invited book chapter) Epub, http://dx.doi.org/10.1016/B978-1-78242-362-1.00009-2.
    • Type: Book Chapters Status: Submitted Year Published: 2015 Citation: Zhong C, You C, Wei P, Zhang Y-HP*. 2015. Simple cloning by prolonged overlap extension PCR with application to the preparation of large-size random gene mutagenesis library in Escherichia coli. In the Book of Methods in Molecular Biology. Revision submitted.
    • Type: Book Chapters Status: Published Year Published: 2015 Citation: Rollin JA, Ye XH, Martin del Campo JS, Adams MWW, Zhang Y-HP*. 2014. Novel hydrogen detection apparatus along with bioreactor Systems. Advances in Biochemical Engineering-Biotechnology Epub, DOI: 10.1007/10_2014_274 (Invited).
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhang Y-HP*. 2015. Production of biofuels and biochemicals by in vitro synthetic biosystems: opportunities and challenges. Biotechnology Advances, Epub, DOI: 10.1016/j.biotechadv.2014.10.009 (Invited Review)
    • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Kim J-E, Zhang Y-HP*. 2015. Biosynthesis of D-xylulose 5-phosphate from D-xylose and polyphosphate through a minimized two-enzyme cascade. Biotechnology and Bioengineering. Epub, DOI: 10.1002/bit.25718.
    • Type: Book Chapters Status: Published Year Published: 2015 Citation: JE Kim, Zhang Y-HP*. 2015. Chapter 3. High-yield production of biohydrogen from carbohydrates and water based on in vitro synthetic (enzymatic) pathways. In Book Hydrogen production from renewable resources, Springer Book Series - Biofuels and Biorefineries Editors: Zhen Fang, Richard L. Smith, Jr., Xinhua Qi. (Springer) (Invited book chapter).


    Progress 10/01/13 to 09/30/14

    Outputs
    Target Audience: Scientific community, biofuels, hydrogen, sustainable agriculture, biotechnology industry, government agencies Changes/Problems: Limited funding and few hands are our obstacles. No matter what happens, our goals will be achieved eventually. What opportunities for training and professional development has the project provided? Two Ph.D. students, two undergraduate students, two postdocs and two visiting scholars recieved the training. How have the results been disseminated to communities of interest? In the past year, the PI, his group and his collaborators published8journalpublications and 3 invited book chapterspertaining to this topic. The PI has given numerous invited talks in international conferences and universities. What do you plan to do during the next reporting period to accomplish the goals? We will produce plenty of artificial starch from biomass and conduct animal test. We will scale up biobattery to power small size electric car. We will create more redox enzymes that use biomimetic cofactors. We will have more applications for demonstration.

    Impacts
    What was accomplished under these goals? Beyond the proof-of-concept experiments pertaining to enzymatic conversion of cellulose to starch (PNAS 2013), we have developed several new methods to decrease production costs. We believe that our technology has no problem in its commercialization. As a result, we will feed the world by using biomass eventually. We have achieved the complete oxidation of hexose in enzymatic fuel cells for the first time and the paper was published in Nature Communications. Plenty of news media highlighted this breakthrough including Time magazine, etc. We achieved high-yield hydrogen production from biomass sugars and increased reaction rates by nearly 20-fold by using mathematical modeling. The manuscript is in revision for publication in PNAS. We have increased reaction rates more than 800 fold to ~160 mmole H2/L/h, which is the highest reaction rates for biohydrogen generation in the world. We have succeeded in engineering cofactor preference of two redox enzymes: glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) working on a low-cost and stable biomimetic cofactor - BCP. Also, we have established a novel high-throughput screening for cofactor engineering and discovered that wild-type hydrogenase can work on BCP.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhu ZG, Tam TK, Sun FF, You C, Zhang Y-HP*. 2014. A high-energy-density sugar biobattery based on a synthetic enzymatic pathway. Nature Communications 5: 3026.
    • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang Y-HP*. 2014. Production of biofuels and biochemicals by in vitro synthetic biosystems: opportunities and challenges. Biotechnology Advances, Epub, DOI: 10.1016/j.biotechadv.2014.10.009 (invited).
    • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Rollin JA, Martin del Campo JS, Myung S, Sun FF, Bakovic AE, Castro RL, Chandrayan S, Wu C-H, Adams MWW, Zhang Y-HP*. 2014. High-yield and high-speed hydrogen production from biomass by in vitro metabolic engineering. Proc. Nat. Acad. Sci. USA. in revision. 2014-17719.
    • Type: Journal Articles Status: Published Year Published: 2014 Citation: Myung S, Rollin JA, You C, Sun FF, Chandrayan S, Adams MWW, Zhang Y-HP*. 2014. In vitro metabolic engineering of hydrogen production at theoretical yield from sucrose. Metabolic Engineering, 24(1): 70-77.
    • Type: Journal Articles Status: Published Year Published: 2014 Citation: You C, Zhang Y-HP*. 2014. Annexation of a high-activity rate-limiting enzyme in a synthetic three-enzyme complex greatly decreases the degree of substrate channeling. ACS Synthetic Biology 3:380-386.
    • Type: Journal Articles Status: Published Year Published: 2014 Citation: Qi P, You C, Zhang Y-HP*. 2014. One-pot enzymatic conversion of sucrose to synthetic amylose by using enzyme cascades. ACS Catalysis 4: 1311-1317.
    • Type: Journal Articles Status: Published Year Published: 2014 Citation: Gao SH, You C, Renneckar S, Bao J, Zhang Y-HP*. 2014. New insights into enzymatic hydrolysis of heterogeneous cellulose by using carbohydrate-binding module 3 containing GFP and carbohydrate-binding module 17 containing CFP. Biotechnology for Biofuels 7(1): 24
    • Type: Journal Articles Status: Published Year Published: 2014 Citation: Ahmad S, Ma H, Akhtar MW, Zhang Y-HP*, Zhang X-Z*. 2014. Directed evolution of Clostridium phytofermentans glycoside hydrolase family 9 endoglucanase for enhanced specific activity on solid cellulosic substrate. Bioenergy Research 7(1): 381-388.
    • Type: Book Chapters Status: Published Year Published: 2014 Citation: Rollin JA, Ye XH, Martin del Campo JS, Adams MWW, Zhang Y-HP*. 2014. Novel hydrogen detection apparatus along with bioreactor Systems. Advances in Biochemical Engineering/Biotechnology Epub, DOI: 10.1007/10_2014_274 (invited).
    • Type: Book Chapters Status: Published Year Published: 2014 Citation: You C, Zhang Y-HP*. 2014. Simple cloning and DNA assembly in E. coli by prolonged overlap extension PCR. Methods of Molecular Biology 1116: 183-192. (Invited book chapter)
    • Type: Book Chapters Status: Awaiting Publication Year Published: 2014 Citation: Zhu ZG, Zhang Y-HP*. 2014. Chemical biotechnology of in vitro biosystems for biomanufacturing. In the book of Chemically Promoted Biotechnology and Bioengineering. Royal Society of Chemistry, UK (Invited book chapter)
    • Type: Book Chapters Status: Awaiting Publication Year Published: 2014 Citation: JE Kim, Zhang Y-HP*. 2014. Chapter 12. Use of carbohydrates for hydrogen storage. In Compendium to Hydrogen Energy - Volume 2: Hydrogen storage, transmission, transportation and infrastructure. Edited by: Ram B. Gupta  Series editors: Angelo Basile & T. Nejat Veziroglu.


    Progress 10/01/12 to 09/30/13

    Outputs
    Target Audience: Scientific community, biofuels industry, biotechnology industry, government agencies. Changes/Problems: We will design new pathways for the demonstration of the potential applications of this cell-free biomanufacturing platform. What opportunities for training and professional development has the project provided? Three Ph.D. students completed their Ph.D. degrees; one Ph.D. student, and three postdocs are in training. How have the results been disseminated to communities of interest? Published 10 peer-refereed journal papers, 3 book chapters, 3 Ph.D. dissertations What do you plan to do during the next reporting period to accomplish the goals? To further decrease biocatalyst cost, we will increase specific activities of key redox enzymes used for this novel biomanufacturing platform. To increase reaction rates, we will optimize enzyme ratios and discover better enzymes. To expand its applications, we will design new pathways for the production of more commodities and value-added fine chemicals.

    Impacts
    What was accomplished under these goals? To decrease biocatalysis cost, we have succeeded in engineering the cofactor preference of two redox enzymes: glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) working on a low-cost and stable biomimetic cofactor - BCP. Also, we have established a novel high-throughput screening for cofactor engineering and discovered that wild-type hydrogenase can work on BCP. To increase reaction rates, we have increased reaction rates more than 300 fold to 160 mmole H2/L/h. To expand the application range of this new platform, we are the first to convert cellulose to starch by using cascade enzymes without sugar loss. The paper published in PNAS was highlighted by Science magazine “Could wood feed the world”. To increase substrate utilization range, we have expanded substrate ranges of enzymatic hydrogen production from cellulose and starch to monomeric sugars, xylose, and glucose through novel pathways. To expand the application range of this new platform, we have achieved the complete oxidation of hexose in enzymatic fuel cells for the first time and the paper has been accepted for publication in Nature Communications.

    Publications

    • Type: Book Chapters Status: Published Year Published: 2013 Citation: You C, Zhang Y-HP. 2013. Simple cloning and DNA assembly in E. coli by prolonged overlap extension PCR. Methods of Molecular Biology 1116; DOI: 10.1007/978-1-62703-764-8_13
    • Type: Journal Articles Status: Published Year Published: 2013 Citation: Myung S, You C, Zhang Y-HP. 2013. Recyclable cellulose-containing magnetic nanoparticles: immobilization of cellulose-binding module-tagged proteins and synthetic metabolon featuring substrate channeling. Journal of Materials Chemistry B. 1:4419-4427
    • Type: Journal Articles Status: Published Year Published: 2013 Citation: Rollin JR, Tam TK, Zhang Y-HP. 2013. A biotechnology paradigm: cell-free biosystems for biomanufacturing. Green Chemistry. 15:1708-1719 (invited).
    • Type: Journal Articles Status: Published Year Published: 2013 Citation: Zhang Y-HP. 2013. Next-generation biorefineries will solve the food, biofuels and environmental trilemma in the energy-food-water nexus. Energy Science and Engineering 1:25-41
    • Type: Journal Articles Status: Published Year Published: 2013 Citation: You C, Chen HG, Myung S, Sathisuksanoh N, Ma H, Zhang XZ, Li JY, Zhang Y-HP. 2013. Enzymatic transformation of non-food biomass to starch. Proceedings of the National Academy of Sciences of the USA. 110: 7182-7189. Highlighted by Science magazine
    • Type: Journal Articles Status: Published Year Published: 2013 Citation: Martin del Campo JS, Rollin JR, Myung S, You C, Chandrayan S, Pati�o R, Adams MWW, Zhang Y-HP. 2013. Dihydrogen production from xylose and water mediated by synthetic cascade enzymes. Angewandte Chemie International Edition 52:4587-4590. (Editors choice paper).
    • Type: Journal Articles Status: Published Year Published: 2013 Citation: Martin del Campo JS, You C, Kim J-E, Pati�o R, Zhang Y-HP. 2013. Discovery and characterization of a novel ATP/polyphosphate xylulokinase from a hyperthermophilic bacterium Thermotoga maritima. Journal of Industrial Microbiology and Biotechnology 40:661-669.
    • Type: Book Chapters Status: Published Year Published: 2013 Citation: Zhu ZG, Tam TK, Zhang Y-HP. 2013. Cell-free biosystems for biomanufacturing in the production of electricity and bioenergy. Advances in Biochemical Engineering/Biotechnolog, Epub, http://dx.doi.org/10.1007/10_2013_201
    • Type: Book Chapters Status: Published Year Published: 2013 Citation: Jandt U, You C, Zhang Y-HP, Zeng A-P. 2013. Compartmentation and metabolic channeling: practical and modeling aspects for multienzymatic biosynthesis. Advances in Biochemical Engineering/Biotechnology Epub, DOI: 10.1007/10_2013_221
    • Type: Journal Articles Status: Published Year Published: 2013 Citation: Myung S, Zhang Y-HP. 2013. Non-complexed four cascade enzyme mixture: simple purification and synergetic co-stabilization. PLoS ONE 8, e61500.
    • Type: Journal Articles Status: Published Year Published: 2013 Citation: Zhang Y-HP, Xu J-H, Zhong J-J. 2013. A new high-energy density hydrogen carrier - carbohydrate - might be better than methanol. International Journal of Energy Research 37:769-779.
    • Type: Journal Articles Status: Published Year Published: 2013 Citation: You C, Zhang Y-HP. 2013. Self-assembly of synthetic metabolons through synthetic scaffoldins: single-step purification, co-immobilization, and substrate channeling. ACS Synthetic Biology 2:102-110 (cover page).
    • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Myung, S. (2013) Cell-free biosystems comprised of synthetic enzymatic pathways: development of building blocks, immobilization of enzymes, stabilization of cascade enzymes, and generation of hydrogen (143 pages).
    • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Zhu, Z. (2013). Enzymatic fuel cells via synthetic enzymatic pathway biotransformation (134 pages).
    • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Rollin, JA (2013). Synthetic enzymatic pathway conversion of cellulosic biomass to hydrogen (110 pages)..


    Progress 10/01/11 to 09/30/12

    Outputs
    OUTPUTS: Cascade Enzyme Factories (CEFs) are believed to represent a new biotechnology paradigm that can produce a variety of products by using numerous enzymes in vitro together;however there are some doubts about DEFs. To address these doubts, we attempted to (i) decrease biocatalyst costs by replacing all mesophilic enzymes with stable recombinant thermoenzymes, (ii) engineer redox enzymes that can utilize low-cost stable biomimetic cofactors, and (iii) increase reaction rates mediated by synthetic enzyme pathways. With regard to the first issue, we have cloned, expressed, and purified all thermoenzymes in the laboratory. With regard to the second issue, we have modified two redox enzymes: glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) by rational design to increase their specificity on different cofactors: NADP, NAD, NMN, and BCP. As a result, engineered 6PGDHs can work on NMN and BCP. We will further enhance their activity. With regard to the third issue, we have increased reaction rates more than 300 fold to 160 mmole H2/L/h. In addition, we have expanded substrate ranges of enzymatic hydrogen production from cellulose and starch to monomeric sugars, xylose, and glucose through novel pathways. Also, we have applied cell-free biosystems concepts to several new applications. We are the first to convert cellulose to starch by using cascade enzymes. We have achieved complete oxidation of hexose in enzymatic fuel cells for the first time. We have designed a synthetic pathway for production of high-value carbohydrates as potential drugs. In the past year, the PI, his group and his collaborators published 13 publications. Two of them are invited reviews or perspectives. The PI has given numerous invited talks in international conferences and universities, such as ETH Zurich, Basel, Switzerland (09/17/2012); Technological University of Munchen (TUM), Straubing, Germany (09/14/2012); Keynote talk of DECHEMA annual meeting, Karlsruhe, Germany (09/11/2012); 6th World Congress of Biocatalysis, Hamburg, Germany (09/04/2012); Chemical Engineering department, Auburn University, Auburn, Alabama (04/04/2012). Three postdocs and six graduate students were working on this project. One graduate received her MS degree in 2012. PARTICIPANTS: Biological Systems Engineering Department of Virginia Tech, Blacksburg, VA: Dr. Chun You (postdoc); Dr. James Galman (postdoc), Dr. Xing Zhang (postdoc), Fangfang Sun (MS graduate), Hehuan Liao (MS graduate), Suwan Myung (Ph.D. candidate), Joe Rollin (Ph.D. candidate), Zhiguang Zhu (Ph.D. candidate), Julia Sanchez Martin Del Campo (visiting Ph.D. candidate) TARGET AUDIENCES: Scientific community, biofuels industry, biotechnology industry, government agencies PROJECT MODIFICATIONS: Not relevant to this project.

    Impacts
    Advances in cascade enzyme factories clearly suggest their great potential in biomanufacturing. We discovered new enzymes featuring both great stability and high activity by using bioinformatics tools to search the exploding genome database of thermophilic microorganisms, sequence comparison, expression prediction, and so on. Now we have better ways to determine desired putative thermoenzymes rather than blind trials. As a result, we will save labor and costs for developing building blocks for synthetic biology projects. We developed new technologies that can co-purify and co-immobilize several cascade enzymes in one-step by using cohesins and dockerins from cellulosomes. Such multi-enzyme complexes, called synthetic metabolons, exhibited 10-20 times higher reaction rates than non-complexed enzyme mixtures at the same enzyme concentration. In addition to cellulosic materials, such enzyme complexes on the surface of nano-magnetic particles could be easily purified by a magnetic force. As a result, we will standardize biocatalytic modules as building modules for synthetic biology projects. The 300-fold increase in hydrogen reaction rate (i.e., 160 mmole H2/L/h) suggests a great potential in further enhancement of hydrogen reaction rates. These reaction rates are fast enough for stationary hydrogen generators. Our advances imply that developing sugar hydrogen fuel vehicles could not be a dream. According to biomass-to-wheel energy analysis, approximately 700 million tons of biomass could be sufficient to replace all gasoline consumption if we increase energy efficiency by approximately three-four times from current systems. This analysis reinforces the importance of increasing energy utilization efficiency. The bioeconomy will bring benefits: promoting rural economy, creating new biomanufacturing jobs, decreasing reliance on imported crude oil, mitigating climate change, increasing national energy security, decreasing water and carbon footprints, and decreasing trade deficits.

    Publications

    • Sun FF, Zhang X-Z, Myung S, Zhang Y-HP. Thermophilic Thermotoga maritima ribolse-5-phosphate isomerase RpiB: optimized heat treatment purification and basic characterization (2012). Protein Expression and Purification 82:302-307.
    • You C, Zhang X-Z, Zhang Y-HP. (2012). Simple Cloning via direct transformation of PCR product (DNA multimer) to Escherichia coli and Bacillus subtilis. Applied and Environmental Microbiology 78(5):1593-1595.
    • Huang SY, Zhang Y-HP, Zhong JJ. (2012). A thermostable recombinant transaldolase with high activity over a broad pH range. Applied Microbiology and Biotechnology 93(6):2403-2410.
    • Liao HH, Myung S, Zhang Y-HP. (2012). One-step purification and immobilization of thermophilic polyphosphate glucokinase from Thermobifida fusca YX: glucose-6-phosphate generation without ATP. Applied Microbiology and Biotechnology 93:1109-1117.
    • You C, Zhang Y-HP. (2012) Cell-free biosystems for biomanufacturing. Advances in Biochemical Engineering_Biotechnology Epub, DOI: 10.1007/10_2012_159 (Invited).
    • You C, Myung S, Zhang Y-HP. (2012). Self-assembly of synthetic metabolons through synthetic scaffoldins: single-step purification, co-immobilization, and substrate channeling. ACS Synthetic Biology Epub, DOI: 10.1021/sb300068g.
    • You C, Myung S, Zhang Y-HP. (2012). Facilitated substrate channeling in a self-assembled trifunctional enzyme complex. Angewandte Chemie International Edition. 51: 8787-8790.
    • You C, Zhang Y-HP. (2012). Easy preparation of a large-size random gene mutagenesis library in Escherichia coli. Analytical Biochemistry 428:7-12.
    • Zhu ZG, Sun FF, Zhang XZ, Zhang Y-HP. 2012. Deep oxidation of glucose in enzymatic fuel cells through a non-natural synthetic enzymatic pathway containing a cascade of two thermostable dehydrogenases. Biosensors & Bioelectronics 36: 110-115.
    • Zhang Y-HP, Chun Y, Chen HG, Feng RL. (2012). Surpassing photosynthesis: high-efficiency and scalable CO2 utilization through artificial photosynthesis. ACS Symposium Series 1097: 275-296.
    • Ye X, Zhang CM, Zhang Y-HP. (2012). Engineering a large protein by combined rational and random approaches: Stabilizing the Clostridium thermocellum cellobiose phosphorylase. Molecular BioSystems 8: 1815-1823.
    • Zhang Y-HP, Xu J-H, Zhong J-J. (2012). A new high-energy density hydrogen carrier - carbohydrate - might be better than methanol. International Journal of Energy Research. Epub, DOI:10.1002/er.2897 (invited).
    • Zhang Y-HP, Huang WD. (2012). Constructing the electricity-carbohydrate-hydrogen cycle for sustainability revolution. Trends in Biotechnology 30: 301-306 (Opinion).