In vitro synthetic enzymatic biosystem mixed purified/partial purified cascade enzymes and cofactors together to convert cheap substrates to value-added products in one-pot. Compared to cell-based production, in vitro biosystems exhibited several advantages such as high product yield, titer and productivity, highly engineering flexibility, and high tolerance towards toxic environments. Through the methods including pathway construction, gene mining, thermodynamic analysis and condition optimization, we developed many in vitro metabolic engineering biosystems for the production of inositol, laminaribiose, and for ATP regeneration in stoichiometric manners from cheap substrates like starch or cellulose, and the inositol production from starch has been applied in a 20 t reactor for industrial purpose. In conclusion, in vitro biosystems have emerged as an alternative approach for biomanufacturing, paralleling with traditional cell-based production..
1. Journal papers
1) Bai, X., Meng, D., Wei, X., Zhou, X., Lu, F., and YOU C*. (2019) Facile synthesis of (？)-vibo-quercitol from maltodextrin via an in vitro synthetic enzymatic biosystem, Biotechnol Bioeng 10.1002/bit.27096
2) Zhang, S., Shi, J., Sun, Y., Wu, Y., Zhang, Y., Cai, Z., Chen, Y., You, C., Han, P., and Jiang, Z. (2019) Artificial Thylakoid for the Coordinated Photoenzymatic Reduction of Carbon Dioxide, ACS Catalysis 9, 3913-3925.
3) Wang, W., Meng, D., Li, Q., Li, Z., and YOU C*. (2019) Characterization of a hyperthermophilic phosphatase from Archaeoglobus fulgidus and its application in in vitro synthetic enzymatic biosystem, Bioresour. Bioprocess. 6. 10.1186/s40643-019-0257-5
4) Sun, S., Wei, X., and YOU C*. (2019) The construction of an in vitro synthetic enzymatic biosystem that facilitates laminaribiose biosynthesis from maltodextrin and glucose, Biotechnol. J. 14, 1800493.
5) Song, Y., Liu, M., Xie, L., YOU C*., Sun, J. *, and Zhang, Y.-H. P. J. * (2019) A Recombinant 12-His Tagged Pyrococcus furiosus Soluble [NiFe]-Hydrogenase I Overexpressed in Thermococcus kodakarensis KOD1 Facilitates Hydrogen-Powered in vitro NADH Regeneration, Biotechnol. J.14, 1800301.
6) Meng, D., Liang, A., Wei, X., and YOU C*. (2019) Enzymatic characterization of a thermostable phosphatase from Thermomicrobium roseum and its application for biosynthesis of fructose from maltodextrin, Appl Microbiol Biot. 10.1007/s00253-019-09917-6
1) Meng, D., Wei, X., Zhang, Y.-H. P. J., Zhu, Z., YOU C*, and Ma, Y. (2018) Stoichiometric conversion of cellulosic biomass by in vitro synthetic enzymatic biosystems for biomanufacturing, ACS Catalysis 8, 9550-9559.
2) Wei,XL., Xie, L., Zhang, Y.-H. P. J., and YOU C* (2018) Stoichiometric Regeneration of ATP by A NAD(P)/CoA-free and Phosphate-balanced in Vitro Synthetic Enzymatic Biosystem, ChemcatChem, DOI.10.1002/cctc.201801562
3) Xie, L., Wei, X., Zhou, X., Meng, D., Zhou, R., Zhang, Y.-H. P. J., Xu, S*., and YOU C*. (2018) Conversion of d-glucose to l-lactate via pyruvate by an optimized cell-free enzymatic biosystem containing minimized reactions, Synthetic and Systems Biotechnology. 3, 204-210.
4) Shi, T., Han, P., YOU C*, and Zhang, Y.-H. P. J.* (2018) An in vitro synthetic biology platform for emerging industrial biomanufacturing: Bottom-up pathway design, Synthetic and Systems Biotechnology. 3, 186-195.
1) YOU C*, Huang R, Zhu ZG, Zhang Y-HP*. 2017 Protein engineering on nicotinamide-based coenzymes with its applications to synthetic biology. Synthetic and Systems Biotechnology 2017;2: 208-218.
2) YOU C, Shi T, Li YJ, Han PP, Zhou XG, Zhang Y-HP*. 2017. An in vitro synthetic biology platform for the industrial biomanufacturing of myo-inositol from starch. Biotechnology and Bioengineering 114: 1855-1864.
3) YOU C*, Zhang Y-HP. 2017. Biomanufacturing by in vitro biosystems containing cascade enzymes. Process Biochemistry. 52: 106-114.
4) 18) Zhong C, YOU C, Wei P, Zhang Y-HP*. 2017. Thermal cycling cascade biocatalysis of myo-inositol synthesis from sucrose. ACS Catalysis. 7: 5992–5999.
1) Kim JE, Huang R, Chen H, YOU C, Zhang Y-HP. 2016. Facile construction of random gene mutagenesis library for directed evolution without the use of restriction enzyme in Escherichia coli. Biotechnol J. 11:1142-1150.
2) Zhou W, YOU C, Ma H, Ma Y, Zhang YH. 2016. One-Pot Biosynthesis of High-Concentration alpha-Glucose 1-Phosphate from Starch by Sequential Addition of Three Hyperthermophilic Enzymes. J Agric Food Chem. 64:1777-1783.
1) Rollin JA, Martin del Campo J, Myung S, Sun F, YOU C, Bakovic A, Castro R, Chandrayan SK, Wu CH, Adams MWW, Senger RS, Zhang Y-HP, 2015. High-yield hydrogen production from biomass by in vitro metabolic engineering: Mixed sugars coutilization and kinetic modeling. Proceedings of the National Academy of Sciences. 112 (16), 4964-4969.
1) 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(6): 380-386
2) 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,
3) Myung S, Rollin J, YOU C, Sun F, Chandrayan S, Adams MWW, Zhang Y-HP. 2014. In vitro metabolic engineering of hydrogen production at theoretical yield from sucrose. Metabolic Engineering 24(0):70-77.
4) Peng Q, YOU C, Zhang Y-HP*, 2014, One-Pot Enzymatic Conversion of Sucrose to Synthetic Amylose by using Enzyme Cascades. ACS Catalysis. 4:1311-1317.
5) Gao SH, YOU C, Renneckar S, Bao J, Zhang Y-HP*. 2014. New insights into enzymatic hydrolysis of heterogeneous cellulose by using CBM3-containing GFP and CBM17-containing CFP. Biotechnology for Biofuels. 7(1):24
1) 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)
2) 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).
3) Martin del Campo JS, Rollin JR, Myung S, YOU C, Chandrayan S, Adams MWW, Zhang Y-HP*. 2013. Dihydrogen production from xylose and water mediated by synthetic cascade enzymes. Angewandte Chemie International Edition 52:4587-4590,
4) 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.
5) Martin del Campo JS, YOU C, Kim J-E, 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.
1) 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
2) YOU C, Zhang X-Z , Sathitsuksanoh N , Lynd LR, Zhang Y-HP*. 2012. Enhanced microbial cellulose utilization of recalcitrant cellulose by an ex vivo cellulosome-microbe complex. Applied and Environmental Microbiology 78(5):1437-1444
3) 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
4) YOU C, Zhang Y-HP*. 2012. Easy preparation of a large-size random gene mutagenesis library in Escherichia coli. Analytical Biochemistry 428:7-12
5) YOU C, Zhang X-Z, Zhang Y-HP*. 2012. Mini-scaffoldin enhanced mini-cellulosome hydrolysis performance on low-accessibility cellulose (Avicel) more than on high-accessibility amorphous cellulose. Biochemical Engineering Journal 63:57-65
6) Xue HP, Zhou JG, YOU C, Huang Q, Lu H *. 2012. Amino acid substitutions in the N-terminus, cord and α-helix domains improved the thermostability of a family 11 xylanase XynR8. Journal of Industrial Microbiology and Biotechnology 39:1279-1288
7) Zhou JG, Bao L, Chang L, Liu Z, YOU C, Lu H *. 2012. Beta-xylosidase activity of a GH3 glucosidase/xylosidase from yak rumen metagenome promotes the enzymatic degradation of hemicellulosic xylans. Letters in Applied Microbiology 54:79-87
1) Zhang Y-HP*, Myung S, YOU C, Zhu ZG, Rollin J. 2011. Toward low-cost biomanufacturing through cell-free synthetic biology: bottom-up design. Journal of Materials Chemistry 21: 18877-18886
1) YOU C, Huang Q, Xue HP, Xu Y, Lu H *. 2010. Potential hydrophobic interaction between two cysteines in interior hydrophobic region improves thermostability of a family 11 xylanase from Neocallimastix patriciarum. Biotechnology and Bioengineering 105(5):861-70
2. Book Chapter
1) YOU C*, Zhang Y-HP*. Ex Vivo Enzymatic Conversion of Non-food Cellulose Biomass to Starch. In: P. C. K. Lau editor. Quality Living Through Chemurgy and Green Chemistry. Berlin, Heidelberg: Springer Berlin Heidelberg; 2016. p. 129-142.
2) YOU C, Zhang Y-HP*. 2013. Cell-free biosystems for biomanufacturing. Advances in Biochemical Engineering/Biotechnology 131:89-119.
3) YOU C, Zhang Y-HP*. 2014. Simple cloning and DNA assembly in E. coli by prolonged overlap extension PCR. Methods of Molecular Biology 1116。
4) Zhang Y-HP*, YOU C, Chen HG, Feng RL. 2012. Surpassing photosynthesis: high-efficiency and scalable CO2 utilization through artificial photosynthesis. ACS Symposium Series 1097: 275-292 (Recent Advances in Post-Combustion CO2 Capture Chemistry), Oxford University Press, UK
5) 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 137:41-65.
6) Zhong C, You C, Wei P, Zhang Y-HP*. 2017. Simple cloning by prolonged overlap extension PCR with application to the preparation of large-size random gene mutagenesis library in Escherichia coli. Methods in Molecular Biology.1472: 49-61.
1) Lu H, YOU C, Huang Q, High temperature and strong alkali resistant xylanase improved gene, genetic engineering bacterial strain thereof and preparation method thereof. Application number: 200810200060.2.
2) Zhang Y-HP, YOU C, A method to produce inositol. Application number:201510184621.4.
3) Zhang Y-HP, YOU C, Zhong C，A method to produce inositol with sucrose as substrate. Application number: 201611035068.9.
4) YOU C, Wei XL. A chassis system and application for ATP regeneration. Application number: 201711047916.2.
5) YOU C, Xie LP. A catalysis reaction system and the preparation method of L-lactic acid. Application number: 201711050815.0.
6) YOU C, Sun SS. A method to produce laminaribiose. Application number: 201711014505.3.
7) YOU C, Meng DD. An enzymic method to produce glucosamine. Application number: 201810772487.3.
8) YOU C, Sun SS. A method to produce laminaribiose with starch as substrate. Application number: 201810897084.1.
9) YOU C, Li Y, Liu S. A method to produce scyllitol. Application number: 201810973696.4.
10) YOU C, Meng DD. A method to produce inositol by the complete phosphorylation of cellulose. Application number: 201810972707.7.
11) YOU C, Li Y, Liu S. A method to produce glucaric acid. Application number: 201811160130.6.
12) YOU C, Li YJ. An enzymic method of C1 compound immobilization by catalyzing xylose . Application number: 201910823381.6.
The general program of the State Natural Science Fund projects;
Sub-project of the grant of the Key Programs from the Chinese Academy.
Second class award, Outstanding Achievement Award of scientific research for universities and colleges, awarded by Ministry of Education of the People's Republic of China, 2012;
Second class award, Shanghai Science and Technology Award for the year 2013.