Scholarly Works, Institute for Critical Technology and Applied Science (ICTAS)
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Browsing Scholarly Works, Institute for Critical Technology and Applied Science (ICTAS) by Department "Biological Systems Engineering"
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- Cell-free protein synthesis energized by slowly-metabolized maltodextrinWang, Yiran; Zhang, Y. H. Percival (2009-06-28)Background Cell-free protein synthesis (CFPS) is a rapid and high throughput technology for obtaining proteins from their genes. The primary energy source ATP is regenerated from the secondary energy source through substrate phosphorylation in CFPS. Results Distinct from common secondary energy sources (e.g., phosphoenolpyruvate - PEP, glucose-6-phosphate), maltodextrin was used for energizing CFPS through substrate phosphorylation and the glycolytic pathway because (i) maltodextrin can be slowly catabolized by maltodextrin phosphorylase for continuous ATP regeneration, (ii) maltodextrin phosphorylation can recycle one phosphate per reaction for glucose-1-phosphate generation, and (iii) the maltodextrin chain-shortening reaction can produce one ATP per glucose equivalent more than glucose can. Three model proteins, esterase 2 from Alicyclobacillus acidocaldarius, green fluorescent protein, and xylose reductase from Neurospora crassa were synthesized for demonstration. Conclusion Slowly-metabolized maltodextrin as a low-cost secondary energy compound for CFPS produced higher levels of proteins than PEP, glucose, and glucose-6-phospahte. The enhancement of protein synthesis was largely attributed to better-controlled phosphate levels (recycling of inorganic phosphate) and a more homeostatic reaction environment.
- A high-energy-density sugar biobattery based on a synthetic enzymatic pathwayZhu, Zhiguang; Tam, Tsz Kin; Sun, Fangfang; You, Chun; Zhang, Y. H. Percival (Springer Nature, 2014-01)High-energy-density, green, safe batteries are highly desirable for meeting the rapidly growing needs of portable electronics. The incomplete oxidation of sugars mediated by one or a few enzymes in enzymatic fuel cells suffers from low energy densities and slow reaction rates. Here we show that nearly 24 electrons per glucose unit of maltodextrin can be produced through a synthetic catabolic pathway that comprises 13 enzymes in an air-breathing enzymatic fuel cell. This enzymatic fuel cell is based on non-immobilized enzymes that exhibit a maximum power output of 0.8 mW cm(-2) and a maximum current density of 6 mA cm(-2), which are far higher than the values for systems based on immobilized enzymes. Enzymatic fuel cells containing a 15% (wt/v) maltodextrin solution have an energy-storage density of 596 Ah kg(-1), which is one order of magnitude higher than that of lithium-ion batteries. Sugar-powered biobatteries could serve as next-generation green power sources, particularly for portable electronics.
- Overcoming Biomass Recalcitrance by Combining Genetically Modified Switchgrass and Cellulose Solvent-Based Lignocellulose PretreatmentSathitsuksanoh, Noppadon; Xu, Bin; Zhao, Bingyu Y.; Zhang, Y. H. Percival (2013-09-27)Decreasing lignin content of plant biomass by genetic engineering is believed to mitigate biomass recalcitrance and improve saccharification efficiency of plant biomass. In this study, we compared two different pretreatment methods (i.e., dilute acid and cellulose solvent) on transgenic plant biomass samples having different lignin contents and investigated biomass saccharification efficiency. Without pretreatment, no correlation was observed between lignin contents of plant biomass and saccharification efficiency. After dilute acid pretreatment, a strong negative correlation between lignin content of plant samples and overall glucose release was observed, wherein the highest overall enzymatic glucan digestibility was 70% for the low-lignin sample. After cellulose solvent- and organic solvent-based lignocellulose fractionation pretreatment, there was no strong correlation between lignin contents and high saccharification efficiencies obtained (i.e., 80-90%). These results suggest that the importance of decreasing lignin content in plant biomass to saccharification was largely dependent on pretreatment choice and conditions.
- Overexpression and simple purification of the Thermotoga maritima 6-phosphogluconate dehydrogenase in Escherichia coli and its application for NADPH regenerationWang, Yiran; Zhang, Y. H. Percival (2009-06-04)Background Thermostable enzymes from thermophilic microorganisms are playing more and more important roles in molecular biology R&D and industrial applications. However, over-production of recombinant soluble proteins from thermophilic microorganisms in mesophilic hosts (e.g. E. coli) remains challenging sometimes. Results An open reading frame TM0438 from a hyperthermophilic bacterium Thermotoga maritima putatively encoding 6-phosphogluconate dehydrogenase (6PGDH) was cloned and expressed in E. coli. The purified protein was confirmed to have 6PGDH activity with a molecular mass of 53 kDa. The kcat of this enzyme was 325 s-1 and the Km values for 6-phosphogluconate, NADP+, and NAD+ were 11, 10 and 380 μM, respectively, at 80°C. This enzyme had half-life times of 48 and 140 h at 90 and 80°C, respectively. Through numerous approaches including expression vectors, hosts, cultivation conditions, inducers, and codon-optimization of the 6pgdh gene, the soluble 6PGDH expression levels were enhanced to ~250 mg per liter of culture by more than 500-fold. The recombinant 6PGDH accounted for >30% of total E. coli cellular proteins when lactose was used as a low-cost inducer. In addition, this enzyme coupled with glucose-6-phosphate dehydrogenase for the first time was demonstrated to generate two moles of NADPH per mole of glucose-6-phosphate. Conclusion We have achieved a more than 500-fold improvement in the expression of soluble T. maritima 6PGDH in E. coli, characterized its basic biochemical properties, and demonstrated its applicability for NADPH regeneration by a new enzyme cocktail. The methodology for over-expression and simple purification of this thermostable protein would be useful for the production of other thermostable proteins in E. coli.
- Renewable Hydrogen Carrier — Carbohydrate: Constructing the Carbon-Neutral Carbohydrate EconomyZhang, Y. H. Percival; Mielenz, Jonathan R. (MDPI, 2011-01-31)The hydrogen economy presents an appealing energy future but its implementation must solve numerous problems ranging from low-cost sustainable production, high-density storage, costly infrastructure, to eliminating safety concern. The use of renewable carbohydrate as a high-density hydrogen carrier and energy source for hydrogen production is possible due to emerging cell-free synthetic biology technology—cell-free synthetic pathway biotransformation (SyPaB). Assembly of numerous enzymes and co-enzymes in vitro can create complicated set of biological reactions or pathways that microorganisms or catalysts cannot complete, for example, C6H10O5 (aq) + 7 H2O (l) à 12 H2 (g) + 6 CO2 (g) (PLoS One 2007, 2:e456). Thanks to 100% selectivity of enzymes, modest reaction conditions, and high-purity of generated hydrogen, carbohydrate is a promising hydrogen carrier for end users. Gravimetric density of carbohydrate is 14.8 H2 mass% if water can be recycled from proton exchange membrane fuel cells or 8.33% H2 mass% without water recycling. Renewable carbohydrate can be isolated from plant biomass or would be produced from a combination of solar electricity/hydrogen and carbon dioxide fixation mediated by high-efficiency artificial photosynthesis mediated by SyPaB. The construction of this carbon-neutral carbohydrate economy would address numerous sustainability challenges, such as electricity and hydrogen storage, CO2 fixation and long-term storage, water conservation, transportation fuel production, plus feed and food production.