In order to meet the energy needs of a growing world in a sustainable manner, new high efficiency, carbon-neutral fuels and chemical feedstocks are required. An emerging approach that shows promise for high efficiency production of renewable fuels and chemicals is the use of purified enzymes combined in one pot to catalyze complex conversions: synthetic pathway biotransformations (SyPaB). An exemplary technology in this burgeoning field is the production of hydrogen from biomass sugars. Lignocellulosic biomass, which includes agricultural residues, energy crops, and industrial waste streams, is an ideal substrate for SyPaB conversion, as it is abundant and cheap, second only to untaxed coal on a $/energy content basis. But the structure of biomass is highly recalcitrant, making high-yield biological conversion difficult to achieve. In order to increase susceptibility to enzymatic digestion, thermochemical pretreatments are applied, with the goals of removing of lignin, the simplest example being soaking in aqueous ammonia (SAA); hemicellulose removal, most often using dilute acid (DA); and increasing cellulose accessibility by cellulose solvent-based pretreatments, such as cellulose solvent- and organic solvent-based lignocellulose fractionation (COSLIF). In a comparison of the lignin removal (SAA) and accessibility increase (COSLIF) approaches, we found that certain levels of lignin removal (~15%) were important, but further lignin removal was less effective at achieving digestibility gains than increasing cellulose accessibility. Pretreated biomass forms an excellent substrate for SyPaB hydrogen generation, due to low cost and high sugar content. Following experiments demonstrating the high yield conversion of sucrose to hydrogen (97%) and SyPaB generation of hydrogen at a rate commensurate with the best biological rates achieved, 157 mmol/L/h. SyPaB was combined with enzymatic hydrolysis to enable the direct conversion of cellulosic biomass, including untreated, DA, and COSLIF corn stover. In addition, an updated kinetic model of the system was used to rationally increase the maximum hydrogen production rate by 70% while minimizing total enzyme loading and without increasing substrate concentration. Together, these results demonstrate the high level of engineering control in cell-free systems, which can enable conversion of a variety of substrates to hydrogen at the highest possible yield and rates as high as any biohydrogen production method. / Ph. D.

The production of hydrogen from low-cost abundant renewable biomass would be vital to sustainable development. Cell-free (in vitro) biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it is essential to produce (purified) enzymes at low costs and stabilize them for long periods to decrease biocatalyst costs. Thermophilic recombinant enzymes as building blocks were discovered and developed: fructose 1,6-bisphosphatase (FBP) from Thermotoga maritime, phosphoglucose isomerase (PGI) from Clostridium thermocellum, triose phosphate isomerase (TIM) from Thermus thermophiles and fructose bisphosphate aldolase (ALD) from T. maritima and T. thermophilus. The recombinant proteins were over-expressed in E. coli, purified and characterized. For purification and stabilization of enzymes, one-step, simple, low-cost purification and immobilization methods were developed based on simple adsorption of cellulose-binding module (CBM)-tagged protein on the external surface of high-capacity regenerated amorphous cellulose. Also, a simple, low-cost purification method of thermophilic enzymes was developed utilizing a combination of heat and ammonium sulfate precipitation. For development of cascade enzymes as building modules (biocatalyst modules), it was discovered that the presence of other enzymes/proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme (e.g., PGI) due to the in vitro macromolecular crowding effect. And substrate channeling among CBM-tagged self-assembled three-enzyme complex (synthetic matabolon) immobilized on the easily-recycled cellulose-containing magnetic nanoparticles can not only increase cascade reaction rates greatly, but also decrease enzyme cost in cell-free biosystems. The high product yield and fast reaction rate of dihydrogen from sucrose was validated in a batch reaction containing fifteen enzymes comprising a non-natural synthetic pathway. The yield of dihydrogen production from 2 mM of sucrose was 96.7 % compared to theoretical yield at 37 °C. The maximum rate was increased 3.1 fold when the substrate concentration was increased from 2 to 50 mM in a fed-batch reaction. The research and development of cell-free biosystems for biomanufacturing require more efforts, especially in low-cost recombinant thermostable enzymes as building blocks, efficient cofactor recycling, enzyme and cofactor stabilization, and fast reaction rates. / Ph. D.

Enzyme-catalyzed biofuel cells would be a great alternative to current battery technology, as they are clean, safe, and capable of using diverse and abundant renewable biomass with high energy densities, at mild reaction conditions. However, currently, three largest technical challenges for emerging enzymatic fuel cell technologies are incomplete oxidation of most fuels, limited power output, and short lifetime of the cell. Synthetic pathway biotransformation is a technology of assembling a number of enzymes coenzymes for producing low-value biocommodities. In this work, it was applied to generate bioelectricity for the first time. Non-natural enzymatic pathways were developed to utilize maltodextrin and glucose in enzymatic fuel cells. Three immobilization approaches were compared for preparing enzyme electrodes. Thermostable enzymes from thermophiles were cloned and expressed for improving the lifetime and stability of the cell. To further increase the power output, non-immobilized enzyme system was demonstrated to have higher power densities compared to those using immobilized enzyme system, due to better mass transfer and retained native enzyme activities. With the progress on pathway development and power density/stability improvement in enzymatic fuel cells, a high energy density sugar-powered enzymatic fuel cell was demonstrated. The enzymatic pathway consisting of 13 thermostable enzymes enabled the complete oxidation of glucose units in maltodextrin to generate 24 electrons, suggesting a high energy density of such enzymatic fuel cell (300 Wh/kg), which was several folds higher than that of a lithium-ion battery. Maximum power density was 0.74 mW/cm2 at 50 deg C and 20 mM fuel concentration, which was sufficient to power a digital clock or a LED light. These results suggest that enzymatic fuel cells via synthetic pathway biotransformation could achieve high energy density, high power density and increased lifetime. Future efforts should be focused on further increasing power density and enzyme stability in order to make enzymatic fuel cells commercially applicable. / Ph. D.