Coenzyme nicotinamide adenine dinucleotide (NAD, including the oxidized form-- NAD+ and reduced form--NADH) and the phosphorylated form--nicotinamide adenine dinucleotide phosphate (NADP, including NADP+ and NADPH) are two of the most important biological electron carriers. Most NAD(P) dependent redox enzymes show a preference of either NADP or NAD as an electron acceptor or donor depending on their unique metabolic roles. In biocatalysis, the low enzymatic activities with unnatural coenzymes have made it difficult to replace costly NADP with economically advantageous NAD or other biomimetic coenzyme for catalysis. This is a significant challenge that must be addressed should in vitro biocatalysis be a viable option for the practical production of low-value biocommodities (i.e., biohydrogen). There is a significant need to first address the coenzyme selectivity of the NADP-dependent dehydrogenases and evolve mutated enzymes that accept biomimetic coenzymes. This is a major focus of this dissertation. Establishment of efficient screening methods to identify beneficial mutants from an enzymatic library is the most challenging task of coenzyme engineering of dehydrogenases. To fine tune the coenzyme preference of dehydrogenases to allow economical hydrogen production, we developed a double-layer Petri-dish based screening method to identify positive mutant of the Moorella thermoacetica 6PGDH (Moth6PGDH) with a more than 4,278-fold reversal of coenzyme selectivity from NADP+ to NAD+. This method was also used to screen the thermostable mutant of a highly active glucose 6-phosphate dehydrogenase from the mesophilic host Zymomonas mobilis. The resulting best mutant Mut 4-1 showed a more than 124-fold improvement of half-life times at 60oC without compromising the specific activity. The screening method was further upgraded for the coenzyme engineering of Thermotaga maritima 6PGDH (Tm6PGDH) on the biomimetic coenzyme NMN+. Through six-rounds of directed evolution and screening, the best mutant showed a more than 50-fold improvement in catalytic efficiency on NMN+ and a more than 6-fold increased hydrogen productivity rate from 6-phosphogluconate and NMN+ compared to those of wild-type enzyme. Together, these results demonstrated the effectiveness of screening methods developed in this research for coenzyme engineering of NAD(P) dependent dehydrogenase and efficient use of the less costly coenzyme in ivSB based hydrogen production. / Ph. D. / NADP and NAD are two of the most important electron carriers in cellular metabolism, and they play distinctive roles in anabolism and catabolism, respectively. Most NAD(P)-dependent dehydrogenases exhibit a strong preference for either NADP or NAD. This coenzyme preference, however, make it nearly impossible to replace the costly NADP with less costly NAD or biomimetic coenzymes in the biocatalysis application. How to engineer dehydrogenases through directed evolution and effective screening method to accept NAD or biomimetic coenzymes, is critical and the focus of this dissertation. The use of in vitro synthetic biosystem (ivSB) to produce hydrogen form starch, is one of the most important in vitro synthetic biology projects, and it depends on NADP coenzyme. With other issues in this system solved, the efficient use of dehydrogenases along with low cost and stable coenzyme is the last obstacle to hydrogen production through industrial biomanufacturing. However, the 6-phosphogluconate dehydrogenase (6PGDH), one of the rate-limiting enzymes in this biosystem, exhibits a strong coenzyme preference for NADP⁺ . For producing low-cost hydrogen, the coenzyme engineering of this dehydrogenase is urgently required. Its activity with less costly NAD or biomimetic coenzymes must be improved. The establishment of an effective screening method is the most challenging task for coenzyme engineering of dehydrogenases. In this research, we developed a Petri-dish double-layer based screening method for coenzyme engineering of thermophilic 6PGDH for activity for NAD⁺ . This screening method was also used to improve the thermostability of a highly active glucose 6-phosphate dehydrogenase from a mesophilic host, where the evolved mutant had a greatly improved thermostability without losing activity. The screening method was further upgraded to develop for coenzyme engineering on biomimetic coenzyme NMN⁺ . The engineered mutant showing a more than 50-fold increase in catalytic efficiency on NMN⁺ was used to develop the first biomimetic coenzyme dependent electron transfer chain for hydrogen production. This screening method is suitable to change the coenzyme selectivity of series of NAD(P)-dependent redox enzymes and show great potential in improving other properties, such as thermostability, substrate scope and optimal pH, of different dehydrogenases. With this method developed, we can efficiently use the low cost stable coenzyme in the biocatalysis, and break the last obstacle to industrial biomanufacturing of hydrogen production.

In order to decrease our dependence on non-renewable petrochemical resources, it is urgently required to establish sustainable biomass-based biorefineries. Replacing fossil fuels with renewable biomass as a raw feedstock for the production of chemicals and biofuels is a main driving force of biorefinering. Almost all kinds of biomass can be converted to biochemicals, biomaterials and biofuels via continuing advances on conversion technologies. In vitro synthetic biology is an emergent biomanufacturing platform that circumvents cellular constraints so that it can implement some biotransformations better than whole-cell fermentation, which spends a fraction of energy and carbon sources for cellular duplication and side-product formation. In this work, the in vitro synthetic (enzymatic) biosystem is used to produce a future carbon-neutral transportation fuel, hydrogen, and two high-value chemicals, a sugar phosphate and a highly marketable sweetener, representing a new portfolio for new biorefineries. Hydrogen gas is a promising future energy carrier as a transportation fuel, offering a high energy conversion efficiency via fuel cells, nearly zero pollutants produced to end users, and high mass-specific and volumetric energy densities compared to rechargeable batteries. Distributed production of cost-competitive green hydrogen from renewable biomass will be vital to the hydrogen economy. Substrate costs contribute to a major portion of the production cost for low-value bulk biocommodities, such as hydrogen. The reconstitution of 17 thermophilic enzymes enabled to construct an artificial enzymatic pathway converting all glucose units of starch, regardless of the branched and linear contents, to hydrogen gas at a theoretic yield (i.e., 12 H2 per glucose), three times of the theoretical yield from dark microbial fermentation. Using a biomimetic electron transport chain, a maximum volumetric productivity was increased by more than 200-fold to 90.2 mmol of H2/L/h at a high starch concentration from the original study in 2007. In order to promote economics of biorefineries, the production of a sugar phosphate and a fourth-generation sweetener is under development. D-xylulose 5-phosphate (Xu5P), which cannot be prepared efficiently by regular fermentation due to the negatively charged and hydrophilic phosphate groups, was synthesized from D-xylose and polyphosphate via a minimized two-enzyme system using a promiscuous activity of xylulose kinase. Under the optimized condition, 32 mM Xu5P was produced from 50 mM xylose and polyphosphate, achieving a 64% conversion yield, after 36 h at 45 °C. L-arabinose, a FDA-approved zero-calorie sweetener, was produced from D-xylose via a novel enzymatic pathway consisting of xylose isomerase, L-arabinose isomerase and xylulose 4-epimerase (Xu4E). Promiscuous activity of Xu4E, a monosaccharide C4-epimerase, was discovered for the first time. Directed evolution of Xu4E enabled to increase the catalytic function of C4-epimerization on D-xylulose as a substrate by more than 29-fold from the wild-type enzyme. Together, these results demonstrate that the in vitro synthetic biosystem as a feasible biomanufacturing platform has great engineering, and can be used to convert renewable biomass resources to a spectrum of marketable products and renewable energy. As future efforts are addressed to overcome remaining challenges, for example, decreasing enzyme production costs, prolonging enzyme lifetime, engineering biomimetic coenzymes to replace natural coenzymes, and so on. This in vitro synthetic biology platform would become a cornerstone technology for biorefinery industries and advanced biomanufacturing (Biomanufacturing 4.0). / Ph. D. / The carbon cycle is the circulation and transformation of carbon back and forth between living things and the environment. With the fixed amount of carbon dioxide in the atmosphere, the carbon cycle has been in the balance of exchanges between living things and the environment. As we evolve with increasing demand on crude oil, however, significant amounts of carbon are being released into the atmosphere much faster than they would have been released naturally. This rapid release is the primary cause of currently observed global warming. In order to decrease our dependence on petrochemical products, the biorefinery was introduced as the sustainable processing of biomass into a spectrum of alternatives to products from petrochemical refineries. Almost all kinds of biomass can be converted to biochemicals, biomaterials and biofuels via continuing advances on conversion technologies. In vitro synthetic biology is an emergent biomanufacturing platform that circumvents whole cell’s constraints, so that it can implement some biotransformations better than whole-cell fermentation spending a significant fraction of energy and carbon sources for cellular duplication and side-product formation. In this work, the in vitro synthetic (enzymatic) biosystem is used to produce a future carbon-neutral transportation fuel, hydrogen gas, and two high-value chemicals, a sugar phosphate and a highly marketable sweetener, representing a new portfolio for new biorefineries. Hydrogen gas is a promising energy carrier as a transportation fuel, offering a high energy conversion efficiency via fuel cells, nearly zero pollutants produced to end users, and high mass-specific and volumetric energy densities compared to rechargeable batteries. Distributed production of cost-competitive green hydrogen will be vital to the hydrogen economy. We demonstrated an in vitro 17-thermophilic enzyme pathway that can convert all glucose units of starch to hydrogen a theoretic yield, which is three times of the theoretical yield from dark microbial fermentation. D-xylulose 5-phosphate (Xu5P), which cannot be prepared efficiently by regular fermentation due to the negatively charged and hydrophilic phosphate groups, was synthesized from D-xylose and polyphosphate via a minimized two-enzyme system using a promiscuous activity of xylulose kinase. This minimal in vitro enzymatic pathway was optimized for improved conversion yield and productivity. L-arabinose, a FDA-approved zero-calorie sweetener, was also produced from D-xylose via a novel enzymatic pathway consisting of xylose isomerase, L-arabinose isomerase and hypothetical enzyme xylulose 4-epimerase (Xu4E), a monosaccharide 4-epimerase that can convert D-xylulose to L-ribulose. Xu4E activities due to substrate promiscuity of some natural 4-epimerases were discovered for the first time. Three rounds of directed evolution have been conducted to increase the catalytic function of carbon 4-epimerization on D-xylulose. As the result, the catalytic activity of Xu4E was improved by more than 29-fold from the wild-type enzyme.