Engineering Cytochrome P450 Enzymes for Improved Activity towards Biomimetic 1,4‐NADH Cofactors

Biotech & Bioengineering

2010

165

137

Cell-free synthetic (enzymatic) pathway biotransformation (SyPaB) is the assembly of a number of purified enzymes (usually more than 10) and coenzymes for the production of desired products through complicated biochemical reaction networks that a single enzyme cannot do. Cell-free SyPaB, as compared to microbial fermentation, has several distinctive advantages, such as high product yield, great engineering flexibility, high product titer, and fast reaction rate. Biocommodities (e.g., ethanol, hydrogen, and butanol) are low-value products where costs of feedstock carbohydrates often account for $30-70% of the prices of the products. Therefore, yield of biocommodities is the most important cost factor, and the lowest yields of profitable biofuels are estimated to be ca. 70% of the theoretical yields of sugar-to-biofuels based on sugar prices of ca. US$ 0.18 per kg. The opinion that SyPaB is too costly for producing low-value biocommodities are mainly attributed to the lack of stable standardized building blocks (e.g., enzymes or their complexes), costly labile coenzymes, and replenishment of enzymes and coenzymes. In this perspective, I propose design principles for SyPaB, present several SyPaB examples for generating hydrogen, alcohols, and electricity, and analyze the advantages and limitations of SyPaB. The economical analyses clearly suggest that developments in stable enzymes or their complexes as standardized parts, efficient coenzyme recycling, and use of low-cost and more stable biomimetic coenzyme analogs, would result in much lower production costs than do microbial fermentations because the stabilized enzymes have more than 3 orders of magnitude higher weight-based total turn-over numbers than microbial biocatalysts, although extra costs for enzyme purification and stabilization are spent.

Section: Cost Analysis and Perspectivesmentioning

confidence: 99%

Section: Cost Analysis and Perspectivesmentioning

confidence: 99%

Production of biocommodities and bioelectricity by cell‐free synthetic enzymatic pathway biotransformations: Challenges and opportunities

Biotech & Bioengineering

2010

165

137

“…But this research area is in its infancy [89,90] because there has not been a sufficiently large market/product opportunity to support further R&D before the invention of SyPaB. Several redox enzymes have been engineered for better performance on biomimetic coenzymes [91][92][93]. For similar reasons, there is a lack of thermostable carbohydrate-metabolized enzymes for the SyPaB projects before the invention of SyPaB.…”

Section: Sypab Challenges and Opportunitiesmentioning

confidence: 99%

“…Clearly, it is highly operative to obtain numerous high-TTN non-membrane enzymes suitable for the SyPaB projects. With developments in (i) engineered oxidoreductases that can use biomimetic NAD factors [91][92][93] and (ii) stable enzymes as building blocks of SyPaB [63,65,66,73], we estimate that ultimate hydrogen production costs may decrease to ~$1.30 per kg of hydrogen (Figure 9a), where carbohydrate accounts for ~95% of its production costs, in part because hydrogen has very low product separation and purification costs and the other chemicals in the reaction can be recycled.…”

Section: Sypab Challenges and Opportunitiesmentioning

confidence: 99%

Renewable Hydrogen Carrier — Carbohydrate: Constructing the Carbon-Neutral Carbohydrate Economy

Mielenz

2011

Energies

Abstract: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

“…The discovery and utilization of more thermostable enzymes suitable for cell-free SyPaB would be a new industrial enzymes gold mine [34]. Costly NAD(P) cofactors are expected to be replaced by low-cost and stable biomimetic analogues [35,36] plus engineered oxidoreductases that can work on the biomimics [37]. Also, several enzymes in the pathways may need further engineering for tolerating toxic metabolites, or increasing enzyme stability, or decreasing product inhibition, or other properties.…”

Section: Closing Remarksmentioning

confidence: 99%

Artificial Photosynthesis Would Unify the Electricity-Carbohydrate-Hydrogen Cycle for Sustainability

2010

Nat Prec

Sustainable development requires balanced integration of four basic human needs -air (O 2 /CO 2 ), water, food, and energy. To solve key challenges, such as CO 2 fixation, electricity storage, food production, transportation fuel production, water conservation or maintaining an ecosystem for space travel, we wish to suggest the electricity-carbohydrate-hydrogen (ECHo) cycle, where electricity is a universal energy carrier, hydrogen is a clean electricity carrier, and carbohydrate is a high-energy density hydrogen (14.8 H 2 mass% or 11-14 MJ electricity output/kg)carrier plus a food and feed source. Each element of this cycle can be converted to the other reversibly & efficiently depending on resource availability, needs, and costs. In order to implement such cycle, here we propose to fix carbon dioxide by electricity or hydrogen to carbohydrate (starch) plus ethanol by cell-free synthetic biology approaches. According to knowledge in the literature, the proposed artificial photosynthesis must be operative. Therefore, collaborations are urgently needed to solve several technological bottlenecks before large-scale implementation.Keywords: artificial photosynthesis, biofuels, cell-free synthetic biology, climate change, CO 2 fixation, electricity storage, food production, hydrogen, ecosystem for space travel, water 3 Sustainable development is a pattern of resource use that aims to meet human needs while preserving the environment. As the human population grows and its needs and desires (e.g., food, energy) expand, the problem of sustaining civilization or even improving the quality of life is challenging for scientists and engineers.To address sustainability challenges associated with air, water, food, and energy, I propose to construct the electricity, carbohydrate, and hydrogen (ECHo) cycle (Fig. 1). In this cycle, electricity is difficult to store but it is a universal energy carrier that can be produced from nonrenewable primary energy sources (e.g., fossil fuels or nuclear energy) or renewable primary energy sources (e.g., solar, wind, biomass, etc.); hydrogen, a clean electricity carrier that can be converted to electricity by fuel cells with high efficiency and without pollutants, is difficult and costly to store and distribute; solid carbohydrate, which is a high-density hydrogen carrier and food source, can be easily produced, stored, and distributed.To implement the ECHo cycle, we must efficiently fix CO 2 in the form of carbohydrates with an energy input of electricity or hydrogen (Fig. 1). When these technologies are developed, we can address the below a number of challenges associated with sustainability: (i) fixing CO 2 to mitigate climate change, (ii) producing food from CO 2 and water through artificial photosynthesis, (iii) generating sustainable transportation biofuel from CO 2 and electricity, (iv) storing electricity as a form of chemical compound(s) on a large scale, (v) conserving water as compared to natural plant photosynthesis, (vi) storing hydrogen in the form of carbohydrate a...