Engineering topics

Jewett, Michael C.

Publications and source records attributed to Jewett, Michael C..

24 records · Page 2

DOE OSTI2021

Editorial: Cell Free Biocatalysis for the Production of Bioproducts

Biological approaches show promise for the sustainable and cost competitive production of commodity fuels and chemicals. A lot of efforts have been devoted to engineering microbial chassis to produce select fuels and chemicals. These approaches have had some level of success exemplified, of course, by the production of ethanol at high yield and titer but also recent successes with commodity chemicals such as 1,4-butanediol, 1,3-propanediol, and even to some extent with sustainable aviation fuel intermediates such as farnesene. However, many of these processes still suffer from low yields, usually below the standard needed to drive a sustainable and atom efficient bioeconomy. Additionally, for many other chemicals and fuel precursors, titers and yields are only a fraction of what they are for ethanol. Indeed, there exist many limiting factors associated with the use of microbial biocatalysts. Some of these factors include the diversion of carbon from products to sustain growth and biomass formation, the production of byproducts or regulatory mechanisms, the toxicity of end products or intermediates, and the difficulty of separating products from growth media.

09 BIOMASS FUELS↗

DOE OSTI2021

An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts

Cell-free systems using crude cell extracts present appealing opportunities for designing biosynthetic pathways and enabling sustainable chemical synthesis. However, the lack of tools to effectively manipulate the underlying host metabolism in vitro limits the potential of these systems. Here, we create an integrated framework to address this gap that leverages cell extracts from host strains genetically rewired by multiplexed CRISPR-dCas9 modulation and other metabolic engineering techniques. As a model, we explore conversion of glucose to 2,3-butanediol in extracts from flux-enhanced Saccharomyces cerevisiae strains. We show that cellular flux rewiring in several strains of S. cerevisiae combined with systematic optimization of the cell-free reaction environment significantly increases 2,3-butanediol titers and volumetric productivities, reaching productivities greater than 0.9 g/L-h. We then show the generalizability of the framework by improving cell-free itaconic acid and glycerol biosynthesis. Our coupled in vivo/in vitro metabolic engineering approach opens opportunities for synthetic biology prototyping efforts and cell-free biomanufacturing.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Analysis of the Innovation Trend in Cell-Free Synthetic Biology

Cell-free synthetic biology is a maturing field that aims to assemble biomolecular reactions outside cells for compelling applications in drug discovery, metabolic engineering, biomanufacturing, diagnostics, and education. Cell-free systems have several key features. They circumvent mechanisms that have evolved to facilitate species survival, bypass limitations on molecular transport across the cell wall, enable high-yielding and rapid synthesis of proteins without creating recombinant cells, and provide high tolerance towards toxic substrates or products. Here, we analyze ~750 published patents and ~2000 peer-reviewed manuscripts in the field of cell-free systems. Three hallmarks emerged. First, we found that both patent filings and manuscript publications per year are significantly increasing (five-fold and 1.5-fold over the last decade, respectively). Second, we observed that the innovation landscape has changed. Patent applications were dominated by Japan in the early 2000s before shifting to China and the USA in recent years. Finally, we discovered an increasing prevalence of biotechnology companies using cell-free systems. Our analysis has broad implications on the future development of cell-free synthetic biology for commercial and industrial applications.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Stepping on the Gas to a Circular Economy: Accelerating Development of Carbon-Negative Chemical Production from Gas Fermentation

Owing to rising levels of greenhouse gases in our atmosphere and oceans, climate change poses significant environmental, economic, and social challenges globally. Technologies that enable carbon capture and conversion of greenhouse gases into useful products will help mitigate climate change by enabling a new circular carbon economy. Gas fermentation using carbon-fixing microorganisms offers an economically viable and scalable solution with unique feedstock and product flexibility that has been commercialized recently. Here, we review the state of the art of gas fermentation and discuss opportunities to accelerate future development and rollout. We discuss the current commercial process for conversion of waste gases to ethanol, including the underlying biology, challenges in process scale-up, and progress on genetic tool development and metabolic engineering to expand the product spectrum. We emphasize key enabling technologies to accelerate strain development for acetogens and other nonmodel organisms.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Toward sustainable, cell-free biomanufacturing

Industrial biotechnology is an attractive approach to address the need for low-cost fuels and products from sustainable resources. Unfortunately, cells impose inherent limitations on the effective synthesis and release of target products. One key constraint is that cellular survival objectives often work against the production objectives of biochemical engineers. Additionally, industrial strains release CO2 and struggle to utilize sustainable, potentially profitable feedstocks. Cell-free biotechnology, which uses biological machinery harvested from cells, can address these challenges with advantages including: (i) shorter development times, (ii) higher volumetric production rates, and (iii) tolerance to otherwise toxic molecules. In this review, we highlight recent advances in cell-free technologies toward the production of non-protein products beyond lab-scale demonstrations and describe guiding principles for designing cell-free systems. Specifically, we discuss carbon and energy sources, reaction homeostasis, and scale-up. Expanding the scope of cell-free biomanufacturing practice could enable innovative approaches for the industrial production of green chemicals.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways

Gas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste. Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic part function, gene expression patterns, and biosynthetic pathway performance in vitro before implementing designs in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here in this paper, we report the development and optimization of a high-yielding (236 ± 24 μg/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map aerobic cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes.

59 BASIC BIOLOGICAL SCIENCES↗