Pavan, Marilene | Search NASA

DOE OSTI2022

Advances in systems metabolic engineering of autotrophic carbon oxide-fixing biocatalysts towards a circular economy

High levels of anthropogenic CO 2 emissions are driving the warming of global climate. If this pattern of increasing emissions does not change, it will cause further climate change with severe consequences for the human population. On top of this, the increasing accumulation of solid waste within the linear economy model is threatening global biosustainability. The magnitude of these challenges requires several approaches to capture and utilize waste carbon and establish a circular economy. Microbial gas fermentation presents an exciting opportunity to capture carbon oxides from gaseous and solid waste streams with high feedstock flexibility and selectivity. Here we discuss available microbial systems and review in detail the metabolism of both anaerobic acetogens and aerobic hydrogenotrophs and their ability to utilize C1 waste feedstocks. More specifically, we provide an overview of the systems-level understanding of metabolism, key metabolic pathways, scale-up opportunities and commercial successes, and the most recent technological advances in strain and process engineering. Finally, we also discuss in detail the gaps and opportunities to advance the understanding of these autotrophic biocatalysts for the efficient and economically viable production of bioproducts from recycled carbon.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Modular cell-free expression plasmids to accelerate biological design in cells

Industrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms—organisms that are easy to use like Escherichia coli—often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have desirable metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes, and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has matured as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institute’s DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression of functional enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for Clostridium autoethanogenum expression with efficiencies ranging from 68% to 90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.

59 BASIC BIOLOGICAL SCIENCES↗