Jewett and collaborators at LanzaTech and the University of South Florida (USF) have optimized and implemented a cyclic reverse beta-oxidation (r-BOX) pathway for the specific production of butanol, butanoic acid, d hexanol and hexanoic acid on three biotechnology platforms. The three platforms include a cell-free system, a heterotrophic model organism (E.coli), and an autotrophic organism (C. autoethanogenum) capable of fixing carbon and using syngas — a mixture of carbon monoxide and hydrogen — as its sole source of carbon and energy.
Jewett and his teammates reported their work in “Cell-free prototyping enables the implementation of optimized reverse beta-oxidation pathways in heterotrophic and autotrophic bacteria“, published on June 1 in Nature Communication. Jewett is the Walter P. Murphy Professor of Chemical and Biological Engineering and director of the Synthetic Biology Center. Other corresponding authors were LanzaTech Vice President of Synthetic Biology Michael Köpke and USF Professor Ramon Gonzalez.
Build on past research Posted in Natural biotechnology where researchers selected, engineered and optimized a bacterial strain, then successfully demonstrated its ability to produce carbon negative acetone and isopropanol, this investigation streamlines the process and broadens the spectrum of molecules that can be produced. A key feature of their work was the creation of an automated liquid handling workflow based on cell-free biosynthesis, which was later adapted for r-BOX. This allowed the team to rapidly screen 762 unique pathway combinations to identify the optimal enzyme sets for improved product selectivity in weeks rather than months.
“Our work forms a new model for the generation and optimization of biochemical pathways for metabolic engineering and synthetic biology,” Jewett said. “This will facilitate the design-build-test cycles of biosynthetic pathways by decreasing the number of strains that need to be designed and the time required to achieve desired process goals.”
“The iterative and modular nature of r-BOX enables the synthesis of products of different chain lengths and functionalities with high carbon and energy efficiency. This allowed us to co-develop bespoke processes for four different products, rather than just one product,” Gonzalez said. “r-BOX can further be extended to an array of other products of interest by building on the developed workflows,” which the authors discussed in a separate article published in the Journal of Industrial Microbiology and Biotechnology.
The next steps, Köpke said, are to scale up production and expand the reach of sustainable chemicals made by biology.
“LanzaTech already operates two commercial facilities that convert industrial gases into ethanol, mitigating hundreds of thousands of tons of greenhouse gas emissions,” Köpke said. “The implementation of r-BOX will increase the flexibility of biological processes to adapt to new markets, expand the range of fossil-derived products that can be replaced by bio-derived alternatives, and improve the economic benefits of co-produced fuels. “