Accelerating biological systems design for sustainable biomanufacturing
A new cell-free platform rapidly identifies optimal enzymes for
sustainable fuels and materials

Date:
June 15, 2020
Source:
Northwestern University
Summary:
A new cell-free platform rapidly identifies optimal enzyme
combinations for sustainable fuels and materials.



FULL STORY ========================================================================== Northwestern University synthetic biologists have developed a new rapid- prototyping system to accelerate the design of biological systems,
reducing the time to produce sustainable biomanufacturing products from
months to weeks.


==========================================================================
As global challenges like climate change, population growth, and energy security intensify, the need for low-cost biofuels and bioproducts -- like medicines and materials -- produced using sustainable resources increases.

Industrial biotechnology, which uses microbial cellular factories to
harness enzyme sets that can convert molecules to desirable chemical
product, has shown potential to address these needs. However, designing, building, and optimizing these pathways in cells remains complex and slow, unable to keep up with the dynamic shifts in needs.

The new platform, called in vitro Prototyping and Rapid Optimization
of Biosynthetic Enzymes (iPROBE), provides a quick and powerful design-build-test framework to discover optimal biosynthetic pathways for cellular metabolic engineering that could impact a range of industries
(or issues) from clean energy to consumer products.

"For the first time, we show that cell-free platforms can inform and
accelerate the design of industrial cellular systems," said Michael
Jewett, Walter P.

Murphy Professor of Chemical and Biological Engineering and Charles
Deering McCormick Professor of Teaching Excellence at the McCormick
School of Engineering, who directs Northwestern's Center for Synthetic
Biology. "We accomplished in approximately two weeks what traditionally
would have taken six to 12 months. Our findings will help accelerate
the pace at which we can enable sustainable biomanufacturing practices."
The platform leverages Northwestern's leadership in cell-free synthetic
biology and comes into play in three recently published studies, each
led by Jewett.

"iPROBE stands to help scientists identify the best sets of enzymes for
a variety of sustainable chemicals and bring them into manufacturing at
scale," Jewett said. "We envision this cell-free system as an engine to
help realize the future bioeconomy." Adopting a cell-free approach


==========================================================================
"In Vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes for
Cell Design," published June 15 in the journal Nature Chemical Biology, describes how iPROBE works.

To manufacture sustainable chemicals, synthetic biologists stitch together protein enzymes to carry out individual molecular transformations,
converting readily available stock -- like glucose or carbon dioxide --
to a new product.

Current testing methods require these enzymes get encoded in DNA, placed
on a single plasmid molecule, and then inserted into a living cell. The
process must be repeated each time to study a different set of enzymes
in hopes of determining the most optimal grouping.

"The result is that the design cycles are just too slow," Jewett said. "We
end up needing hundreds of combined person years of development to bring
a product to market. That's too slow to address challenges like climate
change and other rapidly growing problems we face." iPROBE bypasses
the limitations of engineering living organisms using cell-free protein synthesis to enrich biosynthetic enzymes in test tubes to carry out transformations. Combined with computational design algorithms developed
by Lockheed Martin, the system rapidly studies pathway enzyme ratios,
tuning individual enzymes in the context of the desired multi-step
pathway, screening for high-performance enzymes, and discovering enzymes
with optimal functionalities.

"iPROBE had to be multifaceted and easy to use," said Ashty Karim, first
author on the paper and research fellow and assistant scientific director
in the Jewett Lab. "We set out to design a platform that could test
hundreds of biosynthetic hypotheses without having to re-engineer microbes simply by mixing and matching enzymes." Jewett likened the mix-and-match analysis of different enzyme combinations to making a cocktail.



========================================================================== "Imagine you're a bartender interested in making the perfect mixed
drink. You would want to bring together all of the possible cocktail ingredients that potentially could be used," Jewett said. "iPROBE allows
us to mix and match enzymes in this type of cocktail-based approach
to determine the best combinations to carry out the transformation and synthesis of sustainable chemicals -- but instead of taking months to
years to do, we can do it in days to weeks." Finding the optimal pathways
in Clostridium To validate the iPROBE system, the researchers developed
optimal biosynthetic pathways for 3-hydroxybutyrate (3-HB) and butanol,
two organic compounds in Clostridium autoethanogenum, a bacterium that naturally produces ethanol from metabolized carbon monoxide.

"It was important to us that we demonstrated the practical use of the technology," Karim said. "We had this dream solution to increase the pace
of biotechnology research and development that could only be realized
through the right collaboration." After identifying the optimal pathways
in vitro, the researchers shared them with collaborators at clean energy startup Lanzatech, which specializes in using Clostridium strains to
produce sustainable fuel. Researchers there applied the pathways and
found a 20-fold increase in 3-HB production in Clostridium, bridging
the iPROBE's success in the lab to an industrial setting.

"Working with an organism like Clostridium is difficult; genetic tools
are not as sophisticated, high-throughput workflows are often lacking,
and there exist transformation idiosyncrasies," Jewett said. "To have this process work successfully pushes a new vision for sustainability. What
could be better than turning waste gases from the atmosphere into
sustainable chemicals at scale?" Synthesizing limonene and styrene In a
second paper published in the journal Metabolic Engineering, Jewett and
his team focused on applying iPROBE to optimize the synthesis of limonene,
a member of a class of organic compounds called terpenes. Limonene is
found in the oil of orange and other citrus peels and responsible for
its fruity fragrance. The molecule is not only commonly used to enhance
the smell of household cleaners and manufactured foods, but also has
also shown the potential to help advance sustainable fuels.

In a matter of weeks, iPROBE's cell-free approach led to the exploration
of hundreds of enzyme combinations to synthesize limonene.

"In the past, people have only been able to study 20 or 30 pathways,"
Jewett said. "We demonstrated how iPROBE could be applied to this
particular biosynthetic pathway and scale not just to 100 or 200 pathways,
but 500. It sets a new standard for how cell-free systems can accelerate biological design of an important sustainable chemical." The third
paper, also published in Metabolic Engineering, looked at styrene,
a petroleum-derived molecule commonly used in disposable silverware
and foam packaging. While past efforts have attempted to synthesize
the molecule using living organisms like E. coli, styrene's natural
toxicity limited production capacity. With iPROBE, Jewett and his team synthesized the highest amount of styrene through a biochemical approach
to date without additional process enhancements.

"This advance opens the door to one day moving from production processes reliant on fossil fuels to more sustainable, biosynthetic-based
strategies," Jewett said.

This work was supported by the US Department of Energy's (DOE) Office
of Biological and Environmental Research within DOE's Office of Science
under award number DE-SC0018249.


========================================================================== Story Source: Materials provided by Northwestern_University. Original
written by Alex Gerage.

Note: Content may be edited for style and length.


========================================================================== Journal Reference:
1. Ashty S. Karim, Quentin M. Dudley, Alex Juminaga, Yongbo Yuan,
Samantha
A. Crowe, Jacob T. Heggestad, Shivani Garg, Tanus Abdalla,
William S.

Grubbe, Blake J. Rasor, David N. Coar, Maria Torculas, Michael
Krein, FungMin (Eric) Liew, Amy Quattlebaum, Rasmus O. Jensen,
Jeffrey A.

Stuart, Sean D. Simpson, Michael Ko"pke, Michael C. Jewett. In
vitro prototyping and rapid optimization of biosynthetic
enzymes for cell design. Nature Chemical Biology, 2020; DOI:
10.1038/s41589-020-0559-0 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/06/200615152112.htm

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