Biosensor and optogenetics for systems biology of yeast branched-chain alcohol production and tolerance

Technical Report · Wed Nov 30 04:00:00 EST 2022

DOI:https://doi.org/10.2172/1900526· OSTI ID:1900526

Avalos, Jose L. ^[1]

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1. Princeton University, NY (United States); Princeton University

In this project we combined synthetic biology, systems biology, protein engineering and metabolic engineering to study, control, and improve the production of branched chain alcohols (BCAs), a class of advanced biofuels preferred by the DOE, in the yeast Saccharomyces cerevisiae. This involved the development and application of optogenetic systems as a new modality of dynamic control of native and engineered metabolic pathways, using light as inducible or repressible agent. The optogenetic systems include gene circuits for light control of gene expression, as well as light-assembled synthetic organelles and photo-switchable protein binders to control metabolic and protein function with light at the protein level. In addition, we developed the first genetically encoded biosensor for BCA production in yeast, which we used to design high throughput assays to identify highly productive strains, pathways, and enzymes. We also showed that this biosensor can be functionally co-expressed with optogenetic circuits in the same strain, raising the possibility of establishing, for the first time, computer-interfaced closed-loop controls of engineered metabolic pathways. The yeast gene deletion library was also utilized to conduct the first systems-level study on BCA toxicity in yeast, which uncovered key fundamental principles of yeast sensitivity and tolerance to these alcohols, allowing us to design highly tolerant strains with increased BCA production. This project, thus comprises the development of several new technologies, which we integrated to make new discoveries on the dynamics of BCA production and their mechanisms of cellular toxicity and tolerance, as well as to establish new paradigms to engineer and control metabolic pathways and microbial fermentations with light, for the production of BCAs and other products of interest to the DOE.

• Research Organization: Princeton University, NY (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER)
• DOE Contract Number: SC0019363
• OSTI ID: 1900526
• Report Number(s): DOE-PRINCETON--0019363
• Country of Publication: United States
• Language: English

Related Subjects

2-metyl-1-butanol
59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
Optogenetics
Saccaromyces cerevisiae
biosensors
branched-chain alcohols
cellulosic
isobutanol
isopentantol
metabolic engineering
mitochondrial engineering
monobodiesl
nanobodies
optical binders
synthetic organelles
xylose
yeast gene deletion library