Engineering and Rapid Prototyping for Biology in Extreme Conditions

Author: Meyerowitz, Joseph Toshiro

Year: 2023

Degree: Dissertation (Ph.D.)

Advisor: Murray, Richard M.

Committee Members: Leadbetter, Jared R.; Newman, Dianne K.; Phillips, Robert B.; Murray, Richard M.

Option: Biochemistry and Molecular Biophysics

DOI: 10.7907/9gbb-n831

Abstract

In this thesis we show three projects in which biological systems are engineered for increased robustness to environmental stressors such as toxic small molecules. Several lignocellulose-derived growth inhibitors commonly found in industrial feedstocks for fermentation were used to grow a panel of yeast knockouts for several efflux pumps and detoxifying enzymes. Some specific knockout strains showed slowed growth on specific growth inhibitors, while other knockout strains showed the same growth rate as the wild-type. One efflux pump was identified for vanillin, YHK8, and was overexpressed in yeast. The overexpression strain did not show an improved tolerance to vanillin, and grew more slowly than the wild-type. To regulate the expression of the vanillin pump, a sensor for vanillin was created. The starting enzyme was the wild-type qacR transcription factor, and several variants were generated using computational protein design. The designs were synthesized and tested using in vitro transcription-translation (TX-TL) as part of a rapid prototyping process. This rapid prototyping considerably sped up the design-build-test process. Finally, four bacteria, Pseudomonas synxantha 2-79, Pseudomonas chlororaphis PCL1391, Pseudomonas aureofaciens 30-84, and E. coli are tested against the same lignocellulose growth inhibitors. The Pseudomonas spp. show an improved tolerance to the growth inhibitors. We then develop some ability to engineer and prototype in all three species. A panel of promoter parts were integrated into the P. synxantha genome to produce a collection of test strains. These same promoter parts were also used as DNA templates for TX-TL reactions. The in vivo measurements of promoter strength and in vitro measurements show similar relative strengths between the parts, showing the Pseudomonas-based TX-TL systems can be used for design-build-test activities in these non-model organisms. This alternate approach to developing tolerance, starting with a species that already has a working tolerance to the stressor in question, changes the problem to one of building engineering capabilities in the new chassis.

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