Microbial engineering, characterization, and applications through novel data processing and synthetic biology systems

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2021-08
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Warner, Aric Cody
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Jarboe, Laura R
Shai, Zengyi
Yandeau-Nelson, Marna
Mansell, Thomas
Phillips, Gregory
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Chemical and Biological Engineering
Abstract
Microbes have been an indispensable source of resources for humanity for millennia, providing a wide range of valuable commodities including foods, medicines, fuels, and many other common and exotic materials. However, additional microbial characterization and engineering tools are needed in order to benefit from their full potential in modern applications. Despite the enormity of the collective research that has been performed with model organisms like the nearly ubiquitous, gram-negative bacteria Escherichia coli and the prototypical microbe Saccharomyces cerevisiae (a.k.a. brewer’s yeast), their ultimate utility is still limited by several hurdles: 1) even in these model hosts, there are still a vast number of uncharacterized or under-characterized components in their biology, 2) the organisms themselves cannot sustainably tolerate the harmful conditions required for their use in many promising applications, 3) cultures of individual hosts are not well-suited to meet the demands of these high-value applications—particularly not these model hosts, even with extensive modifications, and 4) we still lack the synthetic and/or molecular biology tools needed to conduct efficient, effective research towards realizing these microbiological applications. The research described herein represents attempts to address aspects of all four of these issues. First, to glean value from existing research regarding insufficiently characterized genes, existing data was mined for promising engineering targets in E. coli. For this purpose, a robust analytical method is demonstrated that identified eight potential targets, with four of which (yajL, yejM, yhdP, and yifB) showing statistically significant improvements in host thermotolerance upon deletion. Second, targeted characterization was performed for specific mutations in both rpoC and ompA from different evolved strains that confer tolerance to a range of industrially relevant, stressful conditions. Third, to ameliorate some common metabolic hurdles of substrate utilization and production efficiency in E. coli, a tri-stable phenotype switch using the native pap & fim epigenetic phase variation operons, which regulate expression of P-pili and Type 1 pili, was designed and constructed to attempt to generate an isogenic consortium of a single K12 MG1655 mutant. Optimizations for circuit design and expression tuning via codon optimization, circuit layout, and expression locus are described. Finally, to improve the time and cost efficiency in the identification of the chromosomal insertion locus following transposon mutagenesis, a casposon-based platform is described for S. cerevisiae. The current limitations of eukaryotic expression of casposons and alternative design strategies are discussed.
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