Engineered CRISPR systems and other novel metabolic engineering tools for production of natural compounds
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Authors
Cress, Brady Fletcher
Issue Date
2016-05
Type
Electronic thesis
Thesis
Thesis
Language
ENG
Keywords
Chemical engineering
Alternative Title
Abstract
Although metabolic engineering has been a distinct discipline for over two decades, coercing simple cells to overproduce certain secondary metabolites often remains a significant challenge. Fortunately, advancing technologies continue to drive the field toward creation of increasingly sophisticated microbial production platforms for the generation of specialty chemicals and pharmaceuticals. The aim of the work presented in this dissertation is to leverage these emerging technologies for development of novel tools at the interface of synthetic biology, metabolic engineering, and analytical chemistry. First, CRISPR interference (CRISPRi) is explored and utilized as a tool for metabolic engineering in Escherichia coli, enabling repression of many endogenous genes simultaneously.
Compared to previous technologies capable of achieving the same goal, CRISPRi is highly specific and easier to engineer, enabling quick assessment of metabolic engineering strategies in many host strains. Next a series of orthogonal promoters, regulated by CRISPRi, were constructed to facilitate orthogonal redirection of metabolic flux in the branched violacein pathway. This work also uncovered, for the first time, valuable insights about how CRISPRi orthogonality changes with context (copy number of DNA target site and number of mismatches between guide RNA and target DNA sequence). As described in the future work section of this dissertation, we have also begun to explore the combinatorial cloning method, Golden Gate Shuffling, for rapid, random assembly of CRISPR arrays, which has enabled us to experimentally explore repression space in E. coli at a much faster pace than in initial efforts. Finally, we also harness E. coli as a starting point for the chemoenzymatic preparation of heavy (perdeuterated) heparin. In this work, we demonstrate that stable-isotope enrichment of incredibly complex pharmaceuticals is readily facilitated using microbes, cultured in a medium enriched in the isotope of interest, for precursor generation. The research presented in this thesis is a significant step forward in expediting and improving our ability to engineer microbes to produce valuable natural products.
Compared to previous technologies capable of achieving the same goal, CRISPRi is highly specific and easier to engineer, enabling quick assessment of metabolic engineering strategies in many host strains. Next a series of orthogonal promoters, regulated by CRISPRi, were constructed to facilitate orthogonal redirection of metabolic flux in the branched violacein pathway. This work also uncovered, for the first time, valuable insights about how CRISPRi orthogonality changes with context (copy number of DNA target site and number of mismatches between guide RNA and target DNA sequence). As described in the future work section of this dissertation, we have also begun to explore the combinatorial cloning method, Golden Gate Shuffling, for rapid, random assembly of CRISPR arrays, which has enabled us to experimentally explore repression space in E. coli at a much faster pace than in initial efforts. Finally, we also harness E. coli as a starting point for the chemoenzymatic preparation of heavy (perdeuterated) heparin. In this work, we demonstrate that stable-isotope enrichment of incredibly complex pharmaceuticals is readily facilitated using microbes, cultured in a medium enriched in the isotope of interest, for precursor generation. The research presented in this thesis is a significant step forward in expediting and improving our ability to engineer microbes to produce valuable natural products.
Description
May 2016
School of Engineering
School of Engineering
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY