Doctoral Dissertations

Date of Award

12-2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Energy Science and Engineering

Major Professor

Adam M Guss

Committee Members

Brian H. Davison, Cong T. Trinh, Erik Zinser, Erik R. Zinser

Abstract

Many organisms naturally possess complex physiological traits that are of interest for biotechnology research. These non-model organisms are not routinely used though, due to a lack of foundational knowledge and genetic tools. The development of genetic tools is limited in part by the inability to efficiently transform DNA into these organisms. One of the major barriers to efficient DNA transformation is the native DNA Restriction-Modification systems within bacteria. DNA Restriction-Modification systems act as an immune system to cut heterologous DNA methylated differently than the host. To overcome these systems, DNA needs to be methylated in the same manner as the host organism prior to transformation. In order to determine the sites targeted for restriction we performed methylome analysis for non-model organisms of interest in collaboration with the Department of Energy’s Joint Genome Institute. This information was used to choose methyltransferases for expression in E. coli to protect DNA for transformation of multiple organisms. Using this approach, we enabled the transformation of eight strains of bacteria including two strains of Megasphaera elsdenii. We are now developing M. elsdenii into a new bioengineering platform for the production of fuels and chemicals. M. elsdenii is a compelling platform organism due to the ability to efficiently produce C-4 and C-6 acids from lactate and glucose. This work acts as a blueprint for the rapid transformation and genetic engineering of non-model organisms.

Download
Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Included in

Biotechnology Commons

Share

COinS