Author
Gedeon, Kamil Sankara
Other Contributors
Collins, Cynthia H.; Koffas, Mattheos A. G.; Garde, Shekhar; Rogers, Karyn;
Date Issued
2019-08
Subject
Chemical engineering
Degree
PhD;
Terms of Use
This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.;
Abstract
The final aim of this work was to engineer co-cultures for improved product titer and increased total sugar consumption as compared to mono-cultures. From this work we showed violacein production solely xylose for the first time in E. coli. We also showed the production of violacein and naringenin from co-cultures in mixed sugar media. By varying inoculation ratios of the two E. coli strains, we successfully determined co-culture conditions that resulted in higher production of violacein and naringenin than their mono-culture counterparts. The final co-culture system serves as a platform for improved production of other biomolecules from lignocellulosic sugars over mono-culture production, while demonstrating that modularization of metabolic pathways in different strains can improve product titer, and sugar consumption.; In order to engineer an E. coli strain that will preferentially consume xylose in the presence of glucose, we first hypothesized the potential genetic changes that would need to be executed. Several strains were evaluated for the growth dynamics and sugar consumption specificity. A set of glucose-specialist and a set of xylose-specialist were determined; the BL, MG, B2, and M4 strains were chosen for further experimentation. As the B2 strain showed poor growth as compared to its wild-type counterpart, studies were performed to improve its growth. CRISPRi was used to target repression of genes in B2; targeting xylR, a gene involved in xylose metabolism, increased its final cell density and increased its consumption of xylose in mixed sugar media. Gene overexpression studies were also performed; overexpression of gatC and tktA, genes involved in xylose metabolism, also increased the B2 strain’s final cell density and increased its consumption of xylose in mixed sugar media.; Lignocellulosic biomass is an abundant waste material that can serve as an ideal carbon source in the process of generating high-value bioproducts from microbial systems. Hydrolysis of lignocellulose results primarily in glucose and xylose, two sugars that can be used by Escherichia coli in its metabolism. Due to carbon catabolite repression (CCR), E. coli will preferentially use glucose over xylose, resulting in poor use of the total carbon source present. In addition to CCR, E. coli growth on xylose results in a lower energy yield from its metabolism, as compared to growth on glucose. Due to these obstacles, the use of lignocellulosic sugars for high value bioproduct formation is inefficient and uneconomical. To solve this problem, we proposed engineering a microbial community for simultaneous conversion of glucose and xylose into desired bioproducts, where each member of the community can express the full pathway for a desired bioproduct. Microbial communities are advantageous as each member can perform specialized tasks, thereby allowing for the greater system to carry out higher complex tasks. Separating the necessary tasks in a microbial community allows for modularity, increasing the potential of the system. In this work we developed a co-culture system of two E. coli strains that convert glucose and xylose, respectively, into our desired bioproducts. We hypothesized that the engineered co-culture system will achieve higher titer due to better carbon conversion.;
Description
August 2019; School of Engineering
Department
Dept. of Chemical and Biological Engineering;
Publisher
Rensselaer Polytechnic Institute, Troy, NY
Relationships
Rensselaer Theses and Dissertations Online Collection;
Access
Restricted to current Rensselaer faculty, staff and students. Access inquiries may be directed to the Rensselaer Libraries.;