Multiplexed transcriptional control strategies for biosynthesis from mixed substrates in Escherichia coli

Metabolic engineering reprograms microbes to produce value-added chemicals. Microbial production has the potential to use renewable feedstocks, such as conventional waste streams. Metabolic engineers already contend with the metabolic burden of recombinant production pathways; utilizing complex input streams only further complicates allocating cellular resources appropriately for biosynthesis. This thesis aims to develop transcriptional control strategies that sense and respond to changing feedstock conditions for biosynthesis and demonstrate the ability to produce a product of interest from mixed substrate feeds. We constructed a galacturonate biosensor with the galacturonate-responsive transcription factor, ExuR, from Bacillus subtilis and determined the best performer from a selection of biosensor variants. After establishing no interactions with the host Escherichia coli native regulatory system, we applied the biosensor to control expression of biosynthetic pathway. It was confirmed that the biosensor activated transcription in the presence of galacturonate, eliminating the need for a chemically-inducible control system. A second, gluconate biosensor was constructed with GntR, from B. subtilis. The two biosensors were shown to be orthogonal and each was used to control the expression of a novel D-glycerate biosynthetic pathway from its cognate substrate. We demonstrated D-glycerate production from single and mixed substrate feeds and showed that mixed substrates in different ratios resulted in fairly consistent titers. Finally, the pairwise orthogonality of various AHL-based QS systems was characterized to establish multiple controllers for autonomous, cell density activated expression. We determined the the las and tra systems demonstrated minimal crosstalk, which agreed with literature findings. This work demonstrates the engineering of expression controllers and a production strain for biosynthesis from mixed substrate feeds.

Name thesis:degree_name

Doctoral

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Chemical Engineering

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2022

Author dc:creator

• Ni, Cynthia

Advisor dc:contributor.advisor

• L. J. Prather, Kristala

Statement dc:rights

• In Copyright - Educational Use Permitted
• Copyright MIT

Licence dc:rights.uri

• http://rightsstatements.org/page/InC-EDU/1.0/

Handle dc:identifier.uri

https://hdl.handle.net/1721.1/145014

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/145014