Redesign of an Escherichia coli Nissle treatment for phenylketonuria using insulated genomic landing pads and genetic circuits to reduce burden

Alexander J Triassi; Brandon D Fields; Catherine E Monahan; Jillian M Means; Yongjin Park; Hamid Doosthosseini; Jai P Padmakumar; Vincent M Isabella; Christopher A Voigt

doi:10.1016/j.cels.2023.05.004

Redesign of an Escherichia coli Nissle treatment for phenylketonuria using insulated genomic landing pads and genetic circuits to reduce burden

Cell Syst. 2023 Jun 21;14(6):512-524.e12. doi: 10.1016/j.cels.2023.05.004.

Authors

Alexander J Triassi¹, Brandon D Fields¹, Catherine E Monahan², Jillian M Means³, Yongjin Park¹, Hamid Doosthosseini¹, Jai P Padmakumar¹, Vincent M Isabella², Christopher A Voigt⁴

Affiliations

¹ Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
² Synlogic Inc., Cambridge, MA 02142, USA.
³ Synlogic Inc., Cambridge, MA 02142, USA. Electronic address: jillian.means@synlogictx.com.
⁴ Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: cavoigt@gmail.com.

PMID: 37348465
DOI: 10.1016/j.cels.2023.05.004

Abstract

To build therapeutic strains, Escherichia coli Nissle (EcN) have been engineered to express antibiotics, toxin-degrading enzymes, immunoregulators, and anti-cancer chemotherapies. For efficacy, the recombinant genes need to be highly expressed, but this imposes a burden on the cell, and plasmids are difficult to maintain in the body. To address these problems, we have developed landing pads in the EcN genome and genetic circuits to control therapeutic gene expression. These tools were applied to EcN SYNB1618, undergoing clinical trials as a phenylketonuria treatment. The pathway for converting phenylalanine to trans-cinnamic acid was moved to a landing pad under the control of a circuit that keeps the pathway off during storage. The resulting strain (EcN SYN8784) achieved higher activity than EcN SYNB1618, reaching levels near when the pathway is carried on a plasmid. This work demonstrates a simple system for engineering EcN that aids quantitative strain design for therapeutics.

Keywords: E. coli Nissle; PKU; genetic circuit; genome editing; gut microbiome; landing pads; phenylketonuria; probiotic; synthetic biology; systems biology.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Anti-Bacterial Agents / metabolism
Escherichia coli* / genetics
Escherichia coli* / metabolism
Genomics
Humans
Phenylketonurias* / genetics
Phenylketonurias* / therapy
Plasmids / genetics

Substances

Anti-Bacterial Agents