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Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
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Genetic Code Expansion in Pseudomonas putida KT2440.

Xinyuan He1, Tianyu Gao2, Yan Chen2

  • 1Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, Nebraska 68588, United States.

ACS Synthetic Biology
|October 26, 2022
PubMed
Summary

We successfully expanded the genetic code in *Pseudomonas putida* KT2440, enabling the incorporation of unnatural amino acids (unAAs) into proteins. This breakthrough advances engineering efforts for biobased chemical production and bioremediation.

Keywords:
Pseudomonas putida KT2440genetic code expansionphotocross-linkingprotein−protein interactionsunnatural amino acids

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Area of Science:

  • Synthetic Biology
  • Microbial Engineering

Background:

  • Pseudomonas putida KT2440 is a promising microbial chassis for biobased production and bioremediation.
  • Current tools for protein complex and enzyme engineering in P. putida KT2440 are limited.

Purpose of the Study:

  • To establish genetic code expansion in P. putida KT2440 for incorporating unnatural amino acids (unAAs).
  • To enable advanced protein engineering and biological process control in this microbial strain.

Main Methods:

  • Orthogonality of two archaeal tRNA synthetase/tRNA pairs was established in P. putida KT2440.
  • Decoding systems were optimized for efficient unAA incorporation via UAG stop codons.
  • Photocross-linking amino acid pBpa was incorporated into GstA and CheY proteins.

Main Results:

  • Four unAAs were incorporated into proteins with 34.6-78% efficiency.
  • Successful demonstration of unAA utility in studying protein-protein interactions.
  • First report of genetic code expansion in P. putida KT2440.

Conclusions:

  • This study provides the first successful genetic code expansion in P. putida KT2440.
  • The established system lays a foundation for future research on enhancing P. putida KT2440's biological functions.
  • This work opens new avenues for protein engineering and microbial strain development.