Genetic Code Expansion in Pseudomonas putida KT2440

Tianyu Gao; Jiantao Guo; Wei Niu

doi:10.1007/978-1-0716-3658-9_13

Genetic Code Expansion in Pseudomonas putida KT2440

Methods Mol Biol. 2024:2760:209-217. doi: 10.1007/978-1-0716-3658-9_13.

Authors

Tianyu Gao¹, Jiantao Guo^{2

3}, Wei Niu^{4

5}

Affiliations

¹ Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, USA.
² Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, USA. jguo4@unl.edu.
³ The Nebraska Center for Integrated Biomolecular Communication (NCIBC), University of Nebraska-Lincoln, Lincoln, NE, USA. jguo4@unl.edu.
⁴ The Nebraska Center for Integrated Biomolecular Communication (NCIBC), University of Nebraska-Lincoln, Lincoln, NE, USA. wniu2@unl.edu.
⁵ Department of Chemical & Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE, USA. wniu2@unl.edu.

PMID: 38468091
DOI: 10.1007/978-1-0716-3658-9_13

Abstract

Emerging microorganism Pseudomonas putida KT2440 is utilized for the synthesis of biobased chemicals from renewable feedstocks and for bioremediation. However, the methods for analyzing, engineering, and regulating the biosynthetic enzymes and protein complexes in this organism remain underdeveloped.Such attempts can be advanced by the genetic code expansion-enabled incorporation of noncanonical amino acids (ncAAs) into proteins, which also enables further controls over the strain's biological processes. Here, we give a step-by-step account of the incorporation of two ncAAs into any protein of interest (POI) in response to a UAG stop codon by two commonly used orthogonal archaeal tRNA synthetase and tRNA pairs. Using superfolder green fluorescent protein (sfGFP) as an example, this method lays down a solid foundation for future work to study and enhance the biological functions of KT2440.

Keywords: Amber suppression; Genetic code expansion; Noncanonical amino acids; Orthogonal tRNA synthetase and tRNA; Pseudomonas putida KT2440.

MeSH terms

Amino Acids / genetics
Amino Acids / metabolism
Amino Acyl-tRNA Synthetases* / metabolism
Genetic Code
Pseudomonas putida* / genetics
Pseudomonas putida* / metabolism
RNA, Transfer / genetics
RNA, Transfer / metabolism

Substances

Amino Acids
RNA, Transfer
Amino Acyl-tRNA Synthetases