# In Vivo Biosynthesis and Direct Incorporation of Noncanonical Amino Acids into Proteins

## TL;DR

This paper reviews engineered cell systems that can produce and incorporate non-standard amino acids into proteins, comparing semi-autonomous and fully autonomous approaches.

## Contribution

The paper provides a critical evaluation and future outlook on autonomous cell systems for noncanonical amino acid biosynthesis and incorporation.

## Key findings

- Autonomous cells reduce process costs and increase protein yields by producing noncanonical amino acids intracellularly.
- Integrated biosynthetic pathways overcome challenges like poor membrane permeability of noncanonical amino acids.
- Semi-autonomous systems depend on external precursors for ncAA production.

## Abstract

Autonomous cells are engineered biological systems capable of biosynthesising and directly incorporating noncanonical amino acids (ncAAs) into proteins. These systems have the potential to extend the applicability of the genetic code to enable large‐scale fermentative production of proteins carrying ncAAs. This work evaluates approaches for the generation of autonomous and semi‐autonomous cells. Semi‐autonomous cells rely on the external addition of a precursor, which is enzymatically converted in vivo to an ncAA that is directly incorporated. In contrast, autonomous cells have a metabolic system that produces and directly incorporates an ncAA in vivo. Through a critical evaluation of the state of the art, the reader is provided with an opinion on the future development of the field.

This review evaluates engineered (semi)autonomous cell systems for the biosynthesis and incorporation of noncanonical amino acids (ncAAs) into proteins. While semi‐autonomous cells convert supplied precursors into ncAAs autonomous cells integrate biosynthetic pathways that produce these building blocks intracellularly. Such integrated approaches significantly reduce process costs, can increase protein yields, and overcome challenges such as the limited membrane permeability of ncAAs.© 2025 WILEY‐VCH GmbH