Komagataeibacter Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion...

Area of Science:

• Synthetic Biology
• Biomaterials Engineering
• Microbial Engineering

Background:

• Cellulose-producing bacteria, particularly from the *Komagataeibacter* genus, are promising hosts for Engineered Living Materials (ELMs).
• Existing genetic engineering methods in these bacteria are limited, hindering synthetic biology applications.
• The ability to genetically modify these bacteria allows for tailored material properties and production.

Purpose of the Study:

• To introduce a standardized modular cloning system, the Komagataeibacter tool kit (KTK), for *Komagataeibacter* species.
• To facilitate the construction of complex multigene constructs for enhanced synthetic biology applications.
• To accelerate progress in the field of Engineered Living Materials (ELMs).

Main Methods:

• Development of the Komagataeibacter tool kit (KTK) based on Golden Gate DNA assembly.
• Creation of basic DNA parts including promoters, fusion tags, and reporter proteins for *Komagataeibacter rhaeticus*.
• Reformatting and functional expression of the *Escherichia coli* curli amyloid fiber system in *K. rhaeticus* using KTK.

Main Results:

• Successful implementation of the KTK system in *Komagataeibacter rhaeticus*.
• Demonstration of modular cloning for building complex genetic constructs.
• Functional expression of the *E. coli* curli system and its modification for protein secretion in *K. rhaeticus*.

Conclusions:

• The KTK provides a standardized and versatile platform for synthetic biology in cellulose-producing bacteria.
• This toolkit enables rapid engineering of *Komagataeibacter* strains for novel biomaterial applications.
• KTK will accelerate innovation in the emerging field of Engineered Living Materials.