An in vivo target mutagenesis system for multiple hosts.

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• In vivo target mutagenesis accelerates protein evolution but is limited to conventional organisms.
• Developing methods for non-conventional hosts is crucial for expanding protein engineering capabilities.

Purpose of the Study:

• To design and validate a novel in vivo target mutagenesis system for multiple microbial hosts.
• To enable efficient protein evolution across diverse bacterial and yeast species.

Main Methods:

• Developed the in vivo target mutagenesis system for multiple hosts (ITMU) using the RSF1010 broad-host-range plasmid.
• Utilized a deaminase-helicase fusion and a primase error-prone DNA polymerase I fusion for high-rate mutagenesis.
• Tested ITMU effectiveness in Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, and Yarrowia lipolytica.

Main Results:

• The ITMU system achieved a mutation rate 1.18 × 10^5-fold higher than the host genome.
• Demonstrated successful in vivo continuous evolution using ITMU in four distinct microbial species.
• Confirmed ITMU's applicability across diverse microbial chassis.

Conclusions:

• The ITMU system is a robust tool for in vivo protein evolution in multiple hosts.
• ITMU provides a viable alternative to existing in vivo continuous evolution systems.
• Facilitates protein engineering in non-conventional microbial hosts, expanding evolutionary possibilities.