Adenine Base Editing System for Pseudomonas and Prediction Workflow for Protein Dysfunction via ABE.

Area of Science:

• Microbiology
• Biotechnology
• Synthetic Biology

Background:

• Pseudomonas species possess metabolic versatility, enabling diverse environmental and industrial applications.
• Efficient genetic manipulation tools are crucial for understanding and harnessing Pseudomonas' potential.
• Adenine base editing (ABE) offers precise genome editing capabilities.

Purpose of the Study:

• To develop a highly efficient adenine base editing system for Pseudomonas species.
• To create a computational workflow for predicting protein dysfunction caused by base editing.
• To demonstrate the utility of these tools in metabolic engineering and non-model Pseudomonas species.

Main Methods:

• Development of the dxABE-PS system utilizing xCas9 3.7 for broad PAM recognition (NG).
• Creation of the DABE-CSP (dysfunction via ABE through CRISPOR-SIFT prediction) workflow.
• Application of DABE-CSP for gene inactivation in Pseudomonas putida KT2440 and development of nxABE for P. chengduensis.

Main Results:

• The dxABE-PS system achieved up to 100% efficiency for A:T to G:C transitions in Pseudomonas.
• DABE-CSP successfully predicted and guided gene inactivation for muconic acid production in P. putida.
• An adapted system, nxABE, was successfully developed for the non-model species P. chengduensis.

Conclusions:

• The developed ABE systems (dxABE-PS and nxABE) significantly enhance genetic manipulation capabilities in Pseudomonas.
• The DABE-CSP workflow aids in predicting the functional impact of base editing, facilitating targeted gene inactivation.
• These advancements collectively accelerate research and biotechnological applications of Pseudomonas species.