Development of an optimized medium, strain and high-throughput culturing methods for Methylobacterium extorquens

Area of Science:

• Microbiology
• Biotechnology
• Metabolic Engineering

Background:

• Methylobacterium extorquens is a well-established model organism for studying methylotrophic metabolism.
• Existing culturing methods for M. extorquens lack efficiency and consistency for high-throughput applications.
• Biofilm formation due to cellulose synthase genes can complicate batch cultures.

Purpose of the Study:

• To develop an optimized, high-throughput batch culture system for M. extorquens.
• To engineer a novel growth medium that promotes faster and more consistent growth.
• To improve the reliability of growth rate estimations in M. extorquens.

Main Methods:

• Genetic modification of M. extorquens strains AM1 and PA1 to remove cellulose synthase genes, preventing biofilm formation.
• Implementation of a high-throughput microtiter plate culturing system utilizing lab automation and open-source software.
• Application of response surface methodologies to optimize growth medium composition, focusing on buffers and metal chelators.

Main Results:

• The engineered M. extorquens strains exhibited reduced biofilm formation, enabling reliable growth rate measurements.
• A new Methylobacterium PIPES (MP) medium, using PIPES buffer and citrate for metal chelation, significantly improved growth rates.
• The MP medium demonstrated robustness against variations in component ingredients, reducing experimental batch effects and enhancing consistency.

Conclusions:

• The developed high-throughput culturing system and MP medium provide a superior method for studying M. extorquens.
• This optimized system facilitates more efficient and reliable research into methylotrophic metabolism.
• The MP medium represents a significant advancement over existing media for M. extorquens cultivation.