'Small volume-big problem': culturing Yarrowia lipolytica in high-throughput micro-formats

Area of Science:

• Synthetic biology
• Microbial physiology
• Biotechnology

Background:

• The 'design-build-test-learn' cycle in synthetic biology is often limited by testing capabilities.
• Down-scaling microbial cultures for parallelization can introduce metabolic footprints, especially in demanding species like Yarrowia lipolytica.
• Y. lipolytica presents unique challenges for micro-scale cultivation, including aerobic metabolism, filamentation, adhesion, flocculation, and media acidification.

Purpose of the Study:

• To review progress in scaling down and parallelizing Y. lipolytica cultures.
• To highlight common challenges and effective strategies for Y. lipolytica micro-cultivation.
• To present a novel, optimized high-throughput cultivation protocol for Y. lipolytica.

Main Methods:

• Analysis of existing literature on Y. lipolytica micro-cultivation.
• Cost analysis of micro-cultivations to identify uncontrolled variables.
• Discussion of various microtiter plate (MTP) formats and micro-/pico-bioreactors for Y. lipolytica.
• Presentation of a newly developed and optimized high-throughput cultivation protocol.

Main Results:

• Down-scaling Y. lipolytica necessitates personalized optimization for accurate phenotypic studies.
• Common challenges in Y. lipolytica micro-cultivation include metabolic perturbations and physical characteristics.
• A detailed, pre-optimized high-throughput cultivation protocol for Y. lipolytica is presented.

Conclusions:

• Overcoming the challenges of Y. lipolytica down-scaling is crucial for advancing synthetic biology projects.
• The presented high-throughput protocol offers a practical guide for researchers working with Y. lipolytica.
• Accurate phenotypic studies of Y. lipolytica require tailored micro-cultivation strategies.