Highly-efficient electrotransformation of the yeast Hansenula polymorpha

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Hansenula polymorpha is a methylotrophic yeast with significant biotechnological potential.
• Efficient genetic manipulation methods are crucial for harnessing its capabilities.
• Existing transformation protocols may not be optimal for H. polymorpha.

Purpose of the Study:

• To develop a highly efficient electroporation-based transformation method for Hansenula polymorpha.
• To optimize key parameters influencing transformation efficiency.
• To establish a robust protocol advantageous for gene cloning.

Main Methods:

• Electroporation using exponential decay electric pulses (7.5 kV/cm, 5 ms).
• Pretreatment of cells with dithiotreitol (DTT).
• Utilizing sucrose as an osmotic stabilizer in an ionic buffer.
• Employing mid-logarithmic phase cells.
• Investigating effects of field strength, pulse duration, and cell concentration.

Main Results:

• Achieved transformation frequencies up to 1.7 x 10(6) transformants per microgram of plasmid DNA.
• Transformation efficiency was significantly enhanced by DTT pretreatment, sucrose stabilization, and mid-log phase cells.
• High transformation frequencies were maintained even with large amounts of plasmid DNA (up to 10 µg).

Conclusions:

• A highly efficient and reproducible electroporation protocol for Hansenula polymorpha has been established.
• The developed method offers significant advantages for gene cloning requiring numerous transformants.
• This optimized protocol facilitates advanced genetic studies and biotechnological applications of H. polymorpha.