A dominant selection system designed for copy-number-controlled gene integration in Hansenula polymorpha DL-1

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Selecting multiple gene integrants in Hansenula polymorpha is challenging.
• Standard selectable markers like the aminoglycoside 3-phosphotransferase (APH) gene can lead to slow growth and spontaneous resistance.
• Efficient gene integration is crucial for optimizing industrial yeast strains.

Purpose of the Study:

• To develop a rapid and copy-number-controlled selection system for H. polymorpha.
• To improve the performance of the APH gene as a selectable marker.
• To enable precise control over the number of integrated gene copies.

Main Methods:

• A vector was constructed containing a telomeric autonomous replication sequence and the bacterial aminoglycoside 3-phosphotransferase (APH) gene.
• Deleted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters of H. polymorpha were fused to the APH gene.
• Promoter deletions were systematically performed to fine-tune gene expression and integration copy number.

Main Results:

• Fusion of the APH gene with a 578-bp GAPDH promoter enabled rapid growth and selection of transformants with up to 15 tandem vector copies.
• Serial deletion of the GAPDH promoter allowed controlled integration copy numbers ranging from 1 to 50.
• Tandemly integrated plasmid copies were mitotically stable for over 150 generations.

Conclusions:

• The developed dosage-dependent selection system facilitates rapid and controlled multiple gene integration in H. polymorpha.
• This system is a powerful tool for developing H. polymorpha as an industrial strain for recombinant protein production.
• Optimized promoter-marker fusions enhance the efficiency and control of genetic engineering in industrial yeast.