Sequential gene deletions in Hypocrea jecorina using a single blaster cassette

Area of Science:

• Molecular Biology
• Mycology
• Genetic Engineering

Background:

• Multiple gene deletions in Hypocrea jecorina (Trichoderma reesei) are hindered by a limited number of selectable markers.
• Efficiently generating strains with multiple gene deletions is crucial for understanding fungal physiology and metabolism.

Purpose of the Study:

• To construct and validate a versatile blaster cassette for successive gene knock-outs in H. jecorina.
• To demonstrate the cassette's utility by deleting the glucokinase (glk1) and hexokinase (hxk1) genes.

Main Methods:

• Construction of a 3.5 kb pyr4 blaster cassette containing the H. jecorina pyr4 marker gene flanked by Streptoalloteichus hindustanus bleomycin (Sh ble) genes.
• Cloning non-coding flanking regions of glk1 and hxk1 into the blaster cassettes for homologous recombination.
• Transformation of pyr4-negative H. jecorina strains and selection for gene deletion and cassette excision using 5-fluoroorotic acid.

Main Results:

• Successful deletion of glk1 or hxk1 in 10-13% of transformants using the blaster cassette system.
• Excision of the pyr4 blaster cassette via homologous recombination between Sh ble elements, creating auxotrophic strains.
• Generation of a double Deltaglk1Deltahxk1 deletion strain completely unable to grow on glucose or fructose.

Conclusions:

• The developed pyr4 blaster cassette enables efficient successive gene deletions in H. jecorina.
• The study provides insights into the roles of glucokinase and hexokinase in H. jecorina's carbon metabolism.
• This methodology significantly advances the genetic manipulation capabilities for H. jecorina research.