Tandem-yeast expression system for engineering and producing unspecific peroxygenase.

Area of Science:

• Biotechnology
• Enzymology
• Molecular Biology

Background:

• Unspecific peroxygenase (UPO) is a versatile biocatalyst with significant biotechnological potential.
• Limitations in heterologous expression systems have hindered industrial UPO applications.
• Previous work evolved UPO from Agrocybe aegerita for secretion in Saccharomyces cerevisiae.

Purpose of the Study:

• To develop a tandem-yeast expression system for UPO engineering and large-scale production.
• To leverage directed evolution for improved UPO secretion and activity.
• To facilitate industrial utilization of UPO biocatalysts.

Main Methods:

• Utilized directed evolution in Saccharomyces cerevisiae to identify beneficial mutations for UPO secretion.
• Established a tandem-yeast system transferring evolved UPO genes to Pichia pastoris.
• Employed the methanol-inducible alcohol oxidase 1 promoter for UPO expression in P. pastoris.
• Compared UPO production in flask and fed-batch fermentations.

Main Results:

• Achieved similar secretion levels (∼8mg/L) in flask fermentations for both yeasts.
• Demonstrated a 27-fold enhanced production of recombinant UPO in Pichia pastoris fed-batch fermentation (217mg/L).
• Confirmed that the P. pastoris UPO variant retained biochemical properties similar to the S. cerevisiae counterpart.

Conclusions:

• The tandem-yeast expression system is effective for engineering and large-scale production of UPO.
• This system overcomes previous limitations, paving the way for industrial UPO applications.
• The developed system supports future engineering and industrial deployment of UPO biocatalysts.