Codon optimization increases steady-state mRNA levels in Aspergillus oryzae heterologous gene expression

Area of Science:

• Biotechnology
• Molecular Biology
• Microbial Engineering

Background:

• Heterologous protein expression in Aspergillus oryzae is crucial for industrial applications.
• Native genes often exhibit suboptimal expression due to codon bias and regulatory elements.
• Mite allergen Der f 7 serves as a model for studying protein expression challenges.

Purpose of the Study:

• To evaluate the impact of codon optimization on Der f 7 expression in A. oryzae.
• To investigate the mechanisms behind increased protein and mRNA levels post-optimization.
• To address issues of aberrant mRNA production and degradation.

Main Methods:

• Codon optimization of the Der f 7 gene using recursive PCR based on A. oryzae codon usage frequency.
• Expression of native and optimized Der f 7 genes, both standalone and fused with glucoamylase (GlaA).
• Analysis of mRNA levels and integrity using 3' rapid amplification of cDNA ends (RACE).

Main Results:

• Codon optimization markedly increased both protein and mRNA production for Der f 7.
• Native constructs showed aberrant mRNA formation due to premature polyadenylation within the coding region.
• Addition of a termination codon in fusion constructs improved mRNA and secreted protein levels.

Conclusions:

• Codon optimization enhances heterologous protein expression in A. oryzae by mitigating cryptic polyadenylation signals.
• This strategy prevents the generation of truncated transcripts, leading to higher steady-state mRNA levels.
• Codon optimization is a valuable tool for improving recombinant protein yields in filamentous fungi.