Modifying the potato tuber storage protein patatin targeting improved thermal stability

Area of Science:

• Plant molecular biology
• Biochemistry
• Protein engineering

Background:

• Patatins are nutritious potato proteins with functional properties, but their low thermostability hinders industrial purification.
• Current patatin purification methods face challenges, especially under acidic conditions, limiting their large-scale application.
• Stabilizing patatin structure via point mutations is a potential strategy to enhance extraction and usability.

Purpose of the Study:

• To assess the suitability of the patatin gene cluster for CRISPR/Cas9 mutagenesis.
• To engineer patatin variants with improved thermostability for industrial applications.
• To investigate the impact of specific mutations on patatin protein properties.

Main Methods:

• CRISPR/Cas9-based mutagenesis targeting the patatin gene cluster with a single-guide RNA.
• In silico analysis of patatin protein structure to design amino acid substitutions.
• Heterologous expression of engineered patatin variants in bacteria.
• Evaluation of mutant patatin thermostability and pH sensitivity.

Main Results:

• CRISPR/Cas9 targeting with a single sgRNA resulted in mutations in over 10% of patatin alleles.
• Four patatin variants with designed amino acid substitutions were generated.
• None of the engineered variants exhibited superior thermostability compared to wild-type patatin.
• One mutant variant showed reduced sensitivity to pH variations, indicating potential for further optimization.

Conclusions:

• CRISPR/Cas9 is effective for gene editing the patatin gene cluster, achieving significant allele modification.
• Engineered patatin variants did not show enhanced thermostability, but one variant displayed improved pH tolerance.
• Further protein engineering efforts are warranted to optimize patatin for industrial applications, focusing on pH stability.