A novel integrative expression vector for Sulfolobus species

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Generating stable microbial strains is crucial for genetic studies and biotechnology.
• Sulfolobus acidocaldarius is an important model organism in archaeal research.

Purpose of the Study:

• To construct a novel shuttle vector, pINEX, for efficient gene integration into the Sulfolobus acidocaldarius chromosome.
• To demonstrate the utility of pINEX by generating a stable alpha-glucosidase-producing strain.

Main Methods:

• Construction of the pINEX shuttle vector with selection markers and recombination sites.
• Gene cloning and ligation of the malA gene into pINEX.
• Chromosomal integration using the "pop-in" and "pop-out" method.
• Verification of gene insertion via colony PCR and sequencing.
• Purification and activity assay of the expressed protein.

Main Results:

• The pINEX vector was successfully constructed and validated.
• A stable Sulfolobus acidocaldarius strain expressing alpha-glucosidase was generated.
• The expressed alpha-glucosidase activity was confirmed.

Conclusions:

• The pINEX vector is a valuable tool for stable gene integration and functional gene analysis in Sulfolobus acidocaldarius.
• This system facilitates the development of engineered microbial strains for research and industrial applications.