iCatch: a new strategy for capturing large DNA fragments using homing endonucleases.

Area of Science:

• Synthetic biology
• Molecular biology
• Genomics

Background:

• Natural genetic materials harbor valuable biosynthetic gene clusters for natural products.
• Synthetic biology relies on DNA assembly standards for reusing genetic materials.
• The iBrick standard facilitates manipulation of large DNA fragments but faces cloning challenges.

Purpose of the Study:

• To develop a novel method for efficiently cloning large biosynthetic gene clusters.
• To create a method compatible with the iBrick DNA assembly standard.
• To enable heterologous expression of microbial natural products.

Main Methods:

• The iCatch method utilizes natural homologous recombination.
• It flanks target gene clusters with specific endonuclease recognition sites (I-SceI and PI-PspI).
• Genome digestion and self-ligation are employed to capture DNA fragments.

Main Results:

• Successfully cloned the actinorhodin biosynthetic cluster from Streptomyces coelicolor.
• Heterologously expressed the cloned cluster in a thermophilic Streptomyces strain.
• Demonstrated iCatch's capability for cloning large DNA sequences (tens of kilobases).

Conclusions:

• iCatch is an effective method for capturing large DNA fragments, particularly biosynthetic gene clusters.
• The cloned fragments are compatible with the iBrick standard for further manipulation.
• This method advances the heterologous expression of microbial natural products and complements large DNA fragment manipulation in synthetic biology.