Efficient genome engineering in Mycolicibacterium neoaurum using Cas9 from Streptococcus thermophilus

Area of Science:

• Microbiology
• Biotechnology
• Synthetic Biology

Background:

• Non-pathogenic mycobacteria, like Mycolicibacterium neoaurum, naturally metabolize phytosterols for steroid production.
• M. neoaurum is a key organism for industrial synthesis of steroid medicine intermediates, such as Pregnadien-20-carboxylic acid (PDC).
• Targeted genetic modification of M. neoaurum can enhance yields of high-value steroid products.

Purpose of the Study:

• To develop a simple and efficient genetic engineering strategy for M. neoaurum.
• To create a customizable CRISPR/sth1Cas9 tool for precise genome editing in M. neoaurum.
• To facilitate the production of high-yield engineering strains for steroid intermediate synthesis.

Main Methods:

• Construction of a customizable plasmid tool (pMSC9) integrating sth1Cas9 protein and sgRNA scaffold.
• Design of editing plasmids with specific spacer sequences for targeted gene disruption.
• Transformation of M. neoaurum, induction of DNA double-strand breaks (DSBs) via CRISPR/sth1Cas9, and repair via NHEJ.
• Curing of editing plasmids and sequential gene deletion using a no-resistance cultivation method.

Main Results:

• Successful development and application of the CRISPR/sth1Cas9 system for M. neoaurum genome editing.
• Demonstration of rapid and targeted gene deletion in M. neoaurum strains.
• Creation of edited M. neoaurum mutants suitable for further genetic manipulation and strain improvement.

Conclusions:

• The CRISPR/sth1Cas9 tool provides a rapid and efficient method for editing M. neoaurum.
• This system significantly enhances the genetic engineering toolbox for M. neoaurum.
• The developed tool supports the advancement of industrial steroid production through improved microbial engineering.