A Programmable CRISPR/Cas9 Toolkit Improves Lycopene Production in Bacillus subtilis.

Area of Science:

• Synthetic Biology
• Microbial Engineering
• Biotechnology

Background:

• Bacillus subtilis is a safe and widely used host for producing recombinant proteins and chemicals.
• Metabolic engineering of B. subtilis is crucial but hindered by limited selective markers, especially for complex pathways.
• Efficient genetic tools are needed to overcome these limitations in B. subtilis.

Purpose of the Study:

• To develop an easy-to-use CRISPR/Cas9-based cloning toolkit for B. subtilis metabolic engineering.
• To address challenges in chromosomal integration, promoter selection, terminator function, and guide RNA targeting.
• To demonstrate the toolkit's utility in optimizing the production of industrially relevant compounds.

Main Methods:

• Utilized CRISPR/Cas9 technology to construct a versatile cloning toolkit for B. subtilis.
• Characterized six promoters (0.9- to 23-fold P43 strength) and seven terminators (>90% efficiency).
• Designed six guide RNA targets and confirmed up to 100% integration efficiency using a GFP reporter.

Main Results:

• Developed a toolkit enabling rapid cloning and one-step subcloning for stable chromosome integration in B. subtilis.
• Demonstrated successful optimization of lycopene production by manipulating 13 key genes in the biosynthetic pathway.
• Identified specific gene clusters (ispG-idi-dxs-ispD positive, dxr-ispE-ispF-ispH negative) impacting lycopene yield.

Conclusions:

• The developed toolkit significantly facilitates pathway assembly and gene expression in B. subtilis.
• This strategy enables rapid engineering of B. subtilis strains for complex chemical and pharmaceutical production.
• The toolkit enhances the industrial application potential of B. subtilis as a biological chassis.