Construction of engineered Arthrobacter simplex with improved performance for cortisone acetate biotransformation

Area of Science:

• Microbiology
• Biotechnology
• Genetic Engineering

Background:

• Arthrobacter simplex 156 is utilized for steroid biotransformation, converting cortisone acetate (CA) to prednisone acetate (PA).
• The ksdD gene, encoding 3-ketosteroid-△(1)-dehydrogenase, is crucial for this bioconversion efficiency.

Purpose of the Study:

• To develop a genetic manipulation system for A. simplex 156 to enhance biotransformation efficiency.
• To create recombinant strains with improved CA to PA conversion rates.

Main Methods:

• Introduction of additional copies of the ksdD gene into A. simplex 156 under the cat promoter.
• Integration of the modified gene into 16S rDNA sites, creating recombinant strains.
• Analysis of recombinant strain properties, including biotransformation efficiency and genetic stability.

Main Results:

• A recombinant strain, A. simplex M158, demonstrated superior CA biotransformation capabilities.
• PA production reached 66.7 g/l (32.9% higher than wild-type) at 83.6 g/l CA substrate concentration.
• Bioconversion time was reduced by 20 hours, and the strain showed genetic stability over 100 generations without antibiotic selection.

Conclusions:

• The engineered A. simplex M158 strain significantly improves prednisone acetate production efficiency.
• Disruption of 16S rRNA operons for gene integration did not impede microbial growth or stability.
• The developed genetic system and recombinant strain are highly suitable for industrial-scale PA production.