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Published on: October 2, 2012
Testosterone 15beta-hydroxylation by solvent tolerant Pseudomonas putida S12
Harald J Ruijssenaars1, Eric M G M Sperling, Peter H G Wiegerinck
1TNO Quality of Life, Julianalaan 67, 2628BC Delft, The Netherlands. harald.ruijssenaars@tno.nl
Journal of Biotechnology
|July 28, 2007
Summary
Engineered whole-cell biocatalysts enhance steroid 15beta-hydroxylation. Co-expression with Fer and CYP106A2 mutation improved testosterone hydroxylation and specificity for industrial applications.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Microbial Engineering
Background:
- Steroid biotransformation is crucial for pharmaceutical synthesis.
- Developing efficient and specific biocatalysts for steroid hydroxylation remains a challenge.
- Solvent-tolerant microorganisms offer advantages for industrial bioprocesses.
Purpose of the Study:
- To construct a solvent-tolerant whole-cell biocatalyst for steroid 15beta-hydroxylation.
- To enhance the activity and specificity of the biocatalyst.
- To enable the application of the biocatalyst in organic solvent systems.
Main Methods:
- Steroid hydroxylase CYP106A2 from Bacillus megaterium was expressed in solvent-tolerant Pseudomonas putida S12.
- Fer, an Fe-S protein from Bacillus subtilis, was co-expressed to enhance activity.
- Site-directed mutagenesis was employed to improve specificity.
Main Results:
- A 16-fold improvement in testosterone hydroxylation was achieved.
- Specificity for 15beta-hydroxylation was enhanced through T248V mutation in CYP106A2.
- The engineered biocatalyst demonstrated solvent tolerance.
Conclusions:
- An optimized whole-cell biocatalyst for steroid 15beta-hydroxylation was successfully developed.
- The engineered biocatalyst exhibits improved activity, specificity, and solvent tolerance.
- This work provides a foundation for industrial steroid biotransformation processes using organic solvents.
