Biological production of optically active muconolactones by Rhodococcus rhodochrous.

Area of Science:

• Microbial metabolism
• Biocatalysis
• Organic synthesis

Background:

• Rhodococcus rhodochrous N75 metabolizes 4-methylcatechol via an ortho-cleavage pathway.
• A mutant strain, CJ30, was developed using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis.
• Strain CJ30 is specifically blocked in the degradation of 3-methyl-muconolactone.

Purpose of the Study:

• To biologically produce optically active (-)-3-methylmuconolactone.
• To investigate the biotransformation capabilities of the mutant strain CJ30.
• To confirm the structure of the produced methylmuconolactone.

Main Methods:

• Mutagenesis of Rhodococcus rhodochrous N75 using NTG to create strain CJ30.
• Cultivation of CJ30 on yeast extract and induction with p-toluate.
• Biotransformation of p-toluate and 4-alkylcatechols using whole cells and cell-free extracts.
• Structural confirmation using Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectrometry.

Main Results:

• Strain CJ30 transformed p-toluate (11.5 mM) to optically active (-)-3-methylmuconolactone with a 53% yield.
• The structure of 3-methylmuconolactone was confirmed via NMR and mass spectrometry.
• Cell-free extracts transformed various 4-alkylcatechols into corresponding 4-alkyl-substituted muconolactones.

Conclusions:

• Optically active (-)-3-methylmuconolactone can be efficiently produced using the engineered Rhodococcus rhodochrous CJ30 strain.
• The modified ortho-cleavage pathway in CJ30 allows for the accumulation of muconolactone derivatives.
• This microbial system offers a viable route for producing substituted muconolactones from alkylcatechols.