Enhanced 3-methylcatechol production by Pseudomonas putida TODE1 in a two-phase biotransformation system

Area of Science:

• Biotechnology
• Biocatalysis
• Metabolic Engineering

Background:

• 3-methylcatechol (3MC) is a valuable chemical intermediate.
• Bioproduction of 3MC from toluene is challenging due to low yields and cell toxicity.
• Pseudomonas putida TODE1 is a genetically engineered strain for toluene biotransformation.

Purpose of the Study:

• To optimize the bioproduction of 3-methylcatechol (3MC) from toluene using engineered Pseudomonas putida TODE1.
• To enhance biocatalyst efficiency and 3MC yield in an aqueous-organic two-phase system.
• To identify optimal conditions including organic solvents, media supplements, and cofactor regeneration.

Main Methods:

• Utilized an aqueous-organic two-phase system with engineered Pseudomonas putida TODE1.
• Screened various organic solvents, with n-decanol showing superior 3MC partitioning and low toxicity.
• Evaluated media supplementation with carbon sources, metal cations (Fe2+), and short-chain alcohols for cofactor regeneration.

Main Results:

• N-decanol was identified as the optimal organic solvent for 3MC partitioning and yield.
• Supplementation with glycerol (4 mM) and Fe2+ (0.4 mM) significantly enhanced toluene dioxygenase (TDO) activity and 3MC production.
• A maximum 3MC concentration of 31.8 mM (166 mM in organic phase) was achieved in small-scale production, and 160.5 mM (333.2 mM in organic phase) in 2-L scale.

Conclusions:

• The optimized bioproduction system using Pseudomonas putida TODE1, n-decanol, glycerol, and Fe2+ represents a significant advancement in 3MC synthesis.
• This study reports the highest 3MC yield achieved to date using a toluene dioxygenase-based biocatalytic system.
• The developed method offers a highly efficient and scalable approach for the industrial production of 3-methylcatechol.