A Two-Phase Enzymatic Cascade for Indigo Synthesis from Anthranilate.

Area of Science:

• Biotechnology
• Synthetic Biology
• Biocatalysis

Background:

• Current indigo dye synthesis relies on petroleum-based feedstocks.
• There is a growing demand for sustainable and bio-based chemical production methods.
• Whole-cell approaches for indigo synthesis face challenges with host toxicity.

Purpose of the Study:

• To develop an enzymatic in vitro synthesis route for indigo dye.
• To establish a five-step enzymatic pathway derived from natural biosynthesis.
• To explore a bio-based alternative to current indigo synthesis methods.

Main Methods:

• Enzymatic assessment by stepwise addition to ensure cascade function.
• Design of a two-phase system accommodating different enzyme melting temperatures.
• Stepwise parameter optimization and process engineering for titer enhancement.

Main Results:

• An indigo titer of 183 mg/L was achieved through optimization.
• Continuous supplementation of phosphoribosyl pyrophosphate was required for indole-3-glycerol phosphate (IGP) formation.
• Challenges were observed in complete IGP uptake and significant byproduct formation in the second phase.

Conclusions:

• An enzymatic cascade using bio-based materials is a potential alternative for sustainable indigo synthesis.
• This method circumvents host toxicity issues associated with whole-cell approaches.
• Further improvements in yield are necessary for economic viability.