Biocatalytic Carboxylic Acid Reduction and Transamination in Cell-Free Lysates at High Substrate Loading
Madan R Gopal1, Nastassja M Corrado1, Wilfred Chen1
1Department of Chemical & Biomolecular Engineering, University of Delaware, Newark, Delaware, USA.
Chemsuschem
|July 21, 2026
Summary
This study presents a novel multienzyme cascade for chemoselective reduction of carboxylic acids. The engineered system enhances product yield and simplifies enzyme preparation for sustainable chemical synthesis.
Area of Science:
- Biocatalysis and Enzyme Engineering
- Synthetic Biology
- Sustainable Chemistry
Background:
- Carboxylic acid reductases (CARs) offer potential for reducing carboxylic acids but face challenges with chemoselectivity and yield, especially in crude enzyme preparations.
- Existing methods often require expensive cofactors and regeneration systems, limiting industrial applicability.
- Developing robust and efficient biocatalytic routes for functional group interconversion is crucial for sustainable chemical manufacturing.
Purpose of the Study:
- To develop a highly chemoselective multienzyme cascade for the reduction of carboxylic acids using crude enzyme lysates.
- To improve the yield and scalability of biocatalytic reductions by optimizing reaction conditions and enzyme stabilization.
- To demonstrate the versatility of the developed platform for synthesizing high-value amines from various carboxylate precursors.
Main Methods:
- Engineered a multienzyme cascade combining a carboxylic acid reductase (CAR) and an ω-transaminase (TA) in crude lysate.
- Utilized engineered Escherichia coli strains for enhanced aldehyde stabilization.
- Optimized reaction parameters, including the ratio of polyphosphate (polyP6) to Mg2+, to overcome substrate loading limitations.
- Tested the platform's portability across different carboxylate substrates.
Main Results:
- Achieved high chemoselectivity for para-xylylenediamine (pXDA) synthesis from terephthalic acid (TPA) using the engineered cascade.
- Overcame limitations in CAR activity at high substrate loadings by modulating polyP6/Mg2+ ratio, enabling substrate loading up to 50 mM TPA.
- Demonstrated successful synthesis of four additional high-value amines from carboxylate precursors.
- Reported high carboxyl group turnover, reaching up to 93.5 mM under tested conditions.
Conclusions:
- The developed multienzyme cascade in crude lysate format offers a promising and sustainable platform for functional group interconversion.
- The combination of improved chemoselectivity, yield, and simplified enzyme preparation addresses key challenges in industrial biocatalysis.
- This approach provides a versatile and scalable method for synthesizing valuable amines from carboxylic acids.
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