Connecting Carbon: Two-enzyme cascade to produce pharmaceutically interesting para-coumaric acids

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Welcome to the first of two articles about “Connection Carbon“

Carbon-carbon bonds are the basis for all vital organic molecules, which makes them attractive building blocks for e.g. pharmaceuticals, fragrances, flavors and many others. The key to an efficient and green production thereof are specific enzymes.

Carbon-carbon bonds represent the backbone of vital organic molecules, including the fatty acids or amino acids in our body or the sugars that keep us moving and our brain working. Nature builds these complex frameworks by connecting smaller building blocks using its most powerful catalysts, the enzymes. But how can enzymes help us to satisfy the worldwide need for carbon-based compounds, such as pharmaceuticals, dietary supplements, fragrances or flavors?

Growing ecological awareness and the need for sustainable or- “greener”- synthetic processes more and more turns attention towards biocatalysis. Using highly selective enzymatic systems instead of chemical catalysts for the production of valuable chemical compounds is extensively studied at the Austrian Centre of Industrial Biotechnology.

Two enzymes, operating simultaneously in a single cell

acib-researchers at the University of Graz recently published a concise two-enzyme cascade for the synthesis of pharmaceutically interesting para-coumaric acid derivatives with excellent positional selectivity and atom economy (82-86%). To access these valuable products, they combine tyrosine phenol lyase (TPL) with tyrosine ammonia lyase (TAL), both enzymes naturally belonging to the metabolic pathway of the amino acid L-tyrosine. The cascade is designed in a way that TPL and TAL operate simultaneously as whole-cell catalysts, which means that in the overall process, the target product can be synthesized without intermediary protection/deprotection steps as often required in classical chemical synthesis.

In more detail, TPL catalyzes the carbon-carbon coupling of simple phenols with pyruvate and ammonia in the first step and the intermediary formed L-tyrosines are subsequently transformed by TAL to the desired para-coumaric acid derivatives via ammonia elimination. Since ammonia is reused by TPL in the next catalytic cycle, water is formed as sole by-product. The optimized process makes use of single enzyme preparations, however the cascade also works with co-expressed enzymes. acib’s scientists at the TU-Graz designed an expression construct for these cascade enzymes that allows for the co-expression of TPL and TAL in a single cell, instead of producing them in two separate host systems. Designing such multi-enzyme constructs is generally challenging and may lead to reduced activities of the involved catalysts. Its realization, however simplifies production and handling of the catalysts as only a single catalyst preparation is required to carry out multiple reaction steps.

This work is based on:
E. Busto, M. Gerstmann, F. Tobola, E. Dittmann, B. Wiltschi, W. Kroutil: Systems biocatalysis: para-alkenylation of unprotected phenols. 2016 Catalysis Science & Technology, DOI 10.1039/C6CY01947A

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