One Step Synthesis of L-DOPA

Background and Unmet Need

The use of L-DOPA by itself and in combination with other drugs for treatment in Parkinson’s disease is a multibillion-dollar market, especially with a growing elderly population worldwide. Furthermore, L-DOPA has significance as a chemical precursor used to synthesize other high-value products such as Opiates (e.g., morphine) and Catecholamines (e.g., dopamine).
Chemical and biochemical methods for L-DOPA production require either toxic starting reagents or produce toxic byproducts. Moreover, chemical-based syntheses are complicated and need multiple steps, limiting overall yield due to the loss of product at each step.
Smaller-scale biochemical production involves the direct use of recombinant enzymes for the direct conversion of Tyrosine to L-DOPA. However, currently available enzymes also react with L-DOPA converting it into different side products, reducing the yield.
Therefore there is a need to synthesize L-DOPA in a non-toxic straightforward procedure without producing side products.

The Solution

Prof. Asaph Aharoni’s group discovered a new enzyme called CYP76AD6, which has a unique capacity to react with Tyrosine and exclusively produce L-DOPA in a single step1.

Technology Essence

The present technology takes advantage of a newly discovered and highly selective Cytochrome P450, CYP76AD6. Tyrosinases or other Cytochromes that synthesize L-DOPA also perform side reactions that generate undesired products, such as dopaquinone or cycloDOPA, whereas CYP76AD6 specifically hydroxylates Tyrosine in a single step to L-DOPA, without side products.
The enzyme CYP76AD6 can be expressed and is functional both within plants and yeast. Further, CYP76AD6 can be produced as a recombinant protein in E. coli to synthesize L-DOPA both inside the cell and in vitro reactions.
Subsequently, CYP76AD6 creates a “Green” chemistry alternative as the only starting material required is the natural amino acid Tyrosine, and the only byproduct is L-DOPA.

Applications and Advantages

• One-step reaction for L-DOPA synthesis from Tyrosine
• Non-toxic and non-hazardous synthesis
• Flexibility in biosynthetic production - multiple possible host systems

Development Status

The team of Prof. Asaph Aharoni identified CYP76AD6 as a novel Cytochrome P450 using transcriptome analysis of the plants red beet (Beta vulgaris) and four o’clocks (Mirabilis jalapa). They further expressed it recombinantly in Nicotiana benthamiana and yeast cells to demonstrate its functionality.

References

Polturak G, Breitel D, Grossman N, et al. Elucidation of the first committed step in betalain biosynthesis enables the heterologous engineering of betalain pigments in plants. New Phytol. 2016;210(1):269-283. doi:10.1111/nph.13796