Resolving the molecular basis underlying convergent evolution of DODA enzymes in the betalain synthesis pathway

Project Information

ConvergentDODA

Grant agreement ID: 101030560

DOI

10.3030/101030560

Project closed

EC signature date 16 April 2021

Start date 1 June 2021

End date 31 May 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 212 933,76

EU contribution

€ 212 933,76

Coordinated by

THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE
United Kingdom

Periodic Reporting for period 1 - ConvergentDODA (Resolving the molecular basis underlying convergent evolution of DODA enzymes in the betalain synthesis pathway)

Reporting period: 2021-06-01 to 2023-05-31

Summary of the context and overall objectives of the project

Betalains are pigments found in plants belonging to the order Caryophyllales where they replace anthocyanins in most families, except for 6 families that produce anthocyanins. Betalains and anthocyanins are mutually exclusive as plants are able to produce betalains or anthocyanins but any plant producing both types of pigments has been described. The phylogenetic distribution of betalains and anthocyanin within Caryophyllales indicates multiple origins of betalains within Caryophyllales, from anthocyanic ancestors.

The betalain biosynthesis pathway is led by 4,5-L-DOPA dioxygenase (DODA), the enzyme that catalyzes the ring cleavage of L-DOPA to yield betalamic acid, the chromophoric and structural unit of betalains. The distribution of betalain-producing families across Caryophyllales has been linked to the convergent evolution of DODA. Thus, there is evidence that betalain-producing plants do not share a common ancestor able to produce betalains but they have evolved independently 3-4 times.

This project aims to understand the molecular modifications that DODA enzymes have experienced through their evolution and how they are related to the betalain-producing capacity detected in Caryophyllales plants. Studying DODA as a symbol of convergent evolution allows us to understand the molecular bases underlying the different approaches that organisms pursue to address similar evolutionary problems.

The overall objectives are:
- Biochemical characterisation of high-activity DODA enzymes
- Characterisation of the ancestral biochemical function of the DODA enzymes
- Identification of candidate residues for high DODA activity using ancestral sequence reconstruction
- Validation of candidate residues using a high-throughput yeast heterologous expression system
- Reconstruction of the evolutionary history of essential residues to understand evolutionary mechanisms for each origin of DODA

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The biochemical characterization of DODA enzymes that belongs to the different origins proposed is performed through their expression in Escherichia coli as heterologous host system. Thus, the subsequent purification of tagged DODA proteins and their in vitro characterization has shown that their molecular differences have had an impact in their performance in terms of stability, oligomeric status, and kinetics. Thus, the affinity of these molecules toward the substrate L-DOPA seems to be related to the origin of the enzymes.

An extensive collection of DODAa coding sequences across Caryophyllales species are used in this project to reconstruct the ancestral trait of betalain-producing Caryophyllales plants. It has allowed us to perform an ancestral reconstruction in the system model Saccharomyces cerevisiae. The expression of DODA genes in yeast has confirmed their independent evolution within Caryophyllales showing that the production of betalains is an acquired ability through the evolution of these enzymes. Thus, the betalain-producing capacities are related to neofunctionalization processes associated with independent duplications among the three lineages detected. The comparison of sequences throughout the different DODA lineages has also allowed the detection of candidate residues responsible for the gain of function. Those residues were individually mutated and the expression of these new sequences in yeast has shown their implication in the production of betalains.

The satisfactory results have been disseminated in the following activities:
a. Results shared with the scientific community:
- International congresses
- Peer-reviewed research articles
- Presentation of results in the lunchtime seminar of the Department of Plant Sciences at the University of Cambridge (United Kingdom)

b. Results communicated to different target public audiences:
- Science on Sundays of the Cambridge University Botanic Garden (Cambridge, United Kingdom)
- Festival of plants of the Cambridge University Botanic Garden

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The study of DODA enzymes and their implication in the betalain synthesis pathway has never been assessed at this level. For the first time, the evolutionary trajectories of high-activity DODA enzymes have been addressed in an interdisciplinary project that combines techniques of molecular biology, biochemistry, and molecular evolution, with an outlook to enhanced industrial approaches. This ambitious project is possible thanks to the unparalleled phylotranscriptomic resources for over 400 species that have been employed to reconstruct the evolutionary trait of Caryophyllales plants.
The expected results of this project will have remarkable importance in the study of convergent evolution at the molecular level. Additionally, the results obtained from the kinetic characterisation of DODA enzymes have helped to find enzymes with extraordinary capacities in the obtention of betalains in biofactories. Thus, these betalains will be applicable in food, cosmetic or pharmaceutical industries due to their health-promoting properties.

Summary of the project