Strategies to increase production of betalain pigments and their use as SynBio tools

Betalains are plant pigments showing a wide range of potential applications in the pharmacological, biotechnological and commodity sectors. They are currently being used as natural food colorants, and intermediates of the biosynthetic pathway exhibit important pharmacological properties (e.g. ʟ-DOPA). Due to this, there is an increasing interest in improving existing plant sources and obtaining new bio-industrial methods for semi-synthetic production. Betalain biosynthesis is achieved in plants via a relatively short metabolic pathway which has been successfully engineered in a diversity of heterologous hosts including plants, bacteria and yeast. Here, we explore a variety of mechanisms to utilise betalain pigments and enhance their bioindustrial production. First, we generated a betalain-based reporter system to visualise arbuscular mycorrhiza symbiosis processes in in Medicago truncatula and Nicotiana benthamiana roots. By expressing the betalain biosynthetic genes under plant symbiosis specific promoters, we were able to effectively restrict betalain production to areas of the root engaging with fungal colonisation. We also showed that this is an efficient tool for the dynamic tracing of root colonisation in vivo over time. This presents an advancement from the current tools used for the visualisation of fungal structures by the symbiosis research community and demonstrates the potential of the betalain pathway for the efficient reporting of other plant physiological processes. Secondly, we explored the existing natural variation in betalain biosynthetic enzymes in order to identify variants exhibiting higher activity than those currently known that could be used to improve betalain production. Initially we focused on the arogenate dehydrogenase (ADH) enzymes in the Caryophyllales, which show relaxed sensitivity to feedback inhibition by product and can produce higher titres of tyrosine, the substrate of the betalain pathway. We interrogated the ADH phylogeny of flowering plants to identify putative ADH homologs with relaxed sensitivity, and found that this phenomenon is not restricted to ADH enzymes of the Caryophyllales, and has likely also occurred repeatedly in other phylogenetically distant clades. Thirdly, we explored the utility of enzymatic diversity held within different origins of betalain pigmentation. Purification and analysis of different ʟ-DOPA 4,5-dioxygenase (DODA) enzymes from different inferred origins in the Caryophyllales revealed distinct kinetic properties and differences in overall pigment production performances in E. coli cultures. Scale-up experiments with the DODAα1 enzyme from Carnegiea gigantea (Cactaceae) in 2 L and 30 L bioreactors allowed for the biotechnological production of unsurpassed titres of betalains. Finally we used ancestral sequence reconstruction and enzyme activity assays in yeast to identify the residues important for high DODA activity in the Globular Inclusion clade, containing the Cactaceae. With this approach, we also confirm that the Globular Inclusion clade represents an independent origin of betalain pigmentation in the Caryophyllales, distinct from other known betalain-producing taxa such as Beta vulgaris.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Timoneda Monfort, Alfonso

Advisor dc:contributor.advisor

• Brockington, Samuel

• betalain
• metabolic engineering
• plant pigments
• synthetic biology

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.88225

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/340788