This project aims to engineer Chlamydomonas reinhardtii to produce betalain pigments, using riboswitches to direct metabolic flux.
Betalain pigments are plant metabolites showing a wide variety of potential applications in the pharmaceutical, agricultural and food industry. Betalains are currently used as food and cosmetic colorants , and can act as potent antioxidant , anti-cancer [3, 4], anti-lipidemic [5, 6] and anti-microbial molecules . Certain intermediates of the betalain metabolic pathway also have pharmacological properties e.g. L-DOPA is used for the treatment of Parkinson’s disease . Given these benefits, it is important to further understand the biosynthetic processes leading to betalain production and accumulation in plants and how this knowledge can be used to engineer new industrial production hosts using model organisms like microalgae.
The betalain pathway consists of three main enzymatic steps starting with the molecule tyrosine (Fig 1) . The first step of the pathway displays an interesting enzymatic redundancy, where at least three cytochrome P450 proteins (CYP76AD1, 5 and 6) are able to perform the conversion of tyrosine to L-DOPA [10, 11]. CYP76AD1 can also catalyse the second reaction of the pathway, from L-DOPA to cyclo-DOPA, necessary for production of red coloration . One molecule of cyclo-DOPA can then spontaneously condense with a molecule of betalamic acid, product of the DODA gene in the third step of the pathway , to give the visible red pigments, betacyanins. Alternatively, betalamic acid can also condense with amino groups to form the fluorescent yellow pigments, betaxanthins.
Different CYP76AD enzymes are important for controlling the types of betalains produced and thus the resulting colour balance from pink to yellow via intermediate hues. Transient transformation of Nicotiana benthamiana with CYP76AD1 and the rest of the betalain biosynthetic machinery results predominantly in betacyanin production (red colour), whereas when CYP76AD6 is substituted for CYP76AD1, plants only produce betaxanthins (yellow colour) . Orange colours in betalain-pigmented species have been shown to result from the presence of both betacyanins and betaxanthins . Silencing of CYP76AD1 expression in beet has been shown to decrease betacyanin content and increase betaxanthins , indicating that modulating CYP76AD1 expression should influence the proportions of these two types of betalain pigments. Altogether, these findings suggest CYP76AD enzymes could have an important regulatory role in determining pigment composition and final coloration. The simplicity of the betalain pathway, the redundancy observed in the first step of betalain biosynthesis and the fact that betalamic acid acts as a shared chromophore for the two final pigment types, makes the betalain pathway an ideal system for visual characterisation of enzymes and their significance for metabolic flux towards either branches of this pathway.
We therefore propose to heterologously express the betalain biosynthetic pathway in Chlamydomonas reinhardtii with metabolic flux artificially regulated towards betacyanin or betaxanthin production. This regulation is possible by the use of characterised riboswitches that respond to exogenous supplementation of culture media with thiamine and hydroxyethyl thiazole (HET). Such riboswitch-driven regulation of the branch point enzymes will allow the ratio of cyclo-DOPA to betalamic acid to be “controlled” and thereby selectively shift the overall coloration produced towards the red or the yellow spectra respectively. The ultimate aim of this project is to engineer a strain of Chlamydomonas reinhardtii that not only will be able to produce betalain pigments but also will be susceptible to external fine tuning towards a desired color mix resulting in particular hues.
This project is due to report in 2018