Optimisation of isoprenoid formation in plants.

Edward Wake

Research output: ThesisDoctoral Thesis

Abstract

Isoprenoids are a highly diverse group of secondary metabolites in plants. Carotenoids are a
class of isoprenoids with health promoting and essential nutritional properties and are valuable
compounds for industrial applications. The production of these molecules in plants has attracted
attention as an alternative to chemical synthesis.
To improve understanding of the regulatory processes governing carotenoid biosynthe-
sis, transgenic tomato (Solanum lycopersicum) lines, overexpressing key pathway enzymes, were
characterised. Tomatoes are typically high in carotenoids. The natural tomato “rr ”-mutant
which lacks a functional fruit phytoene synthase 1 (PSY1, the first committed step in fruit
carotenoid biosynthesis), is a useful tool for such studies. Here, transgenic lines of the rr -mutant,
overexpressing either Psy1 or the green tissue Psy2, were characterised at the molecular and bio-
chemical level, revealing key differences in their regulation. Plastid lipidome quantification also
highlighted the close association of lipid and carotenoid accumulation.
To enhance carotenoid accumulation in tomatoes understanding the contribution of
upstream regulatory processes is required. 1-Deoxy-D-xylulose-5-phosphate synthase (DXS) reg-
ulates the supply of prenyl-lipid precursors (geranylgeranyl pyrophosphate (GGPP)). Therefore,
in this work, E. coli DXS was overexpressed alongside SlPsy1. No additive effects were ob-
served in this line due to non-simultaneous expression/effects. Furthermore, a 28-fold EcDXS
expression, compared to Actin (housekeeping gene), was unable to confer significant increases
in total carotenoids. This suggests conserved, post-translational regulation of the bacterial and
endogenous DXS. Prenyl lipid analysis also revealed the presence of novel GGPP isomers in the
transgenic lines.
The expression of a synthetic enzyme fusion between GGPP synthase (GGPPS) and
PSY was attempted in industrially and nutritionally relevant chassis but the fusion was found
to be only partially functional.
This work has provided novel insights into the differential regulation of PSY1 and PSY2
in fruit, whilst highlighting the importance of both the lipidome and, potentially, GGPP isomers
in carotenoid biosynthesis.
Original languageEnglish
QualificationPh.D.
Awarding Institution
  • Royal Holloway, University of London
Supervisors/Advisors
  • Fraser, Paul, Supervisor
Thesis sponsors
Award date1 Jun 2024
Publication statusUnpublished - 2024

Keywords

  • Tomato
  • Solanum lycopersicum
  • Biotechnology
  • Carotenoids
  • Synthetic Biology
  • Molecular Biology

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