Genetic engineering of the primary/secondary metabolic interface in tobacco BY-2 cells

Thesis

The supply of precursors from primary metabolism is often overlooked when engineering secondary metabolism for increased product yields. This is because precursor supply may be assumed to be non-limiting, and it is considered difficult to engineer primary metabolism, because control of carbon flow (flux) is generally distributed among most enzymes of the pathway.

The aim of this thesis was to increase the production of sterols, part of the isoprenoid class of secondary metabolites, in tobacco (Nicotiana tabacum) Bright Yellow 2 (BY-2) cell cultures. This was achieved by genetically engineering increased activity of mitochondrial citrate synthase, an enzyme of the tricarboxylic acid (TCA) cycle that is involved in the provision of cytosolic acetyl coenzyme A, the primary metabolite precursor to sterols. Metabolic flux analysis revealed that citrate synthase exerts significant control over cyclic TCA cycle flux in BY-2 cells and suggested that increasing the activity of downstream enzymes within secondary metabolism could lead to a further redirection of TCA-cycle-derived precursors into sterol biosynthesis. Attempts were made to achieve this by genetically engineering increased activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), a key enzyme of secondary metabolism involved in sterol biosynthesis. Consistent with previous research, transgenic lines had increased sterol levels. However, the high sterol phenotype was unstable, and attempts to co-express HMGR and citrate synthase genes were unsuccessful.

The thesis demonstrates that increasing the provision of precursors to secondary metabolites can result in increased yields of those secondary metabolites but suggests that in most cases the activity of enzymes within secondary metabolism has a greater effect on those yields. It also reveals that single enzymes can exert significant control of flux within primary metabolism, although the control exerted by specific enzymes probably changes with the demands placed on metabolism.

Authors: Hall-Ponselè, A

• Publication date: 2014
• DOI: 10.5287/ora-b7b08n5kk
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: Oxford University, UK
• Language: English
• Keywords: Metabolic Engineering; Tobacco; Metabolomics; Tobacco BY-2 cells; Secondary Metabolism; Metabolic Flux Analysis; HMGR; 13C-FLUX; Nicotiana tabacum; Plant Cell Culture; Citrate Synthase; Isoprenoid Biosynthesis; Phytosterols; Suspension Cultures; GC-MS; 13C MFA; TCA cycle
• Subjects: Metabolism; Biochemistry; Life Sciences; Plant Sciences; Biology