Construction of a fusion enzyme for astaxanthin formation and its characterisation in microbial and plant hosts: A new tool for engineering ketocarotenoids

Metabolite channeling is a process that enables the efficient biosynthesis of natural products derived from both intermediary and specialised metabolism. The potential of metabolic engineering has, in many cases, been limited by the process of metabolite channeling and associated metabolons. The advent of Synthetic Biology offers new opportunities to create “new to nature” biosynthetic components for the efficient re-wiring of metabolism. In the present study, complementary enzyme activities have been combined through the creation of enzyme fusions in order to overcome leakage of non-endogenous intermediates and pleotropic effects associated with their high levels in plants. The formation of ketocarotenoids has been targeted, representing a branch of carotenoid formation that displays a vast array of diverse co-products. The utility of flexible linker sequences of varying size has been assessed in the construction of 3, 3ˈ -carotene hydroxylase (CRTZ) and 4, 4ˈ -carotene oxygenase (CRTW) enzyme fusions, both originating from the marine bacterium Brevundimonas sp. In vivo colour complementation assays in Escherichia coli have been used to evaluate the potential of the fusion enzymes. Analysis of the carotenoid pigments present in strains generated indicated that the enzyme fusions only possess both catalytic activities when CRTZ is attached as the N-terminal module. Pigment analysis during growth of the strains provided evidence that the fusion enzymes exhibited a reduced synthesis rate compared to individually expressed enzymes, while producing the same levels of the end product astaxanthin. In order to assess the potential of the fusions in a plant system, transient expression in Nicotiana benthamiana was performed. The production of valuable ketocarotenoids was achieved using this plant-based system. This revealed that CRTZ and CRTW, transiently expressed as a fusion, accumulated similar levels of astaxanthin compared to the expression of the individual enzymes, while associated with reduced ketocarotenoid intermediate levels (e.g. phoenicoxanthin, canthaxanthin and 3-OH-echinenone) and a reduced rate of leaf senescence after transformation. The size of the linkers appeared to have no effect upon activity. The potential of the approach to production of valuable plant derived products is discussed.