Engineered plants for the production of the antioxidants arbutin and gallate

Abstract

The shikimate pathway is a crucial metabolic route for the biosynthesis of numerous valuable chemicals. In this study, we engineered the shikimate pathway in plants via expression of microbial enzymes to produce the two important antioxidants gallate and arbutin. The engineered pathways utilize the aromatics protocatechuate and 4-hydroxybenzoate as metabolic intermediates. Through transient expression in Nicotiana benthamiana leaves, we first identified biosynthetic routes for the production of gallate from either chorismate or 3-dehydroshikimate. Gallate production was then achieved in Arabidopsis using a genetic background that overproduces protocatechuate and via expression of a mutated version of the 4-hydroxybenzoate hydroxylase PobA from Pseudomonas sp. Arbutin production was obtained in Arabidopsis using a genetic background that overproduces 4-hydroxybenzoate and via expression of the monooxygenase MNX1 from Candida parapsilosis. The best Arabidopsis transgenic lines accumulated gallate and arbutin in the range of 0.25 and 0.93 dry weight % (dwt%), respectively. Using sorghum for large-scale in planta production, the titers of gallate and arbutin produced from the intermediate 4-hydroxybenzoate reached 0.58 dwt% and 0.50 dwt%, respectively, in mature transgenic plants, surpassing levels typically observed in plants that naturally produce these compounds. Gallate and arbutin were readily extracted from plant tissues using methanol solvent. Analysis of extractive-free biomass showed only trace amounts of gallate and its precursors 4-hydroxybenzoate and protocatechuate crosslinked to cell walls, suggesting that they mainly occur as soluble conjugated forms stored in the vacuole. This study presents alternative synthesis routes using plant hosts for the eco-friendly production of gallate and arbutin.