Metabolic engineering of plant cells for biotransformation of hesperedin into neohesperidin, a substrate for production of the low-calorie sweetener and flavor enhancer NHDC.

yNeohespericlin dihydrochalcone (NHDC) is a serninatural, safe, low-calorie sweetener, bitterness blocker, and flavor enhancer with unique properties and applications for the food, beverage, pharmaceutical, and animal feed industries. Current production is limited by the availability of the substrate neohesperidin, a flavonoid that accumulates to significant levels only in the inedible bitter citrus species. We propose a process to convert hesperidin, a tasteless flavonoid extracted from orange peels that are abundant byproducts of the vast orange juice industry, into neohesperidin using metabolic engineering and biotransformation via three steps: (i) extraction of hesperidin from orange peels, (ii) hydrolysis of sugar moieties, and (iii) biotransformation of hesperidin hydrolysis products into neohesperidin. We overcame the current technological bottleneck in biotransformation of hesperidin hydrolysis products into neohesperidin using metabolically engineered plant cell cultures expressing a recombinant flavanone-7-O-Lglucoside-2-O-Lrhamnosyltransferase. A small-scale production experiment established the feasibility of the proposed process.