High yield anthocyanin biosynthesis in metabolic engineering Escherichia coli
Anthcoyanins, the largest sub-group of flavonoids and a major group of plant pigments, impart the eye-catching hues to most flowers and fruits ranging from pink through red and purple to dark blue. They are widely distributed in the human diet through crops, beans, fruits, vegetables and red wine and have been found to be present in significant amounts in plant-based daily diets. Anthocyanins have been given much attention because of their health promoting properties including anti-oxidative, anti-inflammatory, anti-cancer, anti-obesity, anti-diabetic, and cardioprotective. Due to their commercial value, high-yield anthocyanin biosynthesis has become a subject of great interest for metabolic engineering purposes, both in plants and microorganisms. In the first part of our work, we present therole of anthocyanin stability at various conditions as well as the cofactor contribution, the stability of the final product and intermediate-anthocyanidin, and cofactor UDP-glucose availability in anthocyanin production in Escherichia coli. Various optimization and metabolic engineering strategies including a new fermentation approach established in order to address the product stability issue, the creation of translational fusions in order to address the issue of the intermediate anthocyanidin instability, and rationally modifying the genotype and manipulating metabolic circuit in order to enhance the availability of cofactor UDP-glucose are presented. UDP-glucose is one of the most significant glucosyl donors in a variety of enzymatic reactions including the biosynthesis of simple or complex glucosides, oligosaccharides and polysaccharides, glycoproteins and related macromolecules. It is the starting point of the synthesis of other UDP-sugars, such as UDP-galactose and UDP-glucuronic acid. Although E. coli is capable of synthesizing UDP-glucose, a high regeneration rate of UDP-glucose does not normally occur in wide-type cells without significant metabolic engineering efforts. So, the genotype and metabolic network of host E. coli BL21* were rationally designed for the abundant accumulation of UDP-glucose, a key precursor in anthocyanin biosynthesis. Two methods for improvement of the carbon flux towards the UDP-glucose including overexpression of UDP-glucose synthesis by augmenting both pentose phosphate and nucleotide biosynthesis, and deletion of UDP-glucose competition pathway through gene knockout were adopted. Meanwhile, knockout mutants suggested by a silico model of genome-scale E. coli metabolic network were generated to further improve the intracellular UDP-glucose availability for efficient anthocyanin biosynthesis. With these efforts, we report here that the production of cyanidin 3- O -glucoside was increased to 113 mg/L from its precursor flavan-3-ol (+)-catechin without supplementing UDP-glucose in the fermentation medium. These results demonstrate the efficient production of the core anthocyanins for the first time from a microorganism and built the platform for their commercialization for pharmaceutical and nutraceutical applications. Meanwhile, the metabolic engineering UDP-glucose biosynthesis strategies will have an extended impact in the biosynthesis of various glycosylated natural products such as antibiotics.