As the incidence of diseases, such as diabetes and obesity, increases, there is an increasing global demand for natural sweeteners to replace sugar. Steviol glycosides, compounds extracted from the herbal plant stevia rebaudiana Bertoni, are valuable in the food and beverage industry because they have intense sweetness and no calories. Recently, engineered Escherichia coli (E.coli) was developed to produce steviol glycosides. However, the conversion rate of ent-kaurenoic acid to steviol, which is aglycone of steviol glycosides, was low due to difficulty of functional expression of cytochrome P450 (P450) in E.coli. In this study, to enhance the biosynthesis of steviol in E. coli, engineering of the P450 proteins was carried out, and genomic editing technology was used, to improve NADPH availability. These enhancements produced approximately 4.3 times more steviol than a control in batch fermentation. In addition, the world’s first ent-kaurene biosynthesis pathway was inserted into the E.coli chromosome to reduce the metabolic burden from plasmids. This constructed strain can produce ent-kaurene without adding antibiotics and inducers. The results of this study can be used to develop biosynthetic strains not only steviol but also other P450-derived metabolites.
