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Biosynthesis of the Calorie-Free Sweetener Precursor ent-Kaurenoic Acid from CO2 Using Engineered Cyanobacteria.

To supply the sustainable calorie-free sweetener stevioside, synthetic photosynthetic bacteria were developed to produce ent-kaurenoic acid as a precursor of stevioside directly from CO2. By the use of a combinatorial and modular approach for gene expression, including a cytochrome P450 and the corresponding reductase, engineered Synechoccous elongatus PCC 7942 as a model cyanobacterium enabled the biosynthesis of ent-kaurenoic acid at 2.9 ± 0.01 mg L-1 from CO2. We found that the order of genes for expression was critical, producing ent-kaurenoic acid by balancing gene expressions and accumulation of the toxic intermediate in a cell. The engineered bacteria allowed the complete biosynthesis of ent-kaurenoic acid, and it will be used for stevioside biosynthesis from CO2 as a controlled fermentation.