Abstract
Limonene is a volatile monoterpene compound that is widely used in food additives, pharmaceutical products, fragrances, and toiletries. We herein attempted to perform efficient biosynthesis of limonene in Saccharomyces cerevisiae using systematic metabolic engineering strategies. First, we conducted de novo synthesis of limonene in S. cerevisiae and achieved a titer of 46.96 mg/L. Next, by dynamic inhibition of the competitive bypass of key metabolic branches regulated by ERG20 and optimization of the copy number of tLimS, a greater proportion of the metabolic flow was directed toward limonene synthesis, achieving a titer of 640.87 mg/L. Subsequently, we enhanced the acetyl-CoA and NADPH supply, which increased the limonene titer to 1097.43 mg/L. Then, we reconstructed the limonene synthesis pathway in the mitochondria. Dual regulation of cytoplasmic and mitochondrial metabolism further increased the limonene titer to 1586 mg/L. After optimization of the process of fed-batch fermentation, the limonene titer reached 2.63 g/L, the highest ever reported in S. cerevisiae.
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