New pAraDH2 Promoter for Metabolic Engineering of the Yarrowia lipolytica Yeast

Abstract

The ability to utilize a number of polyhydric alcohols as a sole carbon source has been studied in the Yarrowia lipolytica yeast. The efficiency of the promoter of the Y. lipolytica native AraDH2 gene encoding the enzyme D-mannitol/D-arabitol dehydrogenase was assessed during yeast growth on a minimal medium with different carbon sources. For this purpose, the promoter region of the AraDH2 gene was transcriptionally fused with the green fluorescent protein hrGFP gene, and the construct was used to transform Y. lipolytica. A β-carotene producing strain of Y. lipolytica was created using the described promoter; the strain carried the Mucor circinelloides CarRP and CarB genes encoding the bifunctional enzyme phytoene synthase/lycopene β-cyclase (CarRP) and phytoene dehydrogenase (CarB) as well as the GGPPSs7 gene Synechococcus sp. geranylgeranyl pyrophosphate synthase. The nucleotide sequence encoding the fused CarRP and GGPPSs7 under the regulation of the pAraDH2 promoter and the CarB gene under the control of the pTEF promoter were introduced into the yeast genome. As a result, a transformant was obtained capable of producing 66.3, 121.2, and 148.9 mg/L of β-carotene after 5 days of cultivation in test tubes on media containing glycerol, sucrose, and glucose, respectively. The obtained results testify to the high potential of the pAraDH2 promoter in the field of genetic engineering.