Engineering Yarrowia lipolytica to express secretory invertase with strong FBA1_IN promoter

Abstract

Oleaginous yeast Yarrowia lipolytica is an important host for the production of lipidderived compounds or heterologous proteins. Selection of strong promoters and effective expression systems is critical for heterologous protein secretion. To search for a strong promoter in Y. lipolytica, activities of FBA1, TDH1 and GPM1 promoters were compared to that of TEF1 promoter by constructing GUS reporter fusions. The FBA1 promoter activity was 2.2 and 5.5 times stronger than the TDH1 and GPM1 promoters, respectively. The FBA1 IN promoter (FBA1 sequence of À826 to +169) containing an intron (+64 to +165) showed five-fold higher expression than the FBA1 promoter (À831 to À1). The transcriptional enhancement by the 5′-region within the FBA1 gene was confirmed by GPM1::FBA1 chimeric promoter construction. Using the strong FBA1 IN promoter, four different S. cerevisiae SUC2 expression cassettes were tested for the SUC + phenotype in Y. lipolytica. Functional invertase secretion was facilitated by the Xpr2 prepro-region with an additional 13 amino acids of mature Xpr2, or by the native Suc2 signal sequence. However, these two secretory signals in tandem, or the mature Suc2 with no secretory signal, did not direct secretion of functional invertase. Unlike previously reported Y. lipolytica SUC + strains, our engineered stains secreted most of invertase into the medium. Copyright

Snf1 Is a Regulator of Lipid Accumulation in Yarrowia lipolytica

“…This was then divided into six 25-ml cultures. After incubation for 48 h at 30°C at 250 rpm, a sample from the first time point (day 0) was taken for dry cell weight (DCW) and fatty acid analysis, as described previously (23)(24)(25). Briefly, a 1-ml aliquot was collected by centrifugation, and lipids were extracted.…”

“…Cells were grown in SD medium at a starting OD 600 of 0.1 and harvested at an OD 600 of 2.0 (exponential phase) or after 48 h (stationary phase) (23). RNA samples were prepared as described above.…”

“…We included lipid/energy metabolism-related genes that showed increased expression levels in the microarray analysis as well as the PCK1, FBP1, and ICL1 genes, whose expressions are known to be highly Snf1 dependent in S. cerevisiae (33). We harvested cells grown in SD medium under two different conditions: the exponential phase (OD 600 of 2.0) and the stationary phase (2 days of growth) in SD medium (23). The stationary-phase condition is comparable to cultures grown for 2 days in SD medium for the two-stage flask assays ( Fig.…”

Metabolic Engineering for Expanding the Substrate Range of Yarrowia lipolytica

“…SUC2 expression has also been used to produce citric acid from sucrose [19,34,35]. In a second approach, the stronger FBA1IN promoter was used to express and secrete invertase, via either the XPR2 or the native SUC2 signal peptide [36]. More recently, an improved SUC2 expression cassette was obtained using the strong TEF promoter.…”

Metabolic engineering of Yarrowia lipolytica for industrial applications

“…Wild type (WT) Y. lipolytica strains can use glucose, fructose, glycerol and hydrophobic substrates as carbon sources; strain Po1g can also use xylose and sugarcane bagasse hydrolysate [18]. In addition, Y. lipolytica strains have been engineered to use sucrose [19,20]. When both glucose and fructose are available, Y. lipolytica uses glucose first; over-expression of a hexokinase can help Y. lipolytica to effectively use fructose [21].…”