Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach

Abstract

The development of strong and tunable promoter elements is necessary to enable metabolic and pathway engineering applications for any host organism. Here, we have expanded and generalized a hybrid promoter approach to produce libraries of high-expressing, tunable promoters in the nonconventional yeast Yarrowia lipolytica. These synthetic promoters are comprised of two modular components: the enhancer element and the core promoter element. By exploiting this basic promoter architecture, we have overcome native expression limitations and provided a strategy for both increasing the native promoter capacity and producing libraries for tunable gene expression in a cellular system with ill-defined genetic tools. In doing so, this work has created the strongest promoters ever reported for Y. lipolytica. Furthermore, we have characterized these promoters at the single-cell level through the use of a developed fluorescence-based assay as well as at the transcriptional and whole-cell levels. The resulting promoter libraries exhibited a range of more than 400-fold in terms of mRNA levels, and the strongest promoters in this set had 8-fold-higher fluorescence levels than those of typically used endogenous promoters. These results suggest that promoters in Y. lipolytica are enhancer limited and that this limitation can be partially or fully alleviated through the addition of tandem copies of upstream activation sequences (UASs). Finally, this work illustrates that tandem copies of UAS regions can serve as synthetic transcriptional amplifiers that may be generically used to increase the expression levels of promoters.

Activating and Elucidating Metabolism of Complex Sugars in Yarrowia lipolytica

“…Plasmid pSL16-CEN1-1-227, containing the CYC1 terminator (T CYC1 ) and leucine selection marker, was kindly provided by M. Matsuoka (Sojo University, Japan) (35). Plasmid pSR001 was created by inserting the Y. lipolytica TEF promoter (P TEF ) (36) in front of T CYC1 of pSL16-CEN1-1-227 as described elsewhere (31). The list of primers used in this study is shown in Table S2.…”

Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production

“…Thus, we included the complementing of leucine and uracil biosynthetic capacity both singly and in tandem as targets for this multiplexing. Integrated expression cassettes were driven by our high-strength synthetic UAS1B 16 -TEF constitutive hybrid promoter 23 . Collectively, the combinatorial multiplexing of enzyme overexpressions, fatty-acid inhibition knockouts and other biosynthetic pathways resulted in 57 distinct genotypes that were analysed for lipogenesis capacity compared with the wild-type strain (Supplementary Table 1).…”

“…Primer sequences can be found in the Supplementary Table 4. All Y. lipolytica episomal plasmids were centromeric, replicative vectors derived from plasmid pSl16-Cen1-1(227) 50 after it had been modified to include a multi-cloning site, a hrGFP green fluorescent reporter gene (pIRES-hrGFP, Agilent) driven by the strong UAS1B 16 -TEF promoter 23 and a cyc1 terminator 51 to create plasmid pMCS-UAS1B 16 -TEF-hrGFP. Integrative plasmids were derived from plasmids pUC-S1-UAS1B 16 -Leum or pUC-S1-UAS1B 16 -TEF 25 that contained 5 0 and 3 0 rDNA integrative sequences surrounding the following elements-(from 5 0 to 3 0 ) a uracil section marker surrounded by LoxP sites for marker retrieval, the strong UAS1B 16 -Leum or UAS1B 16 -TEF promoter, AscI and PacI restriction enzyme sites for gene insertion and a XPR2 minimal terminator.…”

“…Yet, the genetic tractability of Y. lipolytica [18][19][20][21][22][23][24] coupled with its modest, innate de novo lipogenesis (B10-15% lipid content in wild type 13,14,[25][26][27] ) make it a potential candidate as a platform organism for superior lipid production.…”

Enhanced membrane protein expression by engineering increased intracellular membrane production

“…All work reported here is based on simple shake flask cultivation with singlecopy strains, thus, further strategies to enhance the yield of the strains developed in this study include optimal oleic acid feeding in controlled bio-fermentors, and exploring the effect of gene copy number increases. In addition, newer generations of stronger promoters for Yarrowia have recently been described [19,20] and will be useful to enhance the obtainable expression levels.…”