title: Using a vector pool containing variable-strength promoters to optimize protein production in Yarrowia lipolytica.
abstract: BACKGROUND
  The yeast Yarrowia lipolytica is an increasingly common biofactory. To enhance protein expression, several promoters have been developed, including the constitutive TEF promoter, the inducible POX2 promotor, and the hybrid hp4d promoter. Recently, new hp4d-inspired promoters have been created that couple various numbers of UAS1 tandem elements with the minimal LEU2 promoter or the TEF promoter. Three different protein-secretion signaling sequences can be used: preLip2, preXpr2, and preSuc2.
  
  
  RESULTS
  To our knowledge, our study is the first to use a set of vectors with promoters of variable strength to produce proteins of industrial interest. We used the more conventional TEF and hp4d promoters along with five new hybrid promoters: 2UAS1-pTEF, 3UAS1-pTEF, 4UAS1-pTEF, 8UAS1-pTEF, and hp8d. We compared the production of RedStar2, glucoamylase, and xylanase C when strains were grown on three media. As expected, levels of RedStar2 and glucoamylase were greatest in the strain with the 8UAS1-pTEF promoter, which was stronger. However, surprisingly, the 2UAS1-pTEF promoter was associated with the greatest xylanase C production and activity. This finding underscored that stronger promoters are not always better when it comes to protein production. We therefore developed a method for easily identifying the best promoter for a given protein of interest. In this gateway method, genes for YFP and α-amylase were transferred into a pool of vectors containing different promoters and gene expression was then analyzed. We observed that, in most cases, protein production and activity were correlated with promoter strength, although this pattern was protein dependent.
  
  
  CONCLUSIONS
  Protein expression depends on more than just promoter strength. Indeed, promoter suitability appears to be protein dependent; in some cases, optimal expression and activity was obtained using a weaker promoter. We showed that using a vector pool containing promoters of variable strength can be a powerful tool for rapidly identifying the best producer for a given protein of interest.
authors.affiliation: Micalis Institute, INRA-AgroParisTech, UMR1319, Team BIMLip: Integrative Metabolism of Microbial Lipids, Université Paris-Saclay, domaine de Vilvert, 78350, Jouy-en-Josas, France. remi.dulermo@ifremer.fr.
authors.forename: Rémi
authors.initials: R
authors.lastname: Dulermo
authors.affiliation: Micalis Institute, INRA-AgroParisTech, UMR1319, Team BIMLip: Integrative Metabolism of Microbial Lipids, Université Paris-Saclay, domaine de Vilvert, 78350, Jouy-en-Josas, France.
authors.forename: François
authors.initials: F
authors.lastname: Brunel
authors.affiliation: Micalis Institute, INRA-AgroParisTech, UMR1319, Team BIMLip: Integrative Metabolism of Microbial Lipids, Université Paris-Saclay, domaine de Vilvert, 78350, Jouy-en-Josas, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Dulermo
authors.affiliation: Micalis Institute, INRA-AgroParisTech, UMR1319, Team BIMLip: Integrative Metabolism of Microbial Lipids, Université Paris-Saclay, domaine de Vilvert, 78350, Jouy-en-Josas, France.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Ledesma-Amaro
authors.affiliation: Micalis Institute, INRA-AgroParisTech, UMR1319, Team BIMLip: Integrative Metabolism of Microbial Lipids, Université Paris-Saclay, domaine de Vilvert, 78350, Jouy-en-Josas, France.
authors.forename: Jérémy
authors.initials: J
authors.lastname: Vion
authors.affiliation: Micalis Institute, INRA-AgroParisTech, UMR1319, Team BIMLip: Integrative Metabolism of Microbial Lipids, Université Paris-Saclay, domaine de Vilvert, 78350, Jouy-en-Josas, France.
authors.forename: Marion
authors.initials: M
authors.lastname: Trassaert
authors.affiliation: Micalis Institute, INRA-AgroParisTech, UMR1319, Team BIMLip: Integrative Metabolism of Microbial Lipids, Université Paris-Saclay, domaine de Vilvert, 78350, Jouy-en-Josas, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Thomas
authors.affiliation: Micalis Institute, INRA-AgroParisTech, UMR1319, Team BIMLip: Integrative Metabolism of Microbial Lipids, Université Paris-Saclay, domaine de Vilvert, 78350, Jouy-en-Josas, France. jean-marc.nicaud@inra.fr.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Nicaud
authors.affiliation: Micalis Institute, INRA-AgroParisTech, UMR1319, Team BIMLip: Integrative Metabolism of Microbial Lipids, Université Paris-Saclay, domaine de Vilvert, 78350, Jouy-en-Josas, France. christophe.leplat.01@gmail.com.
authors.forename: Christophe
authors.initials: C
authors.lastname: Leplat
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D025062:Yarrowia / Q000235:genetics* / Q000378:metabolism*; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D005656:Fungal Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D000516:alpha-Amylases; D005087:Glucan 1,4-alpha-Glucosidase; D043364:Endo-1,4-beta Xylanases
keywords: Glucoamylase; Hybrid promoters; Protein production; RedStar2; Xylanase; Yarrowia lipolytica