title: Deciphering how LIP2 and POX2 promoters can optimally regulate recombinant protein production in the yeast Yarrowia lipolytica.
abstract: BACKGROUND
  In recent years, the non-conventional model yeast species Yarrowia lipolytica has received much attention because it is a useful cell factory for producing recombinant proteins. In this species, expression vectors involving LIP2 and POX2 promoters have been developed and used successfully for protein production at yields similar to or even higher than those of other cell factories, such as Pichia pastoris. However, production processes involving these promoters can be difficult to manage, especially if carried out at large scales in fed-batch bioreactors, because they require hydrophobic inducers, such as oleic acid or methyl oleate. Thus, the challenge has become to reduce loads of hydrophobic substrates while simultaneously promoting recombinant protein production. One possible solution is to replace a portion of the inducer with a co-substrate that can serve as an alternative energy source. However, implementing such an approach would require detailed knowledge of how carbon sources impact promoter regulation, which is surprisingly still lacking for the LIP2 and POX2 promoters. This study's aim was thus to better characterize promoter regulation and cell metabolism in Y. lipolytica cultures grown in media supplemented with different carbon sources.
  
  
  RESULTS
  pPOX2 induction could be detected when glucose or glycerol was used as sole carbon source, which meant these carbon source could not prevent promoter induction. In addition, when a mixture of glucose and oleic acid was used in complex medium, pPOX2 induction level was lower that that of pLIP2. In contrast, pLIP2 induction was absent when glucose was present in the culture medium, which meant that cell growth could occur without any recombinant gene expression. When a 40/60 mixture of glucose and oleic acid (w/w) was used, a tenfold increase in promoter induction, as compared to when an oleic-acid-only medium was observed. It was also clear that individual cells were adapting metabolically to use both glucose and oleic acid. Indeed, no distinct subpopulations that specialized on glucose versus oleic acid were observed; such an outcome would have led to producer and non-producer phenotypes. In medium containing both glucose and oleic acid, cells tended to directly metabolize oleic acid instead of storing it in lipid bodies.
  
  
  CONCLUSIONS
  This study found that pLIP2 is a promoter of choice as compared to pPOX2 to drive gene expression for recombinant protein production by Y. lipolytica used as cell factory.
authors.affiliation: Biotechnology and Bioprocesses, Université libre de Bruxelles, Avenue F.D. Roosevelt 50, 1050, Brussels, Belgium.
authors.forename: Hosni
authors.initials: H
authors.lastname: Sassi
authors.affiliation: Microbial Processes and Interactions, University of Liège-Gembloux AgroBio Tech, Passage des Déportés, 2, B-5030, Gembloux, Belgium.
authors.forename: Frank
authors.initials: F
authors.lastname: Delvigne
authors.affiliation: Microbial Processes and Interactions, University of Liège-Gembloux AgroBio Tech, Passage des Déportés, 2, B-5030, Gembloux, Belgium.
authors.forename: Tambi
authors.initials: T
authors.lastname: Kar
authors.affiliation: Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350, Jouy-en Josas, France.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Nicaud
authors.affiliation: Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350, Jouy-en Josas, France.
authors.forename: Anne-Marie Crutz-Le
authors.initials: AM
authors.lastname: Coq
authors.affiliation: Microbial Processes and Interactions, University of Liège-Gembloux AgroBio Tech, Passage des Déportés, 2, B-5030, Gembloux, Belgium.
authors.forename: Sebastien
authors.initials: S
authors.lastname: Steels
authors.affiliation: Biotechnology and Bioprocesses, Université libre de Bruxelles, Avenue F.D. Roosevelt 50, 1050, Brussels, Belgium. pfickers@ulg.ac.be.|Microbial Processes and Interactions, University of Liège-Gembloux AgroBio Tech, Passage des Déportés, 2, B-5030, Gembloux, Belgium. pfickers@ulg.ac.be.
authors.forename: Patrick
authors.initials: P
authors.lastname: Fickers
mesh_terms: D019149:Bioreactors; D002244:Carbon / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D005656:Fungal Proteins / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D008049:Lipase / Q000235:genetics*; D019301:Oleic Acid / Q000378:metabolism; D009829:Oleic Acids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*; D025062:Yarrowia / Q000235:genetics* / Q000378:metabolism*
chemical_list: D003470:Culture Media; D005656:Fungal Proteins; D009829:Oleic Acids; D011994:Recombinant Proteins; D019301:Oleic Acid; C005576:methyl oleate; D002244:Carbon; C408971:LIP2 protein, Yarrowia lipolytica; D008049:Lipase; D005947:Glucose; D005990:Glycerol
keywords: Carbon source; Co-substrate; LIP2; POX2; Promoter regulation; Recombinant protein; Yarriwia lipolytica