Comprehensive Analysis of a Yeast Lipase Family in the Yarrowia Clade

Muchalin Meunchan; Stéphanie Michely; Hugo Devillers; Jean-Marc Nicaud; Alain Marty; Cécile Neuvéglise

doi:10.1371/journal.pone.0143096

Comprehensive Analysis of a Yeast Lipase Family in the Yarrowia Clade

PLoS One. 2015 Nov 18;10(11):e0143096. doi: 10.1371/journal.pone.0143096. eCollection 2015.

Authors

Muchalin Meunchan^{1

2

3

4}, Stéphanie Michely^{5

6}, Hugo Devillers^{5

6}, Jean-Marc Nicaud^{5

6}, Alain Marty^{1

2

3}, Cécile Neuvéglise^{5

6}

Affiliations

¹ Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077, Toulouse, France.
² INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400, Toulouse, France.
³ CNRS, UMR5504, F-31400, Toulouse, France.
⁴ Department of Biochemistry, Faculty of Science, Khon Kaen University, 123 Mittapap Road, Khon Kaen, 40002, Thailand.
⁵ INRA, UMR 1319 Micalis, F-78352, Jouy-en-Josas, France.
⁶ AgroParisTech, UMR Micalis, F-78352, Jouy-en-Josas, France.

Abstract

Lipases are currently the subject of intensive studies due to their large range of industrial applications. The Lip2p lipase from the yeast Yarrowia lipolytica (YlLIP2) was recently shown to be a good candidate for different biotechnological applications. Using a combination of comparative genomics approaches based on sequence similarity, synteny conservation, and phylogeny, we constructed the evolutionary scenario of the lipase family for six species of the Yarrowia clade. RNA-seq based transcriptome analysis revealed the primary role of LIP2 homologues in the assimilation of different substrates. Once identified, these YlLIP2 homologues were expressed in Y. lipolytica. The lipase Lip2a from Candida phangngensis was shown to naturally present better activity and enantioselectivity than YlLip2. Enantioselectivity was further improved by site-directed mutagenesis targeted to the substrate binding site. The mono-substituted variant V232S displayed enantioselectivity greater than 200 and a 2.5 fold increase in velocity. A double-substituted variant 97A-V232F presented reversed enantioselectivity, with a total preference for the R-enantiomer.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Binding Sites
Butyrates / chemistry
Candida / enzymology
Candida / genetics
Evolution, Molecular*
Fungal Proteins / chemistry
Fungal Proteins / genetics*
Fungal Proteins / metabolism
Gene Expression
Hydrolysis
Industrial Microbiology
Lipase / chemistry
Lipase / genetics*
Lipase / metabolism
Molecular Sequence Data
Mutagenesis, Site-Directed
Phylogeny*
Protein Binding
Sequence Alignment
Sequence Homology, Amino Acid
Stereoisomerism
Substrate Specificity
Synteny
Transcriptome*
Yarrowia / classification
Yarrowia / enzymology
Yarrowia / genetics*

Substances

Butyrates
Fungal Proteins
4-nitrophenyl butyrate
LIP2 protein, Yarrowia lipolytica
Lipase

Grants and funding

This work was performed, in partnership with the SAS PIVERT, within the frame of the French Institute for the Energy Transition (Institut pour la Transition Energétique - ITE) P.I.V.E.R.T. (www.institutpivert.com) selected as an Investment for the Future (“Investissements d’Avenir”). This work was supported as part of the Investments for the Future, by the French Government under the reference ANR-001-01 and by INRA through the AIP-Bioressources YALIP project. Muchalin Meunchan acknowledges the Thailand Research Fund (TRF) together with Khon Kaen University for financial support through Royal Golden Jubilee PhD program (grant number PHD/0282/2549) and French Embassy. Stéphanie Michely has a PhD fellowship funded by the French Minister of Research via the ABIES doctoral school. The funders had no role in study design, data collection and analysis, or preparation of the manuscript.