title: Changes in SAM2 expression affect lactic acid tolerance and lactic acid production in Saccharomyces cerevisiae.
abstract: BACKGROUND
  The great interest in the production of highly pure lactic acid enantiomers comes from the application of polylactic acid (PLA) for the production of biodegradable plastics. Yeasts can be considered as alternative cell factories to lactic acid bacteria for lactic acid production, despite not being natural producers, since they can better tolerate acidic environments. We have previously described metabolically engineered Saccharomyces cerevisiae strains producing high amounts of L-lactic acid (>60 g/L) at low pH. The high product concentration represents the major limiting step of the process, mainly because of its toxic effects. Therefore, our goal was the identification of novel targets for strain improvement possibly involved in the yeast response to lactic acid stress.
  
  
  RESULTS
  The enzyme S-adenosylmethionine (SAM) synthetase catalyses the only known reaction leading to the biosynthesis of SAM, an important cellular cofactor. SAM is involved in phospholipid biosynthesis and hence in membrane remodelling during acid stress. Since only the enzyme isoform 2 seems to be responsive to membrane related signals (e.g. myo-inositol), Sam2p was tagged with GFP to analyse its abundance and cellular localization under different stress conditions. Western blot analyses showed that lactic acid exposure correlates with an increase in protein levels. The SAM2 gene was then overexpressed and deleted in laboratory strains. Remarkably, in the BY4741 strain its deletion conferred higher resistance to lactic acid, while its overexpression was detrimental. Therefore, SAM2 was deleted in a strain previously engineered and evolved for industrial lactic acid production and tolerance, resulting in higher production.
  
  
  CONCLUSIONS
  Here we demonstrated that the modulation of SAM2 can have different outcomes, from clear effects to no significant phenotypic responses, upon lactic acid stress in different genetic backgrounds, and that at least in one genetic background SAM2 deletion led to an industrially relevant increase in lactic acid production. Further work is needed to elucidate the molecular basis of these observations, which underline once more that strain robustness relies on complex cellular mechanisms, involving regulatory genes and proteins. Our data confirm cofactor engineering as an important tool for cell factory improvement.
authors.affiliation: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy. laura.dato@unimib.it.
authors.forename: Laura
authors.initials: L
authors.lastname: Dato
authors.affiliation: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy. n.berterame1@campus.unimib.it.
authors.forename: Nadia Maria
authors.initials: NM
authors.lastname: Berterame
authors.affiliation: Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari Aldo Moro, Via Orabona 4, 70125, Bari, Italy. mariantonietta.ricci@uniba.it.
authors.forename: Maria Antonietta
authors.initials: MA
authors.lastname: Ricci
authors.affiliation: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy. paganoni.paola@gmail.com.
authors.forename: Paola
authors.initials: P
authors.lastname: Paganoni
authors.affiliation: Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari Aldo Moro, Via Orabona 4, 70125, Bari, Italy. luigi.palmieri@uniba.it.
authors.forename: Luigi
authors.initials: L
authors.lastname: Palmieri
authors.affiliation: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy. danilo.porro@unimib.it.
authors.forename: Danilo
authors.initials: D
authors.lastname: Porro
authors.affiliation: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy. paola.branduardi@unimib.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Branduardi
mesh_terms: D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D019344:Lactic Acid / Q000096:biosynthesis*; D008716:Methionine Adenosyltransferase / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D007527:Isoenzymes; D029701:Saccharomyces cerevisiae Proteins; D019344:Lactic Acid; D008716:Methionine Adenosyltransferase