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Changes in SAM2 expression affect lactic acid tolerance and lactic acid production in Saccharomyces cerevisiae.

Dato L, Berterame NM, Ricci MA, Paganoni P, Palmieri L, Porro D, Branduardi P - Microb. Cell Fact. (2014)

Bottom Line: 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.Our data confirm cofactor engineering as an important tool for cell factory improvement.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy. laura.dato@unimib.it.

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.

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Related in: MedlinePlus

Western blot analysis of total Sam2p levels in cells grown in the absence and presence of lactic acid. CEN.PK 113-11C (panel A) and BY4741 SAM2GFP (panel B) cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose without or with the addition of different concentrations of lactic acid (pH 5, pH 3, 12 g/L and 20 g/L lactic acid at pH3) and the Sam2p-GFP levels were evaluated after 16 and 40 hours after inoculation in the total protein fraction, extracted with TCA, using an anti-GFP antibody. Samples were normalised according to cell number. β-actin levels have been detected as control. Bands have been quantified by ImageJ 1.48 software. Histograms refer to the ratio (%) of Sam2p/Actin normalized to the values at pH 5. LA: lactic acid.
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Fig1: Western blot analysis of total Sam2p levels in cells grown in the absence and presence of lactic acid. CEN.PK 113-11C (panel A) and BY4741 SAM2GFP (panel B) cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose without or with the addition of different concentrations of lactic acid (pH 5, pH 3, 12 g/L and 20 g/L lactic acid at pH3) and the Sam2p-GFP levels were evaluated after 16 and 40 hours after inoculation in the total protein fraction, extracted with TCA, using an anti-GFP antibody. Samples were normalised according to cell number. β-actin levels have been detected as control. Bands have been quantified by ImageJ 1.48 software. Histograms refer to the ratio (%) of Sam2p/Actin normalized to the values at pH 5. LA: lactic acid.

Mentions: Figure 1 shows the western blots of the TCA extracts for CEN.PK (panel A) and BY (panel B) strains. Remarkably, in both strains the signal intensity of Sam2p-GFP increased in the presence of lactic acid, particularly in the BY strain (see panels B).Figure 1


Changes in SAM2 expression affect lactic acid tolerance and lactic acid production in Saccharomyces cerevisiae.

Dato L, Berterame NM, Ricci MA, Paganoni P, Palmieri L, Porro D, Branduardi P - Microb. Cell Fact. (2014)

Western blot analysis of total Sam2p levels in cells grown in the absence and presence of lactic acid. CEN.PK 113-11C (panel A) and BY4741 SAM2GFP (panel B) cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose without or with the addition of different concentrations of lactic acid (pH 5, pH 3, 12 g/L and 20 g/L lactic acid at pH3) and the Sam2p-GFP levels were evaluated after 16 and 40 hours after inoculation in the total protein fraction, extracted with TCA, using an anti-GFP antibody. Samples were normalised according to cell number. β-actin levels have been detected as control. Bands have been quantified by ImageJ 1.48 software. Histograms refer to the ratio (%) of Sam2p/Actin normalized to the values at pH 5. LA: lactic acid.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230512&req=5

Fig1: Western blot analysis of total Sam2p levels in cells grown in the absence and presence of lactic acid. CEN.PK 113-11C (panel A) and BY4741 SAM2GFP (panel B) cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose without or with the addition of different concentrations of lactic acid (pH 5, pH 3, 12 g/L and 20 g/L lactic acid at pH3) and the Sam2p-GFP levels were evaluated after 16 and 40 hours after inoculation in the total protein fraction, extracted with TCA, using an anti-GFP antibody. Samples were normalised according to cell number. β-actin levels have been detected as control. Bands have been quantified by ImageJ 1.48 software. Histograms refer to the ratio (%) of Sam2p/Actin normalized to the values at pH 5. LA: lactic acid.
Mentions: Figure 1 shows the western blots of the TCA extracts for CEN.PK (panel A) and BY (panel B) strains. Remarkably, in both strains the signal intensity of Sam2p-GFP increased in the presence of lactic acid, particularly in the BY strain (see panels B).Figure 1

Bottom Line: 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.Our data confirm cofactor engineering as an important tool for cell factory improvement.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy. laura.dato@unimib.it.

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.

Show MeSH
Related in: MedlinePlus