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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

Sam2p-GFP fluorescence distribution during growth in the absence and presence of lactic acid. CEN.PK 113-11C SAM2GFP 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). Epifocal microscope images were taken at 16 and 40 hours after inoculation, corresponding to exponential growth phase and early stationary phase, respectively. Pictures show Sam2p-GFP fluorescence in the green field. White arrows indicate Sam2p-GFP foci. LA: lactic acid.
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Fig2: Sam2p-GFP fluorescence distribution during growth in the absence and presence of lactic acid. CEN.PK 113-11C SAM2GFP 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). Epifocal microscope images were taken at 16 and 40 hours after inoculation, corresponding to exponential growth phase and early stationary phase, respectively. Pictures show Sam2p-GFP fluorescence in the green field. White arrows indicate Sam2p-GFP foci. LA: lactic acid.

Mentions: Yeast cells were grown in the same conditions described above and observed under epifocal microscope at 16 and 40 hours after inoculation. The images of Figure 2, depicting CEN.PK cells, show that the presence of lactic acid had no significant effects on Sam2p-GFP distribution. At 16 h (upper panels) the signal was diffused into the whole cell, with the exclusion of extended dark areas representing the vacuoles and nuclei (based on DAPI staining, not shown). Therefore, the localization appeared to be mainly cytoplasmatic, although a contingent association with membranes cannot be excluded. At 40 h (bottom panels), instead, discrete spots emerging from the diffused fluorescence signal were visible. A similar situation was observed in BY4741 cells (data not shown). The number and dimensions of these foci were highly variable in all cells, irrespective of whether lactic acid was present or not. Therefore the data reported do not allow additional speculations on their relevance to stress tolerance. The nature of the observed Sam2p foci, never reported in literature before, is still unknown, and its biological significance needs to be further investigated.Figure 2


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)

Sam2p-GFP fluorescence distribution during growth in the absence and presence of lactic acid. CEN.PK 113-11C SAM2GFP 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). Epifocal microscope images were taken at 16 and 40 hours after inoculation, corresponding to exponential growth phase and early stationary phase, respectively. Pictures show Sam2p-GFP fluorescence in the green field. White arrows indicate Sam2p-GFP foci. LA: lactic acid.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Sam2p-GFP fluorescence distribution during growth in the absence and presence of lactic acid. CEN.PK 113-11C SAM2GFP 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). Epifocal microscope images were taken at 16 and 40 hours after inoculation, corresponding to exponential growth phase and early stationary phase, respectively. Pictures show Sam2p-GFP fluorescence in the green field. White arrows indicate Sam2p-GFP foci. LA: lactic acid.
Mentions: Yeast cells were grown in the same conditions described above and observed under epifocal microscope at 16 and 40 hours after inoculation. The images of Figure 2, depicting CEN.PK cells, show that the presence of lactic acid had no significant effects on Sam2p-GFP distribution. At 16 h (upper panels) the signal was diffused into the whole cell, with the exclusion of extended dark areas representing the vacuoles and nuclei (based on DAPI staining, not shown). Therefore, the localization appeared to be mainly cytoplasmatic, although a contingent association with membranes cannot be excluded. At 40 h (bottom panels), instead, discrete spots emerging from the diffused fluorescence signal were visible. A similar situation was observed in BY4741 cells (data not shown). The number and dimensions of these foci were highly variable in all cells, irrespective of whether lactic acid was present or not. Therefore the data reported do not allow additional speculations on their relevance to stress tolerance. The nature of the observed Sam2p foci, never reported in literature before, is still unknown, and its biological significance needs to be further investigated.Figure 2

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