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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

Viability determination for cells stressed with lactic lacid. Cells were grown in minimal medium until the exponential phase and then treated with a pulse of lactic acid. After 30 min of incubation, cells were collected and stained with propidium iodide (PI) to detect dead and/or severely damaged cells by flow cytometry. The fluorescence emission was measured through a 670 nm long pass filter (FL3 parameter). For each sample, 25000 cells were analysed. The bar indicates the PI positive subpopulation. Panel A: BY4741 and BY4741 sam2Δ. Panel B: BY4741 [pTEF-L], BY4741 sam2Δ [pTEF-L] and BY4741 [pTEF-L-SAM2].
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Fig7: Viability determination for cells stressed with lactic lacid. Cells were grown in minimal medium until the exponential phase and then treated with a pulse of lactic acid. After 30 min of incubation, cells were collected and stained with propidium iodide (PI) to detect dead and/or severely damaged cells by flow cytometry. The fluorescence emission was measured through a 670 nm long pass filter (FL3 parameter). For each sample, 25000 cells were analysed. The bar indicates the PI positive subpopulation. Panel A: BY4741 and BY4741 sam2Δ. Panel B: BY4741 [pTEF-L], BY4741 sam2Δ [pTEF-L] and BY4741 [pTEF-L-SAM2].

Mentions: The effect of SAM2 deletion and overexpression was also evaluated in terms of cellular viability in the aforementioned strains, i.e. CEN.PK 102-3A and BY4741 wt, SAM2 overexpressing and sam2Δ (complemented or not for leucine auxotrophy). Cells were grown in minimal medium, until the exponential phase was reached, and then treated with a pulse of lactic acid at different concentrations (0, 25, 30, 35, 40 and 45 g/L at pH 3). After 30 minutes the cells were collected, stained with propidium iodide (PI) and analyzed by flow cytometry to identify dead and/or severely compromised cells. Figure 7 shows the histograms obtained for the BY4741 strains, where the left peak corresponds to intact (PI-negative) cells, while the right peak corresponds to the dead/damaged (PI-positive) cells (we currently do not have an interpretation for the bimodal distribution visible in the plots).Figure 7


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)

Viability determination for cells stressed with lactic lacid. Cells were grown in minimal medium until the exponential phase and then treated with a pulse of lactic acid. After 30 min of incubation, cells were collected and stained with propidium iodide (PI) to detect dead and/or severely damaged cells by flow cytometry. The fluorescence emission was measured through a 670 nm long pass filter (FL3 parameter). For each sample, 25000 cells were analysed. The bar indicates the PI positive subpopulation. Panel A: BY4741 and BY4741 sam2Δ. Panel B: BY4741 [pTEF-L], BY4741 sam2Δ [pTEF-L] and BY4741 [pTEF-L-SAM2].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: Viability determination for cells stressed with lactic lacid. Cells were grown in minimal medium until the exponential phase and then treated with a pulse of lactic acid. After 30 min of incubation, cells were collected and stained with propidium iodide (PI) to detect dead and/or severely damaged cells by flow cytometry. The fluorescence emission was measured through a 670 nm long pass filter (FL3 parameter). For each sample, 25000 cells were analysed. The bar indicates the PI positive subpopulation. Panel A: BY4741 and BY4741 sam2Δ. Panel B: BY4741 [pTEF-L], BY4741 sam2Δ [pTEF-L] and BY4741 [pTEF-L-SAM2].
Mentions: The effect of SAM2 deletion and overexpression was also evaluated in terms of cellular viability in the aforementioned strains, i.e. CEN.PK 102-3A and BY4741 wt, SAM2 overexpressing and sam2Δ (complemented or not for leucine auxotrophy). Cells were grown in minimal medium, until the exponential phase was reached, and then treated with a pulse of lactic acid at different concentrations (0, 25, 30, 35, 40 and 45 g/L at pH 3). After 30 minutes the cells were collected, stained with propidium iodide (PI) and analyzed by flow cytometry to identify dead and/or severely compromised cells. Figure 7 shows the histograms obtained for the BY4741 strains, where the left peak corresponds to intact (PI-negative) cells, while the right peak corresponds to the dead/damaged (PI-positive) cells (we currently do not have an interpretation for the bimodal distribution visible in the plots).Figure 7

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