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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

Growth of wild type andsam2Δ leucine nutritionally complemented strains in the absence and presence of lactic acid. Yeast cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose and 50 mg/L of the necessary nutritional supplements at initial pH 3, without (panel A) or with (panel B) 34 and 38 g/l of lactic acid for CEN.PK (left panels) and BY (right panels), respectively. Growth was determined as OD at 660 nm. Light grey squares: parental wild type strains (CEN.PK 102-3A, BY4741). White triangles: sam2Δ cells (CEN.PK 102-3A sam2Δ, BY4741 sam2Δ).
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Fig4: Growth of wild type andsam2Δ leucine nutritionally complemented strains in the absence and presence of lactic acid. Yeast cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose and 50 mg/L of the necessary nutritional supplements at initial pH 3, without (panel A) or with (panel B) 34 and 38 g/l of lactic acid for CEN.PK (left panels) and BY (right panels), respectively. Growth was determined as OD at 660 nm. Light grey squares: parental wild type strains (CEN.PK 102-3A, BY4741). White triangles: sam2Δ cells (CEN.PK 102-3A sam2Δ, BY4741 sam2Δ).

Mentions: Figures 4 and 5 show the growth curves obtained, respectively for the parental strains and for the LEU+ complemented strains. SAM2 deletion had no effect, in all the tested strains, during growth in minimal medium at low pH (Figures 4A and 5A). When cells were stressed with lactic acid, once more no marked differences were observed in the CEN.PK background between the wild type and the deleted strain (Figures 4B and 5B). Interestingly, the BY4741 parental strain sam2Δ turned out to be less sensitive to the stressing agent than the wild type (Figure 4B): the specific growth rate in exponential phase was in fact 45% higher compared to control cells (0.11 ± 0.01 h−1vs 0.16 ± 0.01 h−1, mean and SD from three independent experiments). However, the complementation of leucine auxotrophy made void the positive impact of SAM2 deletion on cellular growth (Figure 5B).Figure 4


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)

Growth of wild type andsam2Δ leucine nutritionally complemented strains in the absence and presence of lactic acid. Yeast cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose and 50 mg/L of the necessary nutritional supplements at initial pH 3, without (panel A) or with (panel B) 34 and 38 g/l of lactic acid for CEN.PK (left panels) and BY (right panels), respectively. Growth was determined as OD at 660 nm. Light grey squares: parental wild type strains (CEN.PK 102-3A, BY4741). White triangles: sam2Δ cells (CEN.PK 102-3A sam2Δ, BY4741 sam2Δ).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Growth of wild type andsam2Δ leucine nutritionally complemented strains in the absence and presence of lactic acid. Yeast cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose and 50 mg/L of the necessary nutritional supplements at initial pH 3, without (panel A) or with (panel B) 34 and 38 g/l of lactic acid for CEN.PK (left panels) and BY (right panels), respectively. Growth was determined as OD at 660 nm. Light grey squares: parental wild type strains (CEN.PK 102-3A, BY4741). White triangles: sam2Δ cells (CEN.PK 102-3A sam2Δ, BY4741 sam2Δ).
Mentions: Figures 4 and 5 show the growth curves obtained, respectively for the parental strains and for the LEU+ complemented strains. SAM2 deletion had no effect, in all the tested strains, during growth in minimal medium at low pH (Figures 4A and 5A). When cells were stressed with lactic acid, once more no marked differences were observed in the CEN.PK background between the wild type and the deleted strain (Figures 4B and 5B). Interestingly, the BY4741 parental strain sam2Δ turned out to be less sensitive to the stressing agent than the wild type (Figure 4B): the specific growth rate in exponential phase was in fact 45% higher compared to control cells (0.11 ± 0.01 h−1vs 0.16 ± 0.01 h−1, mean and SD from three independent experiments). However, the complementation of leucine auxotrophy made void the positive impact of SAM2 deletion on cellular growth (Figure 5B).Figure 4

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