Limits...
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.

Show MeSH

Related in: MedlinePlus

Intracellular adenine nucleotides concentrations in the absence and presence of lactic acid. Yeast cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose at initial pH 3, without (panel A) or with (panel B) 40 g/l of lactic acid. ATP, ADP and AMP were extracted from samples collected during the exponential growth phase and determined by HPLC. Concentrations are expressed per OD of cell culture. The mean and SD for two independent experiments is reported. Left panels: CEN.PK cells; right panels: BY cells; Grey bars: CEN.PK 102-3A [pTEF-L], BY4741 [pTEF-L]; white bars: CEN.PK 102-3A sam2Δ [pTEF-L], BY4741 sam2Δ [pTEF-L]; black bars: CEN.PK [pTEF-L-SAM2], BY4741 [pTEF-L-SAM2].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig8: Intracellular adenine nucleotides concentrations in the absence and presence of lactic acid. Yeast cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose at initial pH 3, without (panel A) or with (panel B) 40 g/l of lactic acid. ATP, ADP and AMP were extracted from samples collected during the exponential growth phase and determined by HPLC. Concentrations are expressed per OD of cell culture. The mean and SD for two independent experiments is reported. Left panels: CEN.PK cells; right panels: BY cells; Grey bars: CEN.PK 102-3A [pTEF-L], BY4741 [pTEF-L]; white bars: CEN.PK 102-3A sam2Δ [pTEF-L], BY4741 sam2Δ [pTEF-L]; black bars: CEN.PK [pTEF-L-SAM2], BY4741 [pTEF-L-SAM2].

Mentions: Our data indicate that the Sam2 protein levels respond to lactic acid in both the CEN.PK and BY4741 yeast strains, but the effects of SAM2 gene deletion and overexpression, at least in terms of growth and cell viability, are only detectable in the BY background. We considered the hypothesis that these differences might be correlated with different AXP pool composition. We therefore measured the adenine nucleotide content of CEN.PK 113-11C and BY4741 wt, SAM2 overexpressing and SAM2 deleted strains, complemented for leucine auxotrophy, during the exponential growth phase on minimal medium with 2% w/v glucose without or with lactic acid (samples were collected at OD ~1 if without and at OD ~0.3 if with lactic acid, respectively). The ATP, ADP and AMP (collectively referred as AXP) intracellular concentrations were determined by HPLC with the method from Ask et al. [50], as described in the Methods section. Data are reported in Figure 8, normalized for culture OD for consistency with the other data.Figure 8


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)

Intracellular adenine nucleotides concentrations in the absence and presence of lactic acid. Yeast cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose at initial pH 3, without (panel A) or with (panel B) 40 g/l of lactic acid. ATP, ADP and AMP were extracted from samples collected during the exponential growth phase and determined by HPLC. Concentrations are expressed per OD of cell culture. The mean and SD for two independent experiments is reported. Left panels: CEN.PK cells; right panels: BY cells; Grey bars: CEN.PK 102-3A [pTEF-L], BY4741 [pTEF-L]; white bars: CEN.PK 102-3A sam2Δ [pTEF-L], BY4741 sam2Δ [pTEF-L]; black bars: CEN.PK [pTEF-L-SAM2], 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

Fig8: Intracellular adenine nucleotides concentrations in the absence and presence of lactic acid. Yeast cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose at initial pH 3, without (panel A) or with (panel B) 40 g/l of lactic acid. ATP, ADP and AMP were extracted from samples collected during the exponential growth phase and determined by HPLC. Concentrations are expressed per OD of cell culture. The mean and SD for two independent experiments is reported. Left panels: CEN.PK cells; right panels: BY cells; Grey bars: CEN.PK 102-3A [pTEF-L], BY4741 [pTEF-L]; white bars: CEN.PK 102-3A sam2Δ [pTEF-L], BY4741 sam2Δ [pTEF-L]; black bars: CEN.PK [pTEF-L-SAM2], BY4741 [pTEF-L-SAM2].
Mentions: Our data indicate that the Sam2 protein levels respond to lactic acid in both the CEN.PK and BY4741 yeast strains, but the effects of SAM2 gene deletion and overexpression, at least in terms of growth and cell viability, are only detectable in the BY background. We considered the hypothesis that these differences might be correlated with different AXP pool composition. We therefore measured the adenine nucleotide content of CEN.PK 113-11C and BY4741 wt, SAM2 overexpressing and SAM2 deleted strains, complemented for leucine auxotrophy, during the exponential growth phase on minimal medium with 2% w/v glucose without or with lactic acid (samples were collected at OD ~1 if without and at OD ~0.3 if with lactic acid, respectively). The ATP, ADP and AMP (collectively referred as AXP) intracellular concentrations were determined by HPLC with the method from Ask et al. [50], as described in the Methods section. Data are reported in Figure 8, normalized for culture OD for consistency with the other data.Figure 8

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