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 lactic acid producing strains. CEN.PK m850 (grey bars) and CEN.PK m850 sam2Δ (white bars) were pre-grown in shake flasks and then transferred in new flasks in minimal medium containing 90 g/L of glucose for the lactic acid production phase. ATP, ADP and AMP were extracted and determined by HPLC, immediately before (0 h) or after the transfer in the production medium, at the indicated times (24 h and 48 h). Nucleotide concentrations are expressed per gram of dry cell weight (DCW). The mean and SD for two independent experiments is reported.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig10: Intracellular adenine nucleotides concentrations in the lactic acid producing strains. CEN.PK m850 (grey bars) and CEN.PK m850 sam2Δ (white bars) were pre-grown in shake flasks and then transferred in new flasks in minimal medium containing 90 g/L of glucose for the lactic acid production phase. ATP, ADP and AMP were extracted and determined by HPLC, immediately before (0 h) or after the transfer in the production medium, at the indicated times (24 h and 48 h). Nucleotide concentrations are expressed per gram of dry cell weight (DCW). The mean and SD for two independent experiments is reported.

Mentions: To test if energetic balance might contribute to the observed differences, the intracellular AXP concentrations were determined in the control and sam2∆ strain during the process already described. Figure 10 shows the mean data and SD relative to cells analyzed immediately before inoculation (indicated as 0 h) and at 24 and 48 hours after the beginning of the production phase, respectively, in two independent experiments. At time 0 h, the ADP and AMP contents were lower whereas the ATP content was higher in the sam2∆ strain compared to the control, despite a high variability in the case of ATP. After inoculation, no differences were found between the two strains. At 24 h, the ADP and AMP concentrations increased in both strains compared to 0 h, while at 48 h all the three species decreased.Figure 10


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 lactic acid producing strains. CEN.PK m850 (grey bars) and CEN.PK m850 sam2Δ (white bars) were pre-grown in shake flasks and then transferred in new flasks in minimal medium containing 90 g/L of glucose for the lactic acid production phase. ATP, ADP and AMP were extracted and determined by HPLC, immediately before (0 h) or after the transfer in the production medium, at the indicated times (24 h and 48 h). Nucleotide concentrations are expressed per gram of dry cell weight (DCW). The mean and SD for two independent experiments is reported.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig10: Intracellular adenine nucleotides concentrations in the lactic acid producing strains. CEN.PK m850 (grey bars) and CEN.PK m850 sam2Δ (white bars) were pre-grown in shake flasks and then transferred in new flasks in minimal medium containing 90 g/L of glucose for the lactic acid production phase. ATP, ADP and AMP were extracted and determined by HPLC, immediately before (0 h) or after the transfer in the production medium, at the indicated times (24 h and 48 h). Nucleotide concentrations are expressed per gram of dry cell weight (DCW). The mean and SD for two independent experiments is reported.
Mentions: To test if energetic balance might contribute to the observed differences, the intracellular AXP concentrations were determined in the control and sam2∆ strain during the process already described. Figure 10 shows the mean data and SD relative to cells analyzed immediately before inoculation (indicated as 0 h) and at 24 and 48 hours after the beginning of the production phase, respectively, in two independent experiments. At time 0 h, the ADP and AMP contents were lower whereas the ATP content was higher in the sam2∆ strain compared to the control, despite a high variability in the case of ATP. After inoculation, no differences were found between the two strains. At 24 h, the ADP and AMP concentrations increased in both strains compared to 0 h, while at 48 h all the three species decreased.Figure 10

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