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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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Growth of wild type, deleted or overexpressingSAM2 BY strains in minimal supplemented medium with lactic acid. BY cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose, 125 mg/L, 500 mg/L, 100 mg/L, 150 mg/L for histidine, leucine, methionine and uracil respectively, at initial pH 3, with 38 g/l of lactic acid. Growth was determined as OD at 660 nm. Panel A. Light grey squares: wild type control strain (BY4741) White triangles: sam2Δ cells (BY4741 sam2Δ). Panel B. Dark grey squares: wild type control strain (BY4741 [pTEF-L]) Black circles: cells overespressing SAM2 (BY4741 [pTEF-L-SAM2]).
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Fig6: Growth of wild type, deleted or overexpressingSAM2 BY strains in minimal supplemented medium with lactic acid. BY cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose, 125 mg/L, 500 mg/L, 100 mg/L, 150 mg/L for histidine, leucine, methionine and uracil respectively, at initial pH 3, with 38 g/l of lactic acid. Growth was determined as OD at 660 nm. Panel A. Light grey squares: wild type control strain (BY4741) White triangles: sam2Δ cells (BY4741 sam2Δ). Panel B. Dark grey squares: wild type control strain (BY4741 [pTEF-L]) Black circles: cells overespressing SAM2 (BY4741 [pTEF-L-SAM2]).

Mentions: It has to be noticed that the final OD reached by the leu− strains in the unstressed condition was lower compared to the values registered for LEU+ complemented strains, possibly indicating that the standard amino acid supplementation (50 mg/L) was not sufficient in the case of leucine. This effect was stronger in the CEN.PK background (Figure 4A). Pronk [49] suggested complementation of the medium with 125 mg/L, 500 mg/L, 100 mg/L, 150 mg/L for histidine, leucine, methionine and uracil respectively. Accordingly, the growth experiments, in which the effect of SAM2 modulation has been observed, were repeated in the presence of lactic acid at pH 3 with the supplemented relevant chemicals (Figure 6). While in this medium SAM2 deletion did not affect the cellular growth in the presence of the stressing agent (panel A), SAM2 overexpression was still detrimental to the cells (panel B). This confirms that Sam2p recombinant overproduction is not beneficial to improve the tolerance to this stress.Figure 6


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, deleted or overexpressingSAM2 BY strains in minimal supplemented medium with lactic acid. BY cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose, 125 mg/L, 500 mg/L, 100 mg/L, 150 mg/L for histidine, leucine, methionine and uracil respectively, at initial pH 3, with 38 g/l of lactic acid. Growth was determined as OD at 660 nm. Panel A. Light grey squares: wild type control strain (BY4741) White triangles: sam2Δ cells (BY4741 sam2Δ). Panel B. Dark grey squares: wild type control strain (BY4741 [pTEF-L]) Black circles: cells overespressing 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

Fig6: Growth of wild type, deleted or overexpressingSAM2 BY strains in minimal supplemented medium with lactic acid. BY cells were grown in shake flasks in minimal (YNB) medium with 2% w/v glucose, 125 mg/L, 500 mg/L, 100 mg/L, 150 mg/L for histidine, leucine, methionine and uracil respectively, at initial pH 3, with 38 g/l of lactic acid. Growth was determined as OD at 660 nm. Panel A. Light grey squares: wild type control strain (BY4741) White triangles: sam2Δ cells (BY4741 sam2Δ). Panel B. Dark grey squares: wild type control strain (BY4741 [pTEF-L]) Black circles: cells overespressing SAM2 (BY4741 [pTEF-L-SAM2]).
Mentions: It has to be noticed that the final OD reached by the leu− strains in the unstressed condition was lower compared to the values registered for LEU+ complemented strains, possibly indicating that the standard amino acid supplementation (50 mg/L) was not sufficient in the case of leucine. This effect was stronger in the CEN.PK background (Figure 4A). Pronk [49] suggested complementation of the medium with 125 mg/L, 500 mg/L, 100 mg/L, 150 mg/L for histidine, leucine, methionine and uracil respectively. Accordingly, the growth experiments, in which the effect of SAM2 modulation has been observed, were repeated in the presence of lactic acid at pH 3 with the supplemented relevant chemicals (Figure 6). While in this medium SAM2 deletion did not affect the cellular growth in the presence of the stressing agent (panel A), SAM2 overexpression was still detrimental to the cells (panel B). This confirms that Sam2p recombinant overproduction is not beneficial to improve the tolerance to this stress.Figure 6

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