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Engineered Pichia pastoris for enhanced production of S-adenosylmethionine.

Kamarthapu V, Ragampeta S, Rao KV, Reddy VD - AMB Express (2013)

Bottom Line: Shake flask cultivation of engineered P. pastoris in BMGY medium supplemented with 1% L-methionine yielded 28 g/L wet cell weight and 0.6 g/L S-adenosylmethionine, whereas control (transformants with vector alone) with similar wet cell weight under identical conditions accumulated 0.018 g/L.The clone cultured in the bioreactor containing enriched methionine medium showed increased WCW (117 g/L) as compared to shake flask cultures and yielded 2.4 g/L S-adenosylmethionine.In spite of expression of SAM 2 gene up to 90 h, S-adenosylmethionine accumulation tended to plateau after 72 h, presumably because of the limited ATP available in the cells at stationery phase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Plant Molecular Biology, Osmania University, Hyderabad 500 007, India.

ABSTRACT
A genetically engineered strain of Pichia pastoris expressing S-adenosylmethionine synthetase gene from Saccharomyces cerevisiae under the control of AOX 1 promoter was developed. Induction of recombinant strain with 1% methanol resulted in the expression of SAM2 protein of ~ 42 kDa, whereas control GS115 showed no such band. Further, the recombinant strain showed 17-fold higher enzyme activity over control. Shake flask cultivation of engineered P. pastoris in BMGY medium supplemented with 1% L-methionine yielded 28 g/L wet cell weight and 0.6 g/L S-adenosylmethionine, whereas control (transformants with vector alone) with similar wet cell weight under identical conditions accumulated 0.018 g/L. The clone cultured in the bioreactor containing enriched methionine medium showed increased WCW (117 g/L) as compared to shake flask cultures and yielded 2.4 g/L S-adenosylmethionine. In spite of expression of SAM 2 gene up to 90 h, S-adenosylmethionine accumulation tended to plateau after 72 h, presumably because of the limited ATP available in the cells at stationery phase. The recombinant P pastoris seems promising as potential source for industrial production of S-adenosylmethionine.

No MeSH data available.


Related in: MedlinePlus

Gain in wet cell weight and accumulation of SAM in the recombinant P. pastoris GS115 cultured in bioreactor at different time points after induction with methanol. a Wet cell weight b SAM content.
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Figure 4: Gain in wet cell weight and accumulation of SAM in the recombinant P. pastoris GS115 cultured in bioreactor at different time points after induction with methanol. a Wet cell weight b SAM content.

Mentions: The clone GS115-SAM2 was cultivated in the bioreactor for production of SAM. The clone was initially grown on glycerol as the sole carbon source for ~28 h. Then feeding with methanol was continued for a further period of 90 h. SDS-PAGE analysis of the recombinant P. pastoris revealed ~ 42 kDa SAM synthetase protein, from 12-90 h induction, with maximum expression of recombinant SAM synthetase from 48-60 h. Accumulation of SAM in the bioreactor also increased gradually from 12 to 72 h and there was no increase in the accumulation after 72 h (Figure 4b). At the end of cultivation of the recombinant P. pastoris, wet cell weight of 117 g/L was achieved (Figure 4a). The recombinant P. pastoris cultured in the bioreactor, enriched with methionine could accumulate 2.4 g/l SAM (Figure 4a) or 0.021 g/g WCW or 0.186 g/g DCW.


Engineered Pichia pastoris for enhanced production of S-adenosylmethionine.

Kamarthapu V, Ragampeta S, Rao KV, Reddy VD - AMB Express (2013)

Gain in wet cell weight and accumulation of SAM in the recombinant P. pastoris GS115 cultured in bioreactor at different time points after induction with methanol. a Wet cell weight b SAM content.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750815&req=5

Figure 4: Gain in wet cell weight and accumulation of SAM in the recombinant P. pastoris GS115 cultured in bioreactor at different time points after induction with methanol. a Wet cell weight b SAM content.
Mentions: The clone GS115-SAM2 was cultivated in the bioreactor for production of SAM. The clone was initially grown on glycerol as the sole carbon source for ~28 h. Then feeding with methanol was continued for a further period of 90 h. SDS-PAGE analysis of the recombinant P. pastoris revealed ~ 42 kDa SAM synthetase protein, from 12-90 h induction, with maximum expression of recombinant SAM synthetase from 48-60 h. Accumulation of SAM in the bioreactor also increased gradually from 12 to 72 h and there was no increase in the accumulation after 72 h (Figure 4b). At the end of cultivation of the recombinant P. pastoris, wet cell weight of 117 g/L was achieved (Figure 4a). The recombinant P. pastoris cultured in the bioreactor, enriched with methionine could accumulate 2.4 g/l SAM (Figure 4a) or 0.021 g/g WCW or 0.186 g/g DCW.

Bottom Line: Shake flask cultivation of engineered P. pastoris in BMGY medium supplemented with 1% L-methionine yielded 28 g/L wet cell weight and 0.6 g/L S-adenosylmethionine, whereas control (transformants with vector alone) with similar wet cell weight under identical conditions accumulated 0.018 g/L.The clone cultured in the bioreactor containing enriched methionine medium showed increased WCW (117 g/L) as compared to shake flask cultures and yielded 2.4 g/L S-adenosylmethionine.In spite of expression of SAM 2 gene up to 90 h, S-adenosylmethionine accumulation tended to plateau after 72 h, presumably because of the limited ATP available in the cells at stationery phase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Plant Molecular Biology, Osmania University, Hyderabad 500 007, India.

ABSTRACT
A genetically engineered strain of Pichia pastoris expressing S-adenosylmethionine synthetase gene from Saccharomyces cerevisiae under the control of AOX 1 promoter was developed. Induction of recombinant strain with 1% methanol resulted in the expression of SAM2 protein of ~ 42 kDa, whereas control GS115 showed no such band. Further, the recombinant strain showed 17-fold higher enzyme activity over control. Shake flask cultivation of engineered P. pastoris in BMGY medium supplemented with 1% L-methionine yielded 28 g/L wet cell weight and 0.6 g/L S-adenosylmethionine, whereas control (transformants with vector alone) with similar wet cell weight under identical conditions accumulated 0.018 g/L. The clone cultured in the bioreactor containing enriched methionine medium showed increased WCW (117 g/L) as compared to shake flask cultures and yielded 2.4 g/L S-adenosylmethionine. In spite of expression of SAM 2 gene up to 90 h, S-adenosylmethionine accumulation tended to plateau after 72 h, presumably because of the limited ATP available in the cells at stationery phase. The recombinant P pastoris seems promising as potential source for industrial production of S-adenosylmethionine.

No MeSH data available.


Related in: MedlinePlus