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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

ESI mass spectrum corresponds to tR at 2.7 min.
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Figure 6: ESI mass spectrum corresponds to tR at 2.7 min.

Mentions: LC/MS analysis of acid-extracted fermented cells exhibited distinct chromatographic peaks at retention times (tR) of 1.4, 1.5, 2.7, 4.2 and 5.8 minutes (Figure 5). The ESI mass spectrum of the chromatographic peak eluted at a tR of 2.7 minutes (Figure 6), and the spectrum showed [M]+ion at m/z 399 as the base peak. The spectrum also showed moderately abundant fragment ion peaks at m/z 355, 298, 136 and highly abundant ion peak at m/z 250. The collision induced dissociation (CID) tandem mass spectrometry (MS/MS) of m/z 399 at 12 eV disclosed characteristic signals at m/z 298, m/z 250, m/z 136, and m/z 102 (Figure 7).


Engineered Pichia pastoris for enhanced production of S-adenosylmethionine.

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

ESI mass spectrum corresponds to tR at 2.7 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: ESI mass spectrum corresponds to tR at 2.7 min.
Mentions: LC/MS analysis of acid-extracted fermented cells exhibited distinct chromatographic peaks at retention times (tR) of 1.4, 1.5, 2.7, 4.2 and 5.8 minutes (Figure 5). The ESI mass spectrum of the chromatographic peak eluted at a tR of 2.7 minutes (Figure 6), and the spectrum showed [M]+ion at m/z 399 as the base peak. The spectrum also showed moderately abundant fragment ion peaks at m/z 355, 298, 136 and highly abundant ion peak at m/z 250. The collision induced dissociation (CID) tandem mass spectrometry (MS/MS) of m/z 399 at 12 eV disclosed characteristic signals at m/z 298, m/z 250, m/z 136, and m/z 102 (Figure 7).

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