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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

SDS-PAGE analysis showing expression of Saccharomyces SAM synthetase in P. pastoris. Lane 1: Control, GS115 transformed with vector alone showing no expression of SAM synthetase. Lane 2: Recombinant P. pastoris showing induced band of SAM synthetase (~ 42 kDa). Lane 3: Pre-stained marker.
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Figure 2: SDS-PAGE analysis showing expression of Saccharomyces SAM synthetase in P. pastoris. Lane 1: Control, GS115 transformed with vector alone showing no expression of SAM synthetase. Lane 2: Recombinant P. pastoris showing induced band of SAM synthetase (~ 42 kDa). Lane 3: Pre-stained marker.

Mentions: Induction of recombinant P. pastoris with 1% methanol at log phase of OD600 = 3.0 and pH of 6.0, resulted in the expression of SAM2 protein of ~ 42 kDa, whereas control GS115 showed no such band (Figure 2). All the 15 clones, expressing SAM2 gene, exhibited enhanced accumulation of the SAM, when compared to the control GS115 transformed with the vector alone. One of the high expressing transformant was selected for further studies, which yielded wet cell weight (28 g/L) and SAM (0.6 g/L or 0.195 g/g DCW). Whereas, the control (transformant with vector alone), under the identical culture conditions with similar wet cell mass, produced 0.018 g/L SAM (Figure 3) or 0.0054 g SAM/g DCW. Further, the clone expressing SAM2 gene showed more than 17 fold higher enzyme activity as compared to control.


Engineered Pichia pastoris for enhanced production of S-adenosylmethionine.

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

SDS-PAGE analysis showing expression of Saccharomyces SAM synthetase in P. pastoris. Lane 1: Control, GS115 transformed with vector alone showing no expression of SAM synthetase. Lane 2: Recombinant P. pastoris showing induced band of SAM synthetase (~ 42 kDa). Lane 3: Pre-stained marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: SDS-PAGE analysis showing expression of Saccharomyces SAM synthetase in P. pastoris. Lane 1: Control, GS115 transformed with vector alone showing no expression of SAM synthetase. Lane 2: Recombinant P. pastoris showing induced band of SAM synthetase (~ 42 kDa). Lane 3: Pre-stained marker.
Mentions: Induction of recombinant P. pastoris with 1% methanol at log phase of OD600 = 3.0 and pH of 6.0, resulted in the expression of SAM2 protein of ~ 42 kDa, whereas control GS115 showed no such band (Figure 2). All the 15 clones, expressing SAM2 gene, exhibited enhanced accumulation of the SAM, when compared to the control GS115 transformed with the vector alone. One of the high expressing transformant was selected for further studies, which yielded wet cell weight (28 g/L) and SAM (0.6 g/L or 0.195 g/g DCW). Whereas, the control (transformant with vector alone), under the identical culture conditions with similar wet cell mass, produced 0.018 g/L SAM (Figure 3) or 0.0054 g SAM/g DCW. Further, the clone expressing SAM2 gene showed more than 17 fold higher enzyme activity as compared to control.

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