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. 2013 Jul 27:3:40.
doi: 10.1186/2191-0855-3-40. eCollection 2013.

Engineered Pichia pastoris for enhanced production of S-adenosylmethionine

Venu Kamarthapu  1 , Srinivas Ragampeta  2 , Khareedu Venkateswara Rao  1 , Vudem Dashavantha Reddy  1
Affiliations

Engineered Pichia pastoris for enhanced production of S-adenosylmethionine

Venu Kamarthapu et al. AMB Express. .

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.

Keywords: Bioreactor; Heterologous host; Pichia pastoris; S-adenosylmethionine synthetase.

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Figures

Figure 1
Figure 1
Confirmation of transformed clones of P. pastoris by colony PCR with AOX and gene specific primers. a: Amplification with AOX primers. Lanes 1, 2, 3, 6, 7 and 8: Clones exhibiting an amplified thick band of ~ 1.4 Kb SAM2 gene and a faint band of 2.2 Kb AOX gene. Lane 4: Untransformed colony exhibiting amplified band of 2.2 Kb AOX gene. Lane 5: DNA Marker (1.0 Kb ladder) showing bands of 250 bp, 500 bp, 750 bp, 1.0 Kb, 1.5 Kb, 2.0 Kb &2.5 Kb. b: Amplification with gene specific primers of SAM2 gene. Lane 1: DNA Marker (1.0 Kb ladder) showing bands of 750 bp, 1 Kb, 1.5 Kb, 2.0 Kb, 2,5 Kb, 3.0 Kb, 3.5 Kb, 4.0 Kb, 5.0 Kb and 6.0 Kb. Lanes 2, 3, 4, 6, 7 and 8: Clones exhibiting amplified band of ~ 1.2 Kb corresponds to SAM2 gene. Lane 5: Untransformed GS115 strain showing no amplification.
Figure 2
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.
Figure 3
Figure 3
Recombinant P. pastoris GS115 expressing SAM2 gene when cultured in shake flask accumulated 33-fold higher SAM content as compared to Vector alone transformed strain of GS115.
Figure 4
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.
Figure 5
Figure 5
Total ion chromatogram using positive ESI-MS detection.
Figure 6
Figure 6
ESI mass spectrum corresponds to tR at 2.7 min.
Figure 7
Figure 7
CID MS/MS spectrum of m/z 399 at 12 eV.
See this image and copyright information in PMC

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