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.
Highlights
S-adenosylmethionine (SAM) is a metabolite of the methionine metabolism
The recombinant plasmid pPIC3.5 K-SAM2 linearized with Sac I was introduced into P. pastoris strain GS115 by electroporation method
Biosynthesis of SAM is catalyzed in cytosol of cell by SAM synthetase using ATP and L-methionine as the substrates
Summary
S-adenosylmethionine (SAM) is a metabolite of the methionine metabolism. In diverse living organisms, it is known to mediate three important metabolic pathways, viz., transmethylation, trans-sulphuration, and aminopropylation (Chiang et al 1996; Bottiglieri 2002; Fontecave et al 2004; Roje 2006) SAM is involved in various other reactions that occur through radical-based catalysis (Grillo and Colombatto 2007). It is known to act as a key physiological compound essential for optimal hepatic function, proper functioning of joints, as well as gastric mucosa protection (Lu 2000; Lu and Mato 2008). As such, it is used extensively in mitigating depression, liver disease and osteoarthritis (Castillo et al 2005; Papakostas et al 2003; Mischoulon et al 2012; Ringdahl and Pandit 2011; Papakostas et al 2012; Harmand et al 1987). Yeast strains have been isolated that could accumulate increased concentration of SAM in comparison with other microorganisms (Shiozaki et al 1984). An efficient positive selection method has been optimized for isolation of SAM accumulating yeast strains with significant increases in SAM content on a dry cell weight basis. The volumetric yield of SAM is low as most of the strains belonged to Saccharomyces cerevisiae and were hardly able to attain high cell density using minimal media (Shobayashi et al 2006)
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