Engineered Pichia pastoris for enhanced production of S-adenosylmethionine

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.

Characterization and designing of an SAM riboswitch to establish a high‐throughput screening platform for SAM overproduction in Saccharomyces cerevisiae

“…The limiting factor of SAM synthesis is l ‐methionine, which is generally added directly to the fermentation medium (Hu et al, 2009; Kanai et al, 2017; Zhao, Shi, et al, 2016). In addition to the overexpression of S ‐adenosylmethionine synthetase, previous studies investigated various other strategies to improve the production of SAM, such as adding more l ‐methionine in the fermentation process (Huang et al, 2012; Kamarthapu et al, 2013; Zhang et al, 2008; Zhao, Hang, et al, 2016; Zhao, Shi, et al, 2016), regulating the intracellular ATP concentration (Chen & Tan, 2018; Chen et al, 2017), or deleting the SPE2 gene (encoding SAM decarboxylase) (Balasundaram et al, 1994), GLC3 gene (encoding a glycogen branching enzyme) (Rowen et al, 1992; Zhao, Hang, et al, 2016) and SAH1 gene (encoding S ‐adenosyl‐ l ‐homocysteine hydrolase) (Ano et al, 2009; Mizunuma et al, 2004) to minimize SAM degradation and consumption (Chen et al, 2016; He et al, 2006; Zhao, Hang, et al, 2016; Zhao, Shi, et al, 2016). These strategies have achieved good results, but excessive supply of the precursors l ‐methionine and ATP, as well as the knockout of bypass pathway genes, will also inhibit cell growth.…”

Efficient production of S‐adenosyl‐l‐methionine from dl‐methionine in metabolic engineered Saccharomyces cerevisiae

“…In previous studies, many screened or genetically engineered yeast strains have been used to enhance SAM production (Chu et al, ; Chen, Wang, Cai, et al, ), such as the deletion of SPE2 encoding the S‐adenosylmethionine decarboxylase (Balasundaram, Dinman, Tabor, & Tabor, ) and GLC3 encoding a glycogen branching enzyme (Rowen, Meinke, & LaPorte, ), which are involved in SAM degradation and glycogen accumulation, respectively (Zhao, Hang, et al, ); the deletion of SAH1 encoding the S‐adenosyl‐ l ‐homocysteine hydrolase, which catalyzes the degradation of S‐adenosyl‐ l ‐homocysteine (Ano et al, ; Mizunuma, Miyamura, Hirata, Yokoyama, & Miyakawa, ) and the overexpression of S ‐adenosylmethionine synthetase (Chen, Wang, Wang, Dou, & Zhou, ; He, Deng, Zheng, & Gu, ; Zhao, Hang, et al, ; Zhao, Shi, et al, ) encoded by SAM2 , which catalyzes the transfer of the adenosyl group of ATP to the sulfur atom of methionine and was the rate‐limiting enzyme for SAM biosynthesis (Thomas, Rothstein, Rosenberg, & Surdin‐Kerjan, ). However, l ‐methionine was fed as the substrate for SAM production in most cases (Huang et al, ; Kamarthapu, Ragampeta, Rao, & Reddy, ; Zhang, Wang, Su, et al, ; Zhao, Hang, et al, ; Zhao, Shi, et al, ). Only one‐third of the price of l ‐methionine (Ren et al, ; Zhang, Gedicke, Kuznetsov, Staroverov, & Seidel‐Morgenstern, ), dl ‐methionine could be the cost‐saving substrate for industrial production of SAM.…”

S-Adenosyl-l-methionine production by Saccharomyces cerevisiae SAM 0801 using dl-methionine mixture: From laboratory to pilot scale

“…In previous studies, a relatively high concentration of SAM, exceeding 1% (w/w), was achieved in the culture of Saccharomyces cerevisiae and Pichia pastoris (see Table 2). However, the drawbacks of using yeast as microbial catalysis are obvious: the expensive L-Met substrate needs to be added, and the SAM productivity is relatively low [13][14][15].…”