Enhancing the production of S-adenosyl-L-methionine in Pichia pastoris GS115 by metabolic engineering
© Yu and Shen; licensee Springer. 2012
Received: 24 September 2012
Accepted: 9 October 2012
Published: 30 October 2012
S-adenosyl-L-methionine is an important bioactive molecule participating in a number of biochemical reactions including the transmethylation and transsulphuration reactions of proteins and the biosynthesis of aliphatic polyamines. Strategies of metabolic engineering were used to alter the metabolic flux for enhancing the production of S-adenosyl-L-methionine (SAM) in Pichia pastoris GS115. These strategies include the over-expression of Sam 2 by knock-in technique and the disruption of Cbs by knock-out technique. Three strains, ZJGSU1 with knock- in of Sam 2, ZJGSU2 with knock-out of Cbs and ZJGSU3 with both knock-in of Sam 2 and knock -out of Cbs, were constructed for the effective production of SAM. Yields of SAM in strains ZJGSU1 and ZJGSU2 were 32- and 5-fold higher than in the original strain P. pastoris GS115, respectively. The strain ZJGSU3 had a dramatic increase in the SAM yield, and it was 46-fold higher compared to the original strain. These results indicate that there is a strong synergistic effect on the production of SAM by combining knock-in with knock-out techniques. The yield of SAM in ZJGSU3 strain was 4.37 g/L in a 3 L fermentor. This study provides deep insight into the effective industrial production of SAM in future.
S-adenosyl-L-methionine (SAM ) plays a significant role in many biological processes since it is a major methyl group donor in the transmethylation and transsulphuration reactions of proteins, nucleic acids, polysaccharides and fatty acids (Cantoni,1953; Meister et al.1984). SAM is very effective in the treatment of osteoarthritis, affective disorders and liver diseases (Barcelo et al.1990; Lieber,1999). Recently, SAM as an intracellular bioactive small molecule has deserved more attentions due to its critical roles in human health and has been successfully used in human therapy for the depressive syndrome and the osteoarthritis (Barcelo et al.1990; Cimino et al.1984; Osman et al.1993).
SAM is prepared commercially by the extraction of yeast cells cultured in media supplied with L-methionine (L-Met) (Schlenk et al.1965; Shiomi et al.1990). Two isozymes of SAM synthase, SAM1 synthase and SAM2 synthase which are respectively encoded by genes Sam 1 and Sam 2, have been identified in Saccharomyces cerevisiae. Sam 1 transcription can be inhibited with excessive L-Met while Sam 2 not (Thomas et al.1988). S. cerevisiae has been one of the most commonly used strain for the production of SAM so far (Liu et al.2006; Schlenk et al.1965; Shen et al.2008; Shiomi et al.1990; Wang and Tan,2008; Wang et al. 1965). However, it is not a best choice for over-expressing SAM synthase and overproducing SAM due to the presence of ethanol during culture, which often makes SAM2 ineffective and hence lowers the yield of SAM. In addition, many strains belonging to Saccharomyces sake and Kluyveromyces lactis have also been studied for the production of SAM (Mincheva et al.2002; Shiozaki et al.1989).
In the present study, three genetically modified P. pastoris strains, ZJGSU1 with over- expression of Sam 2, ZJGSU2 with disruption of Cbs and ZJGSU3 with both over-expression of Sam 2 and disruption of Cbs, were constructed and yields of SAM in these three strains in flasks were determined and compared. Over-expression of Sam 2 and disruption of Cbs in P. pastoris GS115 show a significantly synergistic effect on the production of SAM. The SAM production in ZJGSU3 strain were also investigated in a 3L fermentor.
Materials and methods
Oligonucleotide sequences used as primers
Construction of the plasmid pPIC9K-Sam2 and introduction of Sam2 into P. pastoris GS115
Knock-out of Cbs in P. pastoris GS115
The activities of SAM synthase in four strains
Enzyme activity (U/mL)
P. pastoris culture for the SAM production in flasks
Four strains, P. pastoris GS115, ZJGSU1, ZJGSU2 and ZJGSU3, were respectively inoculated into a 5 mL YPD medium and incubated at 30°C for 16 h as seed cultures. A 1mL aliquot of the seed culture was added to 100 mL BMGY medium (yeast extract 1%, peptone 2%, glycerol 1%, 10×YNB10%, 0.1M potassium phosphate buffer, pH 6.0) supplemented with 1% L-Met in 500 mL flasks and cultured at 30°C for 4 d. 1.5% (v/v) of methanol as an inducer and carbon source was added daily for three times after one day of growth. After 96 h of culture, 1 mL of sample was taken out for determining the yield of SAM.
Fermentation of the ZJGSU3 strain in a 3 L fermentor
The ZJGSU3 strain was grown in a BMGY medium at 30°C for 24 h, 100 mL of cultures were inoculated in a 3 L fermentor containing 1.8 L of minimal salts (PTM) fermentation medium: H3PO4 (85% stock), 27 mL/L; CaSO4, 0.93 g/L; K2SO4, 18.2 g/L; MgSO4·7H2O, 14.9 g/L; KOH, 4.13 g/L; Glycerol, 40 g/L and 4.4 mL/L of PTM1 trace metals solution (P. pastoris fermentation manual of the Invitrogen). Initial fermentation conditions were as follows: dissolved oxygen (DO) was maintained above 20% by the automatic control of agitation, pH was 5 (adjusted with ammonium hydroxide), temperature was set at 30°C. Cells were grown until glycerol depleted completely. This was indicated by a dramatic increase in the DO to 100%. Glycerol feeding was then initiated to increase the cell biomass under limited conditions: 500 mL of 50% glycerol containing 6 mL of PTM trace salts was fed at 60 mL/h. Following the depletion of glycerol, methanol feeding was initiated at a rate of 10 mL/h and increased gradually to a final rate of 21 mL/h. Meanwhile, 500 mL of the saturated L-Met was fed at a rate of 6 mL/h. Samples were taken out at different times for determining the yield of SAM and biomass.
Determination of the yield of SAM
The yield of SAM was determined as described by Lin et al. (2004). 1 mL of the sample was centrifuged at 12,000 rpm for 10 min, and washed twice with the deionized water, then mixed with 1 mL of 1.5 M HClO4 at 4°C for 1.5 h. The supernatant was collected after centrifugation at 12,000 rpm for 5 min and then went through a 0.22 μm filtration membrane before analysis. A 15 μL of the extracted SAM sample was injected into a high performance liquid chromatography (HPLC) system (Agilent, USA) using a C18 column (Hypersil BDS column, 4.6 mm×250 mm, 5 μm) with a mobile phase composed of 0.01 M ammonium formate (pH3.0) at a flow rate of 0.8 mL/min. Peak area analysis was performed based on the standard calibration curve of SAM. Due to the instability of SAM, its p-toluenesulfonate salt (Sigma) was used as standard sample.
Determination of wet cell weight
mL of the sample was taken out and centrifuged at 12,000 rpm for 10 min, and then washed three times with the distilled water. The wet cell weight was determined using a electronic balance.
Determination of the activity of SAM synthase
The activity of SAM synthase was determined as described by Li et al. (2002). Cells in 1mL of the sample were harvested by centrifugation at 6000 rpm for 5 min, and washed immediately with ice-cold lytic buffer (50 mM potassium phosphate at pH 7.4, 5% (v/v) glycerol, 5 mM mercaptoethanol, 1 mM EDTA), and then centrifuged at 12,000 rpm for 5 min. Cell pellets were resuspended in 1 mL of the lytic buffer and disrupted by the ultrasonic treatment. The supernatant was collected by centrifugation at 12,000 rpm for 10 min and used as the sample to assay the activity of SAM synthase as follows:1 mL of the reaction mixture, containing 20 mM L-Met, 20 mM ATP, 8 mM reduced glutathione, 20 mM MgCl2, 100 mM KCl, 150 mM Tris -HCl and the supernatant with an appropriate concentration, was incubated at 37°C for 1 h. 0.5 mL of 20% HClO4 was then added to the reaction mixture. The resultant precipitation was removed by centrifugation at 12,000 rpm for 10 min. The supernatant was used for determining the yield of SAM by the HPLC. One unit of the activity of SAM synthase was defined as the amount of enzyme required to catalyze the transformation of 1μmol of L-Met into SAM per minute at 37°C.
Identification of the introduction of Sam2 or the disruption of Cbs and their respective effect on the production of SAM
Over 5000 transformants grew on MD plates after the electro transformation of pPIC9K-sam2 into P. pastoris GS115. Transformants having a better growth rate on MD plates were streaked on YPD plates containing different concentrations of G418 (0.5, 1.0, 2.0, 3.0, 4.0 mg/mL). 25 transformants having a faster growth rate were picked from YPD plates with G418 at a final concentration of 4 mg/mL. These transformants were cultured in flasks for evaluating the yield of SAM by HPLC. The transformant which produced the highest yield of SAM was chosen finally and named ZJGSU1. According to the manual of the “multi-copy of P. pastoris expression kit” from the Invitrogen, multiple integrated copies can lead to the increase in the G418 resistance level from 0.5 mg/mL (1–2 copies) to 4 mg/mL (7–12 copies). Thus, the copy number of the Sam 2 gene in ZJGSU1 strain was estimated to be 7–12 based on its resistance to G418 at a final concentration of 4 mg/mL. The yield of SAM in ZJGSU1 strain reached 0.8 g/L.
Effect of both knock-in of Sam2 and knock-out of Cbs on the production of SAM
Analysis of the activity of SAM synthase
The activities of SAM synthase in strains GS115, ZJGSU1, ZJGSU2 and ZJGSU3 were determined. The result is showed in Table2. The activity of SAM synthase in ZJGSU3 was up to 705 U/ mL, and was nearly twice than that in ZJGSU2 strain and 18-fold higher than that in the original strain GS115. The activity of SAM synthase in ZJGSU1 strain was 544 U/mL, 14-fold higher than that in the original strain GS115. These results further confirm that enhancing the activity of SAM synthase facilitates the production of SAM. In addition, by comparison of the activities of SAM synthase from four strains, it was also found that the knock-out of Cbs could increase the expression of Sam 2, and hence enhanced the activity of SAM synthase and increased the yield of SAM.
Fermentation of SAM in a 3L fermentor
The results of the present study demonstrate that both introduction of Sam 2 and disruption of Cbs in P. pastoris GS115 by knock-in and knock-out techniques have a obviously synergistic effect on the production of SAM, resulting in a significant increase in the yield of SAM. The yields of SAM in ZJGSU3 strain with both introduction of Sam 2 and disruption of Cbs were 1.2 g/L in flasks and 4.37 g/L in a 3 L fermentor, implying that it has a commercial prospect for the large scale industrial production of SAM in future. To our knowledge, this is the first report with regard to the production of SAM by both introduction of Sam 2 and disruption of Cbs in P. pastoris GS115 strain using the plasmid pPIC9K as the vector.
Previously, the reports pertinent to the production of SAM focused on the S. cerevisiae strain (Liu et al.2006; Schlenk et al.1965; Shen et al.2008; Shiomi et al.1990; Wang and Tan,2008; Wang et al. 1965), S. sake (Shiozaki et al.1989) and K. lacti (Mincheva et al.2002). The traditional fermentation is a main way for the production of SAM in those strains. With the development of the genetic and metabolic engineering, enhancing yields of valuable bioactive chemicals by the modern biotechnology becomes an important trend. The yield of SAM was enhanced by altering its metabolic flux in P. pastoris GS115 strain in our study. S. cerevisiae contains two SAM synthase genes, Sam 1 and Sam 2. Sam 1 is repressed by the excessive L-Met, whereas Sam 2 is not. Because L-Met is an important precursor for the effective accumulation of SAM, Sam 2 is chosen for being introduced into P. pastoris GS115 strain for enhancing the yield of SAM in this study. In the previous studies, Le et al. (2002) and Yu et al. (2003) reported that the knock-in of SAM synthase gene resulted in the increase in the yield of SAM in Pichia. Li et al. (2002) also reported a constructed strain with knock-out of Cbs, but did not investigate the knock-out effect on the production of SAM. Chan and Appling (2003) reported that the deletion of Cbs in S. cerevisiae did not cause a high accumulation of SAM, and the effect of the production of SAM in wild type and mutant strains was not also compared. In our study, the knock-out of Cbs in P. pastoris GS115 strain not only increases the yield of SAM, but also enhances the expression of SAM synthase, and so alters the metabolic flux of SAM and facilitates its further accumulation. Furthermore, a synergistic effect of both introduction of Sam 2 and disruption of Cbs in the P. pastoris GS115 strain by knock-in and knock-out techniques on the production of SAM is for the first time reported in our study. Compared to S. cerevisiae, P. pastoris is a better candidate for the industrial production of recombinant proteins and valuable biochemical molecules. It can grow easily to a high cell density in a minimal salts medium with methanol as a sole carbon source and ammonium sulfate as a sole nitrogen source. The genetically modified P. pastoris (ZJGSU3) reaches a wet cell weight of 247 g/L and produces 4.37 g/L of SAM in a 3 L fermentor.
In conclusion, a genetically modified strain ZJGSU3 for the effective production of SAM was obtained by both knock-in of Sam 2 and knock-out of Cbs in the P. pastoris GS115 strain. This strain shows a great potential for the industrial production of SAM. Continuous efforts should be given to further optimize cultural conditions for the large-scale production of SAM in the fermentor and purify it for the application in the fields of biomedicines, chemical engineering and pharmaceuticals.
We thank the National Natural Science Foundation of China (No.31171658) and the Zhejiang Province Natural Science Foundation (No.Y4080060) for generous granting this study.
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