- Email: [email protected]
Oxygen vectors used for S-adenosylmethionine production in recombinant Pichia pastoris with sorbitol as supplemental carbon source
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 105, No. 4, 335–340. 2008 DOI: 10.1263/jbb.105.335
© 2008, The Society for Biotechnology, Japan
Oxygen Vectors Used for S-Adenosylmethionine Production in Recombinant Pichia pastoris with Sorbitol as Supplemental Carbon Source Jian-Guo Zhang,1 Xue-Dong Wang,1* Ji-Ning Zhang,1 and Dong-Zhi Wei1 State Key Laboratory of Bioreactor Engineering, College of Biotechnology, East China University of Science and Technology, No. 130 Meilong Road, Shanghai 200237, People’s Republic of China1 Received 4 September 2007/Accepted 27 December 2007
In order to increase the yield of S-adenosylmethionine (SAM) in recombinant Pichia pastoris, a strategy of adding oxygen vectors and supplemental carbon sources was described. Three organic solutions were used as oxygen vectors for SAM accumulation at different concentrations and addition times. Firstly, n-hexane (0.5%) or n-heptane (1.0%) was added after 72 h of cultivation to improve SAM production. Carbon metabolism was scarce during the induction phase because of low methanol concentration. Secondly, sorbitol (1.2%), selected from three candidates (glycerol, lactic acid, and sorbitol), was used as the supplemental carbon source. The yield of SAM was improved significantly (53.26%) at 1.0% n-heptane added at 72 h (48 h induction), 1.2% sorbitol added at 72, 96, and 120 h of cultivation and 1.0% methanol added every 24 h during cultivation. [Key words: S-adenosylmethionine, fermentation, oxygen vectors, Pichia pastoris, supplemental carbon source]
S-Adenosylmethionine (SAM), a metabolite in a biological body, is used as a drug to treat liver diseases, depression, osteoarthritis, fibromyalgia, and Alzheimer’s diseases because it has biological functions: a methyl donor, the precursor of aminopropyl groups, and the trans-sulfuration pathway (1). SAM presents a unique feature in which all its constituent parts have a chemical use (2). SAM synthetase catalyzes ATP and L-methionine for SAM production. In recent years, SAM has been produced commercially by chemical synthesis and enzymatic synthesis in vitro (3). Research on SAM accumulation by microorganisms has been carried out for its potential industrial production. Shiozaki et al. optimized components in a medium enriched with L-methionine for wild type Saccharomyces sake for SAM accumulation in 1986 (4). In recombinant microorgnisms, SAM was produced as catalyzed by high-activity recombinant SAM synthetase expressed and cystathionine synthase knocked out in Pichia pastoris simultaneously (5) or by adding glycerol intermittently during the induction phase of P. pastoris fermentation (6). The advantages of recombinant P. pastoris expressing SAM synthetase for SAM accumulation are as follows: (i) High-activity SAM synthetase is expressed in recombinant P. pastoris. However, inclusion bodies are formed when a heterologous gene is expressed in Escherichia coli; (ii) P. pastoris has a high-cell-density fermentation (130 DCW g/l). P. pastoris can accumulate volumetric SAM plentifully; (iii) The P. pastoris fermentation process is simple and can be referred to the Pichia fermentation process guideline (Invitrogen, Carlsbad, CA, USA); (iv) The simplicity of
techniques required for molecular genetic manipulation and availability of commercial expression kits. Over 500 heterogenous genes, which came from viruses, prokaryotic, and eukaryotic miroorganisms, protists, plants, and invertebrates including humans, are expressed in P. pastoris (7, 8); (v) The P. pastoris fermentation medium is basal salt medium, which is advantageous for bioprocessing in bioreactors industrially owning to its low cost and definite component; (vi) In the induction phase, methanol is a cheap substrate for SAM accumulation. Oxygen limitation is the common problem encountered during P. pastoris fermentation. However, oxygen limitation can be overcome by improving the oxygen transfer coefficient (KLa) (9, 10). On the other hand, improvement of KLa can be realized by adding oxygen vectors (11). Oxygen vectors are hydrophobic liquids in which oxygen has higher solubility than in water. The mechanisms of KLa improvement by oxygen vector addition was clarified by Rols et al. (12). There were many reports indicating that oxygen vectors can increase KLa significantly to improve microorganism metabolism for metabolite production (13–17). The advantages of oxygen vectors are effective oxygen solubility, low cost, and low possibility of danger. In this study, a recombinant P. pastoris, haboring SAM2 from Saccharomyces cerevisiae, was cultivated in L-methionine-enriched medium to express SAM synthetase for SAM production (Fig. 1). Methanol was used as the carbon source and inducer of SAM synthetase expression. A low methanol concentration was controlled accurately so as not to cause methanol toxicity to P. pastoris. As SAM is a metabolite in the recombinant P. pastoris, ATP is the limiting factor when SAM is accumulated in recombinant P. pastoris with a high
* Corresponding author. e-mail: [email protected] phone: +86-21-64253156 fax: +86-21-64250068 335
336
ZHANG ET AL.
FIG. 1. Biosynthesis of SAM in recombinant Pichia pastoris. Methanol induced SAM2 expression to form SAM synthetase. At the same time, methanol was oxidized to water and carbon dioxide with ATP released. With L-methionine added to the medium, SAM was formed from ATP and L-methionine by catalysis of SAM synthetase.
activity of SAM synthetase (6), although SAM synthetase was reported as the bottleneck of SAM accumulation in wildtype strains (4). Improvement of cellular carbon metabolism is important for SAM production. Therefore, a nonrepressive carbon source should be used during the induction phase to compensate for carbon scarcity. The purpose of this work was to improve cellular carbon metabolism for SAM production by adding oxygen vectors and a nonrepressive carbon source.
MATERIALS AND METHODS Strain construction To remove the α-factor signal, LA-Taq DNA polymerase was used with pPIC9k as the template to amplify the pPIC9K fragment with two restriction sites (BamHI and EcoRI). The PCR fragment was digested with BamHI and EcoRI in the double digestion system for SAM2 ligation. SAM2 was amplified from the genomic DNA of S. cerevisiae using two primers: 5′-CA GGATCCACCATGTCCAAGAGCAAAACT-3′ and 5′-GCGG CCGCGAATTCAGCCTAGCATAAAGAAA-3′, which incorporated BamHI and EcoRI restriction sites, respectively. The primers were designed in accordance with the Genebank sequence number M23368 (18). Amplification was performed in a final volume of 50 µl, and the reaction mixture contained 100 ng of genomic DNA, 25 pmol of each primer (Invitrogen), 200 µmol/l of dNTP, 1 × PCR buffer, and 1 U of Taq-TM DNA polymerase (Takara Biotechnology, Dalian, China). The following PCR program was used for the amplification of about 1200 bp DNA fragment: 5 min of denaturation at 94°C; 10 cycles of denaturation (30 s at 94°C), annealing (30 s at 42°C), and polymerization (90 s at 72°C); 20 cycles of denaturation (30 s at 94°C), annealing (30 s at 55°C), and polymerization (90 s at 72°C) followed by an additional polymerization (10 min at 68°C). The amplified DNA fragment was digested with BamHI and EcoRI in the double digestion system and inserted into the expression vector, the pPIC9K fragment. The plasmid pPIC9KSAM2 was transformed in P. pastoris GS115 using a Multi-Copy Pichia expression kit in accordance with the manufacturer’s instruction (Invitrogen) after linearization by SacI, and the recombinant P. pastoris was screened using YPD-G418 agar plates (10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose, 15 g/l agar powder, 100 µg/ml G418) for flask cultivation. Medium BMGY medium consists of 10 g/l yeast extract, 20 g/l peptone, 100 mmol/l potassium phosphate buffer (pH 7.0), 13.4 g/l yeast nitrogen base, 400 µg/l biotin, 20 g/l glycerol, 50 mmol/l L-methionine. Culture condition Cells were transferred from YPD agar plates into 250 ml flasks, which contained 50 ml of BMGY and in-
J. BIOSCI. BIOENG.,
cubated at 30°C and 200 rpm for 18–20 h. Cells were used as the inoculum for flask cultivation. Flask cultivation was performed in 250 ml flasks containing 25 ml of BMGY medium at 30°C and 200 rpm with 4% BMGY inoculum. One percent methanol (0.25 ml) was added into BMGY after 24 h of cultivation to induce SAM synthetase for SAM production every day. In our experiments, all experiments by flask cultivation were carried out with three trials. The error bar was obtained using the standard deviation function (Office 2003, Microsoft). The error bar shows the deviation along a curve. Biomass Cells (5 ml) were harvested by centrifugation the broth at 6000 rpm for 3 min, then washed twice with deionized water, and then dried to a constant weight at 90°C. The optical density of the broth (100-fold diluted) was monitored using a spectrophotometer (U-1100; Hitachi, Tokyo) at 600 nm. The dry cell weight (DCW) of a sample was obtained from the experimentally determined calibration curve between cell optical density and the DCW. Methanol concentration The cell supernatant (0.5 ml) obtained by centrifugation at 6000 rpm for 3 min was mixed with the isopyknic mixture of 1-butanol and 1-propanol (4: 1) to extract methanol. The superstratum was transferred to a new eppendorf tube after placement in room temperature for 15 min and centrifugation at 12,000 rpm for 1 min. Sodium sulfite was used to remove water from the superstratum. The superstratum (1 µl) was obtained for analysis by centrifugation at 12,000 rpm for 1 min. Methanol concentration was determined by gas chromatography (GC 6890N; Agilent, Santa Clara, CA, USA) using a DB-WAX column. The temperatures of injection and detector were 200°C and 250°C, respectively. The column temperature profile was as follows: 100°C for 1 min, which increased at a rate of 20°C/min until the temperature reached 200°C. Yield of SAM The yield of SAM was determined by mixing 0.5 ml of fermentation broth with isopyknic 20% HClO4 and storing the mixture for 8 h. The superstratum was analyzed after centrifugation of the mixture at 12,000 rpm for 3 min and filtration using a membrane filter (φ 0.22 µm). SAM was analyzed by HPLC (HP1100; Agilent) using a Hypersil SCX column (4.6 × 250 mm, 5 µm). The mobile phase was 0.5 mol/l ammonium formate (pH 4.0). Data were quantified by Agilent chemstation. SAM synthetase P. pastoris, used for SAM synthetase measurement, was cultured in BMGY without L-methioine because L-methionine is a substrate in the determining reaction. P. pastoris (5 ml) cells were collected by centrifugation at 6000 rpm for 3 min, and washed immediately with lysis buffer (50 mmol/l PBS pH 7.4, 1 mmol/l phenylmethanesulfonyl fluoride, 1 mmol/l EDTA, 5% [v/v] glycerol), then precipitated at 12,000 rpm for 5 min. The resulting cell pellets were suspended in 1 ml of lysis buffer and disrupted by ultrasonication for 3 s at 6 s intervals (total 10 min). Finally, cell debris was removed by centrifugation at 12,000 rpm for 10 min, the resulting supernatant was used to measure the activity of SAM synthetase in accordance with the method of Shiozaki et al. (19). One unit of enzyme activity was defined as the amount of actibity required to catalyze the transformation of 1 µmol of L-methionine into SAM per minute at 37°C. Sorbitol concentration Sorbitol was measured by mixing 0.6 ml of 5% CuSO4, 0.56 ml of 10% NaOH, 1.0 ml of supernatant broth, and 3.0 ml of deionized water in this sequence. Then, the mixture was stated in room temperature for 15 min. The optical density of the mixture was assayed using a spectrophotometer (U-1100; Hitachi) at 655 nm after centrifugation at 4000 rpm for 3 min. Sorbitol concentration was determined using regression equation for sorbitol solution and optical density.
S-ADENOSYLMETHIONINE PRODUCTION IN P. PASTORIS
VOL. 105, 2008
337
RESULTS AND DISCUSSION Curves of cell growth, SAM production, activity of SAM synthetase, and methanol concentration of P. pastoris in flask cultivation The recombinant P. pastoris was cultivated to clarify the pattern of cell growth, yield of SAM, activity of SAM synthetase, and methanol concentration during 144 h of fermentation (Fig. 2). The recombinant P. pastoris grew rapidly during the first 24 h of cultivation, and then grew slowly untill the end, as DCW remained constant after 24 h of cultivation. One percent methanol was added to the medium to induce SAM synthetase every 24 h after 24 h of cultivation. The yield of SAM increased rapidly in the earlier stage of induction (24–72 h of cultivation) and then became slow and even stable untill the end. Therefore, the yield of SAM after 144 h of cultivation was used as the indicator to define SAM perturbations under different conditions. The activity of SAM synthetase existed, and did not disappear during the induction phase (Fig. 2). Therefore, it was ATP that was the limiting factor as SAM yield became stable after 72 h of cultivation, although SAM synthetase activity increased with time. Thus, a large amount of the carbon source should be available in P. pastoris during SAM accumulation because ATP production is closely related to carbon metabolism. Methanol concentration was low, and decreased slowly during the induction phase. Effects of oxygen vectors on cell growth, SAM production, and methanol concentration Three oxygen vectors (n-hexane, n-heptane, and n-dodecane) at different final concentrations (0.5%, 1.0%, 2.0%, 4.0%, and 6.0%) at the beginning of flask cultivation were used respectively to improve SAM production. The concentrations of oxygen vectors should be determined because KLa is affected by oxygen vector concentration (12). The effects of oxygen vectors at different concentrations on cell growth, SAM production, and methanol concentration were investigated after 144 h of cultivation (Fig. 3). Firstly, it was found that DCW increased up to
FIG. 2. Time courses of cell growth (A), SAM production (B), SAM synthetase (C), and methanol concentration (D) during 144 h of cultivation of Pichia pastoris.
4% n-hexane addition (Fig. 3A) and 1% n-heptane addition (Fig. 3B). n-Dodecane did not induce a significant change in DCW, as shown in Fig. 3C. Secondly, regarding the yield of SAM, 0.5% n-hexane addition and 1.0% n-heptane addition increased SAM yield to 4.88% and 3.96%, respectively. The yield of SAM decreased as n-dodecane concentration increased (Fig. 3C). Thirdly, methanol concentration was assayed to determine the effects of oxygen vectors on methanol utilized by P. pastoirs. The lowest methanol concentration was found at 0.5% n-hexane addition and 1.0% n-heptane addition. Whereas, the yield of SAM was the highest at the concentrations of oxygen vectors added (Fig. 3A, B). However, for n-dodecane addition, methanol concentration increased as the yield of SAM decreased (Fig. 3C). It was concluded that P. pastoris utilized methanol efficiently in the
FIG. 3. Effects of addition of three oxygen vectors (n-hexane, n-heptane, and n-dodecane) at the beginning of fermentation on cell growth, SAM production, and methanol concentration after 144 h of cultivation.
338
ZHANG ET AL.
FIG. 4. Effects of time of adding two oxygen vectors on DCW, yield of SAM, and methanol concentration after 144 h of cultivation. (A) n-Hexane (0.5%), (B) n-heptane (1.0%).
presence of 0.5% n-hexane or 1.0% n-heptane for SAM production. Effects of time of adding oxygen vectors on cell growth, SAM production, and methanol concentration The time of adding oxygen vectors should be defined for the following two reasons. The first reason was that oxygen vectors were used to increase the yield of SAM after 72 h of cultivation, which is the time when the yield of SAM was stable (Fig. 1). The second was that the time of immersing cells into oxygen vectors should be as short as possible because of the oxygen vectors’ negative effect on cell viability. Oxygen vectors are organic solutions that are used for cell disruption in intracellular product extraction. n-Hexane and n-heptane, at the final concentrantions of 0.5% and 1.0%, respectively, were added to the medium at different cultivation times (0, 24, 48, 72, 96, and 120 h). n-Dodecane was excluded in this experiment owing to its negative effect on the yield of SAM. Figure 4 shows the effects of time of adding 0.5% n-hexane and 1.0% n-heptane on cell growth, SAM production, and methanol concentration. In Fig. 4A, DCW did not change significantly regardless of the time of adding 0.5% n-hexane. As shown in Fig. 4B, DCW increased by 8.64% compared with the control for addition at 48 h, and then decreased after addition at 72 h. The same pattern of SAM yield decrease before 72 h of cultivation and then increase is observed in Fig. 4. The yields of SAM were the lowest (1.06 and 0.87 g/l) when 0.5% n-hexane or 1.0% n-heptane was added at 48 h of cultivation, while they were the highest (1.28 and 1.25 g/l) when 0.5% n-hexane or 1.0% n-heptane was added at 72 h of cultivation. SAM is an important metabolite and it is used for biosynthesis of many compounds in microorganisms. The yield of SAM in microorganisms is considered as a pool of metabolism (18). As shown in Fig. 1, the yield of SAM is stable after 48 h of induction in recombinant P. pastoris. This is because SAM synthesis and SAM consumption were balanced after 48 h of induction. As shown in Fig. 4, the variability of SAM production indicated the different effects of oxygen vectors on the balance between SAM biosynthesis and consumption. At 24 h or 48 h of adding oxygen vectors, SAM decomposition may be more rapid than SAM bio-
J. BIOSCI. BIOENG.,
FIG. 5. Effects of amount of supplemental carbon sources (glycerol, lactic acid, and sorbitol) each added three times (72, 96, and 120 h) on cell growth after 144 h of cultivation. Symbols: filled squares, glycerol; empty circles, lactic acid; empty triangles, sorbitol.
synthesis. Therefore, the yield of SAM was lower than that of control, and vice versa. n-Hexane and n-heptane were organic solutions from there chemical bases. Some as yet undiscovered functions except those of oxygen vector might have occurred in microorganisms when a vector was added at 48 h of cultivation. Therefore, there was an imbalance between the yield of SAM and methanol concentration. That is, methanol concentration was not high when n-heptane was added at 48 h, although the yield of SAM was the lowest. SAM synthetases in recombinant P. pastoris, when oxygen vectors were added at 72 h of cultivation, were assayed (56.01, 58.18, and 28.95 U/l for the control, 0.5% n-hexane and 1.0% n-heptane) after 144 h of cultivation. It was clear that carbon metabolism was enhanced when oxygen vectors were added at 72 h of cultivation. Addition of supplemental carbon sources without repression of SAM production Nonrepressive carbon sources were added to enhance cell carbon metabolism that is inhibited by the toxicity of high-concentration methanol to P. pastoris. Three carbon sources (glycerol, lactic acid, and sorbitol) were used: glycerol was reported to be nonrepressive compared with glucose (20); lactic acid was used in Xie’s study for its function of improving methanol consumption (21); it was described that sorbitol is a nonrepressive carbon source in P. pastoris fermentation (22). Three carbon sources, besides 1% (0.25 ml) methanol added every 24 h, were added at 72, 96, and 120 h of cultivation continuously at different final concentrations of 0.2%, 0.4%, 0.8%, 1.2% during 72–120 h cultivation. Figures 5 and 6 show the effects of supplemental carbon source addition on cell growth and SAM production, respectively. The nonrepressive effect on cell growth was observed when glycerol or sorbitol was added. As for lactic acid, DCW decreased as the concentration of lactic acid added increased. This might be due to the decrease in pH in the medium following lactic acid addition to the medium. However, pH can not be maintained accurately all the time in flask cultivation. The yield of SAM decreased significantly after glycerol or lactic acid was added. The yield of SAM did not decrease when 0.8% or 1.2% sorbitol was added. From the view point of supplemental carbon source selection for SAM production, glycerol and lactic acid were excluded in flask cultivation. Therefore, sorbitol
VOL. 105, 2008
S-ADENOSYLMETHIONINE PRODUCTION IN P. PASTORIS
339
FIG. 6. Effects of amount of supplemental carbon sources (glycerol, L-lactic acid, and sorbitol) each added three times (72, 96, and 120 h) on SAM production after 144 h of cultivation. Symbols: filled squares, glycerol; empty circles, lactic acid; empty triangles, sorbitol.
FIG. 8. Effects of sorbitol and oxygen vectors on carbon source consumption and SAM synthetase.
FIG. 7. Synergetic effects of sorbitol and oxygen vectors on cell growth and SAM production. Control: 1.0% methanol was added at 24, 48, 72, 96, and 120 h of cultivation; sorbitol: 1.0% methanol was added at 24, 48, 72, 96, and 120 h of cultivation and 1.2% sorbitol was added at 72, 96, and 120 h of cultivation; sorbitol-hexane: 1.0% methanol was added at 24, 48, 72, 96, and 120 h of cultivation, 1.2% sorbitol was added at 72, 96, and 120 h of cultivation and 0.5% hexane was added at 72 h of cultivation; sorbitol-heptane: 1.0% methanol was added at 24, 48, 72, 96, and 120 h of cultivation, 1.2% sorbitol was added at 72, 96, and 120 h of cultivation and 1.0% heptane was added at 72 h of cultivation.
was used as the supplemental carbon source for SAM accumulation with oxygen vector addition in the following experiment. Effects of sorbitol and oxygen vectors added synchronously on SAM production To enhance SAM production, 1.2% sorbitol was added at 72, 96, and 120 h of cultivation continuously, 1.0% methanol was added every 24 h, and 0.5% n-hexane or 1.0% n-heptane was added at 72 h of cultivation to validate the potential effect of oxygen vectors on the yield of SAM. DCW did not change and the yield of SAM increased significantly as the result of the synergetic effects of oxygen vectors and sorbitol (Fig. 7). The highest yield of SAM (1.41 g/l) was obtained when 1.2% sorbitol and 1.0% n-heptane were added, which showed a 53.26% increase compared with the control (0.92 g/l). Sorbitol and methanol concentrations were the lowest (Fig. 8), which showed the largest amount of carbon source consumed at 1.2% sorbitol and 1.0% n-heptane addition. DCW did not in-
crease significantly, although 3.52% sorbitol was consumed in the control. Other metabolites might be synthesized because dissolved oxygen limitation occurred commonly in flask experiments, inculding P. pastoris cultivation. SAM synthetase did not change markedly in the four kinds of cultivation (Fig. 8). Many studies indicated the enhancement of heterologous protein expression by improving consumption of nutrients in the medium (23). Methanol consumption has the major impact on heterologous protein expression in P. pastoris because methanol is the inducer of heterologous protein expression and the substrate for cell growth. Plantz et al. (24) clarified that the energy state is the critical factor during recombinant P. pastoris fermentation. In this work, SAM was accumulated in recombinant P. pastoris with a high activity of SAM synthetase. Methanol and sorbitol were consumed by P. pastoris to form ATP, which was used as the substrate of SAM synthetase and SAM production. SAM synthetase expression is a multistep metabolism from ATP, and SAM accumulation is one step of catalysis by SAM synthetase. Therefore, the yield of SAM was increased and SAM synthetase did not change markedly as sorbitol and methanol were consumed by P. pastoris in the presence of oxygen vectors. In this study, oxygen vectors were used to increase the yield of SAM for there function of high oxygen solubility. Large amounts of methanol and sorbitol were consumed by P. pastoirs in the presence of oxygen vectors. The yield of SAM was increased by 53.26% when sorbitol was used as the supplemental carbon source and 1.0% n-heptane was used as the oxygen vector. Addition of supplemental carbon source and oxygen vectors is a strategy to overcome energy scarcity because methanol concentration is accurately controlled to be low during P. pastoris fermentation. REFERENCES 1. Shelly, C. L.: S-Adenosylmethionine. Int. J. Biochem. Cell. Biol., 32, 391–395 (2000). 2. Fontecave, M., Atta, M., and Mulliez, E.: S-Adenosyl-
340
3. 4. 5.
6.
7. 8. 9.
10. 11. 12.
13.
J. BIOSCI. BIOENG.,
ZHANG ET AL.
methionine: nothing goes to waste. Trends Biochem. Sci., 29, 243–249 (2004). Matos, J. R., Raushel, F. M., and Wong, C. H.: S-Adenosylmethionine studies on chemical and enzymatic synthesis. Biotechnol. Appl. Biochem., 9, 39–52 (1987). Shiozaki, S., Shimizu, S., and Yamada, H.: Production of S-adenosyl-l-methionine by cultivation of wild Saccharomyces sake. J. Biotechnol., 4, 345–354 (1986). He, J. Y., Deng, J. J., Zheng, Y. H., and Gu, J.: A synergistic effect on the production of S-adenosyl-l-methionine in Pichia pastoris by knocking in of S-adenosyl-l-methionine synthase and knocking out of cystathionine synthase. J. Biotechnol., 126, 519–527 (2006). Hu, X. Q., Chu, J., Zhang, S. L., Zhuang, Y. P., Wang, Y. H., Zhu, S., Zhu, Z. G., and Yuan, Z. Y.: A novel feeding strategy during the production phase for enhancing the enzymatic synthesis of S-adenosyl-l-methionine by methylotrophic Pichia pastoris. Enzyme Microb. Technol., 40, 669–674 (2007). Cereghino, J. L. and Cregg, J. M.: Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev., 24, 45–66 (2000). Macauley-Patrick, S., Fazenda, M. L., McNeil, B., and Harvey, L. M.: Heterologous protein production using the Pichia pastoris expression system. Yeast, 22, 249–270 (2005). Cunha, A. E., Clemente, J. J., Gomes, R., Pinto, F., Thomaz, M., Miranda, S., Pinto, R., Moosmayer, D., Donner, P., and Carrondo, M. J.: Methanol induction optimization for scFv antibody fragment production in Pichia pastoris. Biotechnol. Bioeng., 86, 458–467 (2004). Zhang, W., Zheng, J. L., and Agblevor, F. A.: Microbubble fermentation of recombinant Pichia pastoris for human serum albumin production. Process Biochem., 40, 2073–2078 (2005). Jia, S., Wang, M., Kahar, P., Park, Y., and Okabe, M.: Enhancement of yeast by addition of oxygen vectors in air-lift bioreactor. J. Ferment. Bioeng., 84, 176–178 (1997). Rols, J. L., Condoret, J. S., Fonade, C., and Gome, G.: Mechanism of enhanced oxygen transfer in fermentation using emulsified oxygen-vectors. Biotechnol. Bioeng., 35, 427–435 (1990). Liu, Y. S. and Wu, J. Y.: Use of n-hexadecane as an oxygen
14. 15. 16. 17. 18.
19. 20.
21.
22. 23.
24.
vector to improve Phaffia rhodozyma growth and carotenoid production in shake-flask cultures. J. Appl. Microbiol., 101, 1033–1038 (2006). Giridhar, R. and Srivastava, A. K.: Productivity enhancement in L-sorbose fermentation using oxygen vector. Enzyme Microb. Technol., 27, 537–541 (2000). Xu, F., Yuan, Q. P., and Zhu, Y.: Improved production of lycopene and β-carotene by Blakeslea trispora with oxygenvectors. Process Biochem., 42, 289–293 (2007). Wang, J.: Enhancement of citric acid production by Aspergillus niger using n-dodecane as an oxygen-vector. Process Biochem., 35, 1079–1083 (2000). Menge, M., Mukherjee, J., and Scheper, T.: Application of oxygen vectors to Claviceps purpurea cultivation. Appl. Microbiol. Biotechnol., 55, 411–416 (2001). Thomas, D., Rothstein, R., Rosenberg, N., and SurdinKerjan, Y.: SAM2 encodes the second methionine S-adenosyl transferase in Saccharomyces cerevisiae: physiology and regulation of both enzymes. Mol. Cell. Biol., 8, 5132–5139 (1988). Shiozaki, S., Shimizu, S., and Yamada, H.: Unusual intracelluar accumulation of S-adenosyl-l-methionine by microorganisms. Agric. Biol. Chem., 48, 2293–2300 (1984). Tschopp, J. F., Brust, P. F., Cregg, J. M., Stillman, C. A., and Gingeras, T. R.: Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res., 15, 3859–3876 (1987). Xie, J., Zhou, Q., Du, P., Gan, R., and Ye, Q.: Use of different carbon sources in cultivation of recombinant Pichia pastoris for angiostatin production. Enzyme Microb. Technol., 36, 210–216 (2005). Inan, M. and Meagher, M. M.: Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. J. Biosci. Bioeng., 92, 585–589 (2001). Chauhan, A. K., Arora, D., and Khanna, N.: A novel feeding strategy for enhanced protein production by fed-batch fermentation in recombinant Pichia pastoris. Process Biochem., 34, 139–145 (1999). Plantz, B. A., Sinha, J., Villarete, L., Nickerson, K. W., and Schlegel, V. L.: Pichia pastoris fermentation optimization: energy state and testing a growth-associated model. Appl. Microbiol. Biotechnol., 72, 297–305 (2006).
© 2008, The Society for Biotechnology, Japan
Oxygen Vectors Used for S-Adenosylmethionine Production in Recombinant Pichia pastoris with Sorbitol as Supplemental Carbon Source Jian-Guo Zhang,1 Xue-Dong Wang,1* Ji-Ning Zhang,1 and Dong-Zhi Wei1 State Key Laboratory of Bioreactor Engineering, College of Biotechnology, East China University of Science and Technology, No. 130 Meilong Road, Shanghai 200237, People’s Republic of China1 Received 4 September 2007/Accepted 27 December 2007
In order to increase the yield of S-adenosylmethionine (SAM) in recombinant Pichia pastoris, a strategy of adding oxygen vectors and supplemental carbon sources was described. Three organic solutions were used as oxygen vectors for SAM accumulation at different concentrations and addition times. Firstly, n-hexane (0.5%) or n-heptane (1.0%) was added after 72 h of cultivation to improve SAM production. Carbon metabolism was scarce during the induction phase because of low methanol concentration. Secondly, sorbitol (1.2%), selected from three candidates (glycerol, lactic acid, and sorbitol), was used as the supplemental carbon source. The yield of SAM was improved significantly (53.26%) at 1.0% n-heptane added at 72 h (48 h induction), 1.2% sorbitol added at 72, 96, and 120 h of cultivation and 1.0% methanol added every 24 h during cultivation. [Key words: S-adenosylmethionine, fermentation, oxygen vectors, Pichia pastoris, supplemental carbon source]
S-Adenosylmethionine (SAM), a metabolite in a biological body, is used as a drug to treat liver diseases, depression, osteoarthritis, fibromyalgia, and Alzheimer’s diseases because it has biological functions: a methyl donor, the precursor of aminopropyl groups, and the trans-sulfuration pathway (1). SAM presents a unique feature in which all its constituent parts have a chemical use (2). SAM synthetase catalyzes ATP and L-methionine for SAM production. In recent years, SAM has been produced commercially by chemical synthesis and enzymatic synthesis in vitro (3). Research on SAM accumulation by microorganisms has been carried out for its potential industrial production. Shiozaki et al. optimized components in a medium enriched with L-methionine for wild type Saccharomyces sake for SAM accumulation in 1986 (4). In recombinant microorgnisms, SAM was produced as catalyzed by high-activity recombinant SAM synthetase expressed and cystathionine synthase knocked out in Pichia pastoris simultaneously (5) or by adding glycerol intermittently during the induction phase of P. pastoris fermentation (6). The advantages of recombinant P. pastoris expressing SAM synthetase for SAM accumulation are as follows: (i) High-activity SAM synthetase is expressed in recombinant P. pastoris. However, inclusion bodies are formed when a heterologous gene is expressed in Escherichia coli; (ii) P. pastoris has a high-cell-density fermentation (130 DCW g/l). P. pastoris can accumulate volumetric SAM plentifully; (iii) The P. pastoris fermentation process is simple and can be referred to the Pichia fermentation process guideline (Invitrogen, Carlsbad, CA, USA); (iv) The simplicity of
techniques required for molecular genetic manipulation and availability of commercial expression kits. Over 500 heterogenous genes, which came from viruses, prokaryotic, and eukaryotic miroorganisms, protists, plants, and invertebrates including humans, are expressed in P. pastoris (7, 8); (v) The P. pastoris fermentation medium is basal salt medium, which is advantageous for bioprocessing in bioreactors industrially owning to its low cost and definite component; (vi) In the induction phase, methanol is a cheap substrate for SAM accumulation. Oxygen limitation is the common problem encountered during P. pastoris fermentation. However, oxygen limitation can be overcome by improving the oxygen transfer coefficient (KLa) (9, 10). On the other hand, improvement of KLa can be realized by adding oxygen vectors (11). Oxygen vectors are hydrophobic liquids in which oxygen has higher solubility than in water. The mechanisms of KLa improvement by oxygen vector addition was clarified by Rols et al. (12). There were many reports indicating that oxygen vectors can increase KLa significantly to improve microorganism metabolism for metabolite production (13–17). The advantages of oxygen vectors are effective oxygen solubility, low cost, and low possibility of danger. In this study, a recombinant P. pastoris, haboring SAM2 from Saccharomyces cerevisiae, was cultivated in L-methionine-enriched medium to express SAM synthetase for SAM production (Fig. 1). Methanol was used as the carbon source and inducer of SAM synthetase expression. A low methanol concentration was controlled accurately so as not to cause methanol toxicity to P. pastoris. As SAM is a metabolite in the recombinant P. pastoris, ATP is the limiting factor when SAM is accumulated in recombinant P. pastoris with a high
* Corresponding author. e-mail: [email protected] phone: +86-21-64253156 fax: +86-21-64250068 335
336
ZHANG ET AL.
FIG. 1. Biosynthesis of SAM in recombinant Pichia pastoris. Methanol induced SAM2 expression to form SAM synthetase. At the same time, methanol was oxidized to water and carbon dioxide with ATP released. With L-methionine added to the medium, SAM was formed from ATP and L-methionine by catalysis of SAM synthetase.
activity of SAM synthetase (6), although SAM synthetase was reported as the bottleneck of SAM accumulation in wildtype strains (4). Improvement of cellular carbon metabolism is important for SAM production. Therefore, a nonrepressive carbon source should be used during the induction phase to compensate for carbon scarcity. The purpose of this work was to improve cellular carbon metabolism for SAM production by adding oxygen vectors and a nonrepressive carbon source.
MATERIALS AND METHODS Strain construction To remove the α-factor signal, LA-Taq DNA polymerase was used with pPIC9k as the template to amplify the pPIC9K fragment with two restriction sites (BamHI and EcoRI). The PCR fragment was digested with BamHI and EcoRI in the double digestion system for SAM2 ligation. SAM2 was amplified from the genomic DNA of S. cerevisiae using two primers: 5′-CA GGATCCACCATGTCCAAGAGCAAAACT-3′ and 5′-GCGG CCGCGAATTCAGCCTAGCATAAAGAAA-3′, which incorporated BamHI and EcoRI restriction sites, respectively. The primers were designed in accordance with the Genebank sequence number M23368 (18). Amplification was performed in a final volume of 50 µl, and the reaction mixture contained 100 ng of genomic DNA, 25 pmol of each primer (Invitrogen), 200 µmol/l of dNTP, 1 × PCR buffer, and 1 U of Taq-TM DNA polymerase (Takara Biotechnology, Dalian, China). The following PCR program was used for the amplification of about 1200 bp DNA fragment: 5 min of denaturation at 94°C; 10 cycles of denaturation (30 s at 94°C), annealing (30 s at 42°C), and polymerization (90 s at 72°C); 20 cycles of denaturation (30 s at 94°C), annealing (30 s at 55°C), and polymerization (90 s at 72°C) followed by an additional polymerization (10 min at 68°C). The amplified DNA fragment was digested with BamHI and EcoRI in the double digestion system and inserted into the expression vector, the pPIC9K fragment. The plasmid pPIC9KSAM2 was transformed in P. pastoris GS115 using a Multi-Copy Pichia expression kit in accordance with the manufacturer’s instruction (Invitrogen) after linearization by SacI, and the recombinant P. pastoris was screened using YPD-G418 agar plates (10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose, 15 g/l agar powder, 100 µg/ml G418) for flask cultivation. Medium BMGY medium consists of 10 g/l yeast extract, 20 g/l peptone, 100 mmol/l potassium phosphate buffer (pH 7.0), 13.4 g/l yeast nitrogen base, 400 µg/l biotin, 20 g/l glycerol, 50 mmol/l L-methionine. Culture condition Cells were transferred from YPD agar plates into 250 ml flasks, which contained 50 ml of BMGY and in-
J. BIOSCI. BIOENG.,
cubated at 30°C and 200 rpm for 18–20 h. Cells were used as the inoculum for flask cultivation. Flask cultivation was performed in 250 ml flasks containing 25 ml of BMGY medium at 30°C and 200 rpm with 4% BMGY inoculum. One percent methanol (0.25 ml) was added into BMGY after 24 h of cultivation to induce SAM synthetase for SAM production every day. In our experiments, all experiments by flask cultivation were carried out with three trials. The error bar was obtained using the standard deviation function (Office 2003, Microsoft). The error bar shows the deviation along a curve. Biomass Cells (5 ml) were harvested by centrifugation the broth at 6000 rpm for 3 min, then washed twice with deionized water, and then dried to a constant weight at 90°C. The optical density of the broth (100-fold diluted) was monitored using a spectrophotometer (U-1100; Hitachi, Tokyo) at 600 nm. The dry cell weight (DCW) of a sample was obtained from the experimentally determined calibration curve between cell optical density and the DCW. Methanol concentration The cell supernatant (0.5 ml) obtained by centrifugation at 6000 rpm for 3 min was mixed with the isopyknic mixture of 1-butanol and 1-propanol (4: 1) to extract methanol. The superstratum was transferred to a new eppendorf tube after placement in room temperature for 15 min and centrifugation at 12,000 rpm for 1 min. Sodium sulfite was used to remove water from the superstratum. The superstratum (1 µl) was obtained for analysis by centrifugation at 12,000 rpm for 1 min. Methanol concentration was determined by gas chromatography (GC 6890N; Agilent, Santa Clara, CA, USA) using a DB-WAX column. The temperatures of injection and detector were 200°C and 250°C, respectively. The column temperature profile was as follows: 100°C for 1 min, which increased at a rate of 20°C/min until the temperature reached 200°C. Yield of SAM The yield of SAM was determined by mixing 0.5 ml of fermentation broth with isopyknic 20% HClO4 and storing the mixture for 8 h. The superstratum was analyzed after centrifugation of the mixture at 12,000 rpm for 3 min and filtration using a membrane filter (φ 0.22 µm). SAM was analyzed by HPLC (HP1100; Agilent) using a Hypersil SCX column (4.6 × 250 mm, 5 µm). The mobile phase was 0.5 mol/l ammonium formate (pH 4.0). Data were quantified by Agilent chemstation. SAM synthetase P. pastoris, used for SAM synthetase measurement, was cultured in BMGY without L-methioine because L-methionine is a substrate in the determining reaction. P. pastoris (5 ml) cells were collected by centrifugation at 6000 rpm for 3 min, and washed immediately with lysis buffer (50 mmol/l PBS pH 7.4, 1 mmol/l phenylmethanesulfonyl fluoride, 1 mmol/l EDTA, 5% [v/v] glycerol), then precipitated at 12,000 rpm for 5 min. The resulting cell pellets were suspended in 1 ml of lysis buffer and disrupted by ultrasonication for 3 s at 6 s intervals (total 10 min). Finally, cell debris was removed by centrifugation at 12,000 rpm for 10 min, the resulting supernatant was used to measure the activity of SAM synthetase in accordance with the method of Shiozaki et al. (19). One unit of enzyme activity was defined as the amount of actibity required to catalyze the transformation of 1 µmol of L-methionine into SAM per minute at 37°C. Sorbitol concentration Sorbitol was measured by mixing 0.6 ml of 5% CuSO4, 0.56 ml of 10% NaOH, 1.0 ml of supernatant broth, and 3.0 ml of deionized water in this sequence. Then, the mixture was stated in room temperature for 15 min. The optical density of the mixture was assayed using a spectrophotometer (U-1100; Hitachi) at 655 nm after centrifugation at 4000 rpm for 3 min. Sorbitol concentration was determined using regression equation for sorbitol solution and optical density.
S-ADENOSYLMETHIONINE PRODUCTION IN P. PASTORIS
VOL. 105, 2008
337
RESULTS AND DISCUSSION Curves of cell growth, SAM production, activity of SAM synthetase, and methanol concentration of P. pastoris in flask cultivation The recombinant P. pastoris was cultivated to clarify the pattern of cell growth, yield of SAM, activity of SAM synthetase, and methanol concentration during 144 h of fermentation (Fig. 2). The recombinant P. pastoris grew rapidly during the first 24 h of cultivation, and then grew slowly untill the end, as DCW remained constant after 24 h of cultivation. One percent methanol was added to the medium to induce SAM synthetase every 24 h after 24 h of cultivation. The yield of SAM increased rapidly in the earlier stage of induction (24–72 h of cultivation) and then became slow and even stable untill the end. Therefore, the yield of SAM after 144 h of cultivation was used as the indicator to define SAM perturbations under different conditions. The activity of SAM synthetase existed, and did not disappear during the induction phase (Fig. 2). Therefore, it was ATP that was the limiting factor as SAM yield became stable after 72 h of cultivation, although SAM synthetase activity increased with time. Thus, a large amount of the carbon source should be available in P. pastoris during SAM accumulation because ATP production is closely related to carbon metabolism. Methanol concentration was low, and decreased slowly during the induction phase. Effects of oxygen vectors on cell growth, SAM production, and methanol concentration Three oxygen vectors (n-hexane, n-heptane, and n-dodecane) at different final concentrations (0.5%, 1.0%, 2.0%, 4.0%, and 6.0%) at the beginning of flask cultivation were used respectively to improve SAM production. The concentrations of oxygen vectors should be determined because KLa is affected by oxygen vector concentration (12). The effects of oxygen vectors at different concentrations on cell growth, SAM production, and methanol concentration were investigated after 144 h of cultivation (Fig. 3). Firstly, it was found that DCW increased up to
FIG. 2. Time courses of cell growth (A), SAM production (B), SAM synthetase (C), and methanol concentration (D) during 144 h of cultivation of Pichia pastoris.
4% n-hexane addition (Fig. 3A) and 1% n-heptane addition (Fig. 3B). n-Dodecane did not induce a significant change in DCW, as shown in Fig. 3C. Secondly, regarding the yield of SAM, 0.5% n-hexane addition and 1.0% n-heptane addition increased SAM yield to 4.88% and 3.96%, respectively. The yield of SAM decreased as n-dodecane concentration increased (Fig. 3C). Thirdly, methanol concentration was assayed to determine the effects of oxygen vectors on methanol utilized by P. pastoirs. The lowest methanol concentration was found at 0.5% n-hexane addition and 1.0% n-heptane addition. Whereas, the yield of SAM was the highest at the concentrations of oxygen vectors added (Fig. 3A, B). However, for n-dodecane addition, methanol concentration increased as the yield of SAM decreased (Fig. 3C). It was concluded that P. pastoris utilized methanol efficiently in the
FIG. 3. Effects of addition of three oxygen vectors (n-hexane, n-heptane, and n-dodecane) at the beginning of fermentation on cell growth, SAM production, and methanol concentration after 144 h of cultivation.
338
ZHANG ET AL.
FIG. 4. Effects of time of adding two oxygen vectors on DCW, yield of SAM, and methanol concentration after 144 h of cultivation. (A) n-Hexane (0.5%), (B) n-heptane (1.0%).
presence of 0.5% n-hexane or 1.0% n-heptane for SAM production. Effects of time of adding oxygen vectors on cell growth, SAM production, and methanol concentration The time of adding oxygen vectors should be defined for the following two reasons. The first reason was that oxygen vectors were used to increase the yield of SAM after 72 h of cultivation, which is the time when the yield of SAM was stable (Fig. 1). The second was that the time of immersing cells into oxygen vectors should be as short as possible because of the oxygen vectors’ negative effect on cell viability. Oxygen vectors are organic solutions that are used for cell disruption in intracellular product extraction. n-Hexane and n-heptane, at the final concentrantions of 0.5% and 1.0%, respectively, were added to the medium at different cultivation times (0, 24, 48, 72, 96, and 120 h). n-Dodecane was excluded in this experiment owing to its negative effect on the yield of SAM. Figure 4 shows the effects of time of adding 0.5% n-hexane and 1.0% n-heptane on cell growth, SAM production, and methanol concentration. In Fig. 4A, DCW did not change significantly regardless of the time of adding 0.5% n-hexane. As shown in Fig. 4B, DCW increased by 8.64% compared with the control for addition at 48 h, and then decreased after addition at 72 h. The same pattern of SAM yield decrease before 72 h of cultivation and then increase is observed in Fig. 4. The yields of SAM were the lowest (1.06 and 0.87 g/l) when 0.5% n-hexane or 1.0% n-heptane was added at 48 h of cultivation, while they were the highest (1.28 and 1.25 g/l) when 0.5% n-hexane or 1.0% n-heptane was added at 72 h of cultivation. SAM is an important metabolite and it is used for biosynthesis of many compounds in microorganisms. The yield of SAM in microorganisms is considered as a pool of metabolism (18). As shown in Fig. 1, the yield of SAM is stable after 48 h of induction in recombinant P. pastoris. This is because SAM synthesis and SAM consumption were balanced after 48 h of induction. As shown in Fig. 4, the variability of SAM production indicated the different effects of oxygen vectors on the balance between SAM biosynthesis and consumption. At 24 h or 48 h of adding oxygen vectors, SAM decomposition may be more rapid than SAM bio-
J. BIOSCI. BIOENG.,
FIG. 5. Effects of amount of supplemental carbon sources (glycerol, lactic acid, and sorbitol) each added three times (72, 96, and 120 h) on cell growth after 144 h of cultivation. Symbols: filled squares, glycerol; empty circles, lactic acid; empty triangles, sorbitol.
synthesis. Therefore, the yield of SAM was lower than that of control, and vice versa. n-Hexane and n-heptane were organic solutions from there chemical bases. Some as yet undiscovered functions except those of oxygen vector might have occurred in microorganisms when a vector was added at 48 h of cultivation. Therefore, there was an imbalance between the yield of SAM and methanol concentration. That is, methanol concentration was not high when n-heptane was added at 48 h, although the yield of SAM was the lowest. SAM synthetases in recombinant P. pastoris, when oxygen vectors were added at 72 h of cultivation, were assayed (56.01, 58.18, and 28.95 U/l for the control, 0.5% n-hexane and 1.0% n-heptane) after 144 h of cultivation. It was clear that carbon metabolism was enhanced when oxygen vectors were added at 72 h of cultivation. Addition of supplemental carbon sources without repression of SAM production Nonrepressive carbon sources were added to enhance cell carbon metabolism that is inhibited by the toxicity of high-concentration methanol to P. pastoris. Three carbon sources (glycerol, lactic acid, and sorbitol) were used: glycerol was reported to be nonrepressive compared with glucose (20); lactic acid was used in Xie’s study for its function of improving methanol consumption (21); it was described that sorbitol is a nonrepressive carbon source in P. pastoris fermentation (22). Three carbon sources, besides 1% (0.25 ml) methanol added every 24 h, were added at 72, 96, and 120 h of cultivation continuously at different final concentrations of 0.2%, 0.4%, 0.8%, 1.2% during 72–120 h cultivation. Figures 5 and 6 show the effects of supplemental carbon source addition on cell growth and SAM production, respectively. The nonrepressive effect on cell growth was observed when glycerol or sorbitol was added. As for lactic acid, DCW decreased as the concentration of lactic acid added increased. This might be due to the decrease in pH in the medium following lactic acid addition to the medium. However, pH can not be maintained accurately all the time in flask cultivation. The yield of SAM decreased significantly after glycerol or lactic acid was added. The yield of SAM did not decrease when 0.8% or 1.2% sorbitol was added. From the view point of supplemental carbon source selection for SAM production, glycerol and lactic acid were excluded in flask cultivation. Therefore, sorbitol
VOL. 105, 2008
S-ADENOSYLMETHIONINE PRODUCTION IN P. PASTORIS
339
FIG. 6. Effects of amount of supplemental carbon sources (glycerol, L-lactic acid, and sorbitol) each added three times (72, 96, and 120 h) on SAM production after 144 h of cultivation. Symbols: filled squares, glycerol; empty circles, lactic acid; empty triangles, sorbitol.
FIG. 8. Effects of sorbitol and oxygen vectors on carbon source consumption and SAM synthetase.
FIG. 7. Synergetic effects of sorbitol and oxygen vectors on cell growth and SAM production. Control: 1.0% methanol was added at 24, 48, 72, 96, and 120 h of cultivation; sorbitol: 1.0% methanol was added at 24, 48, 72, 96, and 120 h of cultivation and 1.2% sorbitol was added at 72, 96, and 120 h of cultivation; sorbitol-hexane: 1.0% methanol was added at 24, 48, 72, 96, and 120 h of cultivation, 1.2% sorbitol was added at 72, 96, and 120 h of cultivation and 0.5% hexane was added at 72 h of cultivation; sorbitol-heptane: 1.0% methanol was added at 24, 48, 72, 96, and 120 h of cultivation, 1.2% sorbitol was added at 72, 96, and 120 h of cultivation and 1.0% heptane was added at 72 h of cultivation.
was used as the supplemental carbon source for SAM accumulation with oxygen vector addition in the following experiment. Effects of sorbitol and oxygen vectors added synchronously on SAM production To enhance SAM production, 1.2% sorbitol was added at 72, 96, and 120 h of cultivation continuously, 1.0% methanol was added every 24 h, and 0.5% n-hexane or 1.0% n-heptane was added at 72 h of cultivation to validate the potential effect of oxygen vectors on the yield of SAM. DCW did not change and the yield of SAM increased significantly as the result of the synergetic effects of oxygen vectors and sorbitol (Fig. 7). The highest yield of SAM (1.41 g/l) was obtained when 1.2% sorbitol and 1.0% n-heptane were added, which showed a 53.26% increase compared with the control (0.92 g/l). Sorbitol and methanol concentrations were the lowest (Fig. 8), which showed the largest amount of carbon source consumed at 1.2% sorbitol and 1.0% n-heptane addition. DCW did not in-
crease significantly, although 3.52% sorbitol was consumed in the control. Other metabolites might be synthesized because dissolved oxygen limitation occurred commonly in flask experiments, inculding P. pastoris cultivation. SAM synthetase did not change markedly in the four kinds of cultivation (Fig. 8). Many studies indicated the enhancement of heterologous protein expression by improving consumption of nutrients in the medium (23). Methanol consumption has the major impact on heterologous protein expression in P. pastoris because methanol is the inducer of heterologous protein expression and the substrate for cell growth. Plantz et al. (24) clarified that the energy state is the critical factor during recombinant P. pastoris fermentation. In this work, SAM was accumulated in recombinant P. pastoris with a high activity of SAM synthetase. Methanol and sorbitol were consumed by P. pastoris to form ATP, which was used as the substrate of SAM synthetase and SAM production. SAM synthetase expression is a multistep metabolism from ATP, and SAM accumulation is one step of catalysis by SAM synthetase. Therefore, the yield of SAM was increased and SAM synthetase did not change markedly as sorbitol and methanol were consumed by P. pastoris in the presence of oxygen vectors. In this study, oxygen vectors were used to increase the yield of SAM for there function of high oxygen solubility. Large amounts of methanol and sorbitol were consumed by P. pastoirs in the presence of oxygen vectors. The yield of SAM was increased by 53.26% when sorbitol was used as the supplemental carbon source and 1.0% n-heptane was used as the oxygen vector. Addition of supplemental carbon source and oxygen vectors is a strategy to overcome energy scarcity because methanol concentration is accurately controlled to be low during P. pastoris fermentation. REFERENCES 1. Shelly, C. L.: S-Adenosylmethionine. Int. J. Biochem. Cell. Biol., 32, 391–395 (2000). 2. Fontecave, M., Atta, M., and Mulliez, E.: S-Adenosyl-
340
3. 4. 5.
6.
7. 8. 9.
10. 11. 12.
13.
J. BIOSCI. BIOENG.,
ZHANG ET AL.
methionine: nothing goes to waste. Trends Biochem. Sci., 29, 243–249 (2004). Matos, J. R., Raushel, F. M., and Wong, C. H.: S-Adenosylmethionine studies on chemical and enzymatic synthesis. Biotechnol. Appl. Biochem., 9, 39–52 (1987). Shiozaki, S., Shimizu, S., and Yamada, H.: Production of S-adenosyl-l-methionine by cultivation of wild Saccharomyces sake. J. Biotechnol., 4, 345–354 (1986). He, J. Y., Deng, J. J., Zheng, Y. H., and Gu, J.: A synergistic effect on the production of S-adenosyl-l-methionine in Pichia pastoris by knocking in of S-adenosyl-l-methionine synthase and knocking out of cystathionine synthase. J. Biotechnol., 126, 519–527 (2006). Hu, X. Q., Chu, J., Zhang, S. L., Zhuang, Y. P., Wang, Y. H., Zhu, S., Zhu, Z. G., and Yuan, Z. Y.: A novel feeding strategy during the production phase for enhancing the enzymatic synthesis of S-adenosyl-l-methionine by methylotrophic Pichia pastoris. Enzyme Microb. Technol., 40, 669–674 (2007). Cereghino, J. L. and Cregg, J. M.: Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev., 24, 45–66 (2000). Macauley-Patrick, S., Fazenda, M. L., McNeil, B., and Harvey, L. M.: Heterologous protein production using the Pichia pastoris expression system. Yeast, 22, 249–270 (2005). Cunha, A. E., Clemente, J. J., Gomes, R., Pinto, F., Thomaz, M., Miranda, S., Pinto, R., Moosmayer, D., Donner, P., and Carrondo, M. J.: Methanol induction optimization for scFv antibody fragment production in Pichia pastoris. Biotechnol. Bioeng., 86, 458–467 (2004). Zhang, W., Zheng, J. L., and Agblevor, F. A.: Microbubble fermentation of recombinant Pichia pastoris for human serum albumin production. Process Biochem., 40, 2073–2078 (2005). Jia, S., Wang, M., Kahar, P., Park, Y., and Okabe, M.: Enhancement of yeast by addition of oxygen vectors in air-lift bioreactor. J. Ferment. Bioeng., 84, 176–178 (1997). Rols, J. L., Condoret, J. S., Fonade, C., and Gome, G.: Mechanism of enhanced oxygen transfer in fermentation using emulsified oxygen-vectors. Biotechnol. Bioeng., 35, 427–435 (1990). Liu, Y. S. and Wu, J. Y.: Use of n-hexadecane as an oxygen
14. 15. 16. 17. 18.
19. 20.
21.
22. 23.
24.
vector to improve Phaffia rhodozyma growth and carotenoid production in shake-flask cultures. J. Appl. Microbiol., 101, 1033–1038 (2006). Giridhar, R. and Srivastava, A. K.: Productivity enhancement in L-sorbose fermentation using oxygen vector. Enzyme Microb. Technol., 27, 537–541 (2000). Xu, F., Yuan, Q. P., and Zhu, Y.: Improved production of lycopene and β-carotene by Blakeslea trispora with oxygenvectors. Process Biochem., 42, 289–293 (2007). Wang, J.: Enhancement of citric acid production by Aspergillus niger using n-dodecane as an oxygen-vector. Process Biochem., 35, 1079–1083 (2000). Menge, M., Mukherjee, J., and Scheper, T.: Application of oxygen vectors to Claviceps purpurea cultivation. Appl. Microbiol. Biotechnol., 55, 411–416 (2001). Thomas, D., Rothstein, R., Rosenberg, N., and SurdinKerjan, Y.: SAM2 encodes the second methionine S-adenosyl transferase in Saccharomyces cerevisiae: physiology and regulation of both enzymes. Mol. Cell. Biol., 8, 5132–5139 (1988). Shiozaki, S., Shimizu, S., and Yamada, H.: Unusual intracelluar accumulation of S-adenosyl-l-methionine by microorganisms. Agric. Biol. Chem., 48, 2293–2300 (1984). Tschopp, J. F., Brust, P. F., Cregg, J. M., Stillman, C. A., and Gingeras, T. R.: Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res., 15, 3859–3876 (1987). Xie, J., Zhou, Q., Du, P., Gan, R., and Ye, Q.: Use of different carbon sources in cultivation of recombinant Pichia pastoris for angiostatin production. Enzyme Microb. Technol., 36, 210–216 (2005). Inan, M. and Meagher, M. M.: Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. J. Biosci. Bioeng., 92, 585–589 (2001). Chauhan, A. K., Arora, D., and Khanna, N.: A novel feeding strategy for enhanced protein production by fed-batch fermentation in recombinant Pichia pastoris. Process Biochem., 34, 139–145 (1999). Plantz, B. A., Sinha, J., Villarete, L., Nickerson, K. W., and Schlegel, V. L.: Pichia pastoris fermentation optimization: energy state and testing a growth-associated model. Appl. Microbiol. Biotechnol., 72, 297–305 (2006).