High level extracellular expression of inulin fructotransferase in Pichia pastoris for DFA III production
Rongrong Zhana,b, Wanmeng Mua,b, Bo Jianga,b,*, Yungao Lia,b, Liuming Zhoua,c, Tao Zhanga,b * Correspondence to: Bo jiang, State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, 214122 Wuxi, Jiangsu, China. E-mail: [email protected] a State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China b Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China c Roquette America, Keokuk, IA 52632, USA
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.6931
This article is protected by copyright. All rights reserved
Abstract BACKGROUND: Inulin fructotransferase (IFTase) catalyses inulin conversion to difructose anhydride (DFA III), which is a natural low-calorie sweetener. Although heterologous expression of IFTase was achieved in Escherichia coli, the extracellular enzyme activity was very low which limited the commercialization of IFTase. RESULTS: Active IFTase of about 43 kDa molecular mass of subunit was extracellularly expressed by P. pastoris, and was greatly regulated by IFTase gene copy number integrated into the P. pastoris genome and methanol concentration in the induction phase. Under the optimized culture conditions, the multicopy P. pastoris exhibited a maximum extracellular IFTase activity of 105.4 U mL−1 in a 5 L fermenter, which was 8.9-fold higher than in shake flasks, and 5.3-fold of that obtained with wild-type strain. CONCLUSION: IFTase was expressed in eukaryotic Pichia pastoris system for the first time and achieved high level extracellular expression using high cell density fed-batch cultivation strategy. It demonstrated that P. pastoris was a good candidate for potential DFA III production as a novel IFTase expression system. Keywords: Difructose anhydride; Extracellular expression; Fed-batch fermentation; Inulin fructotransferase; Pichia pastoris
This article is protected by copyright. All rights reserved
Introduction Difructose anhydride (DFA III) is a cyclic disaccharide consisting of two fructose units linked by their reducing carbons. DFA III is a natural and low-calorie sweetener with half the sweetness and one fifteenth the calories of sucrose.1 It increases the absorption of calcium at low doses, in both the small and large intestines of rats and humans.2,3 Inulin fructotransferase (IFTase; EC 4.2.2.18), catalysing inulin conversion to DFA III with a β-2,1-fructans depolymerisation reaction,4 is considered to be the most promising enzyme for the production of DFA III. DFA III-forming IFTase was firstly described in Arthrobacter ureafaciens5 and was produced industrially only by Nippon Beet Sugar Mfg. Co., Ltd.6 In order to obtain excellent strains for industrial production, studies have been focused on the IFTase heterologous expression in Escherichia coli since 1997.7 Although the IFTase activity was greatly improved, most of the recombinant IFTase exhibited intracellular enzyme activity.8,9 Till now, only few IFTase achieved extracellular expression in E. coli, and the activity was very low, for example, the activity of recombinant IFTase from A. globiformis C11-1 and Arthrobacter sp L68-1 was just 1.5 U mL−1 and 8 U mL−1, respectively.10,11 It was presumably because that the native signal-peptide of IFTase could not be completely removed in E. coli and the remaining parts had a negative effect on the folding of the following protein.12 Compared with intracellular expression, secretory production of proteins has several advantages, such as few contaminating proteins, no space limitation for the accumulation of the protein, simple downstream processing steps and little harm against host cells.13
Therefore, exploring new expression system for high level extracellular IFTase production was of great significance for the industrial production of IFTase and DFAIII. With the successful α-factor single peptide of Saccharomyces cerevisiae, the methylotrophic Pichia pastoris expression system was able to avoid the intracellular accumulation of recombinant protein and achieve extracellular expression of foreign proteins at high levels.14 Meanwhile, compared with bacterial expression systems, it has provided several advantages, such as accurate execution of post-translational modifications which conserve protein function, extremely high densities cultivation (500 OD600 U mL−1) in simple mineral salts medium, and low levels secretion of unwanted endogenous proteins.15–17 These advantages make recombinant protein production in P. pastoris popular for scientific research and industrial application.18–20 However, the studies on IFTase expression in P. pastoris systems have not been reported. The IFTase gene from Arthrobacter aurescens SK 8.001, encoding a 410-amino acid mature protein was used in this work.21 In our previous study, the P. pastoris expression vector pPIC9K-IFTase was constructed.22 The present study was focused on exploring the feasibility of IFTase extracellular expression in P. pastoris and optimising the growth conditions of P. pastoris to obtain the maximum expression of recombinant IFTase in shake flasks and then scale up further in a lab scale fermenter. MATERIALS AND METHODS Materials, strains and medium DFA III was purchased from Wako Pure Chemical Industries (Osaka, Japan). strain P. pastoris GS115 were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals
were of analytical grade and used as received. MD solid medium:1.34 g L−1 YNB, 4 mg L−1 biotin, 20 g L−1 glucose and 20 g L−1 agar. YPD solid medium: 10 g L−1 yeast extract, 20 g L−1 peptone, 20 g L−1 glucose and 20 g L−1 agar. BMGY medium: 1.34 g L−1 yeast nitrogen base (YNB), 4 mg L−1 biotin, 10 g L−1 yeast extract, 20 g L−1 peptone, 0.1 M potassium phosphate buffer (pH 6.0) and 10 g L−1 glycerol. BMMY medium (pH 6.0): BMGY medium replacing glycerol with 5 mL L−1 methanol. Basal salts medium: 40 g L−1 glycerol, 18 g L−1 K2SO4, 4.13 g L−1 KOH, 14.9 g L−1 MgSO4 · 7H2O, 27 mL L−1 H3PO4, and 0.93 g L−1 CaSO4. PTM1: 6 g L−1 CuSO4 · 5H2O, 0.09 g L−1 KI, 3 g L−1 MnSO4 · H2O, 0.02 g L−1 H3BO3, 0.2 g L−1 MoNa2O4 · 2H2O, 0.5 g L−1 CoCl2, 20 g L−1 ZnCl2, 65 g L−1 FeSO4 · 7H2O, 0.2 g L−1 biotin, and 5.0 mL L−1 H2SO4. Screening of recombinant P. pastoris GS115-IFTase The recombinant expression vector pPIC9K-IFTase was linearised by digestion with the restriction enzyme Sal I and transformed into the yeast P. pastoris GS115 by electroporation. Successful transformations were preliminarily selected by growth on MD solid medium. The MD-positive transformants were further screened by growth on YPD solid medium containing the antibiotic G418 and confirmed by polymerase chain reaction (PCR). The empty vector pPIC9K was used as negative control. Expression of the recombinant IFTase P. pastoris GS115-IFTase transformants were inoculated into 25 mL BMGY medium and incubated at 30 °C with shaking (200 rpm). When an optical density at 600 nm (OD600) of 6 was obtained, the cells were harvested by centrifugation and resuspended to OD600 = 1 in a 1 L shake flask containing 100 ml BMMY medium with 5 mL L−1 methanol and the cells
incubated with shaking. Every 24 h, a 1 mL sample was collected for analysis and methanol was added to the remaining culture to a final concentration of 5 mL L−1. Enzyme assay To measure IFTase activity, the IFTase-containing solution (0.2 mL) at a suitable dilution, distilled water (0.3 mL), 0.1 M citrate buffer (pH 6.0, 0.5 mL) and 20 g L−1 inulin solution (1.0 mL) were mixed. The mixture was incubated at 60 °C for 15 min, then the reaction was halted by incubating in boiling water for 10 min. The concentration of DFA III produced was determined by high-performance liquid chromatography (HPLC) using a water Sugur-PakTM 1 column (6.5 mm × 300 mm, USA) and refractive index detector (Shodex RI101). The HPLC conditions and the definition of IFTase activity used are consistent with Hang et al.23 One unit of IFTase was defined as the amount of enzyme which could produce 1 μmol DFA III from inulin per min at pH 5.5 and 60 °C. Measurement of alcohol oxidase activity The recombinant P. pastoris was constructed under the control of the alcohol oxidase promoter (pAOX) for extracellular expression of IFTase. Alcohol oxidase (AOX) can be induced by methanol, which can further achieve induction of foreign proteins. The intracellular AOX activity was assayed by measuring H2O2 production during the oxidation of methanol. The standard assay mixture contained 100 μmol L−1 sodium phosphate buffer (pH 7.0), 4.3 μmol L−1 phenol, 5 units peroxidase mL−1, 1 μmol L−1 4-aminoantipyrine and 400 μmol L−1 methanol. Samples (100 μL) were immediately mixed with 2.9 mL standard assay mixture at 37 °C for 10 min. The reaction rate was measured by the increase in absorbance at 500 nm using a temperature-controlled UV2450 spectrophotometer. One AOX
activity was defined as formation of 1 μmol L−1 of H2O2 per minute at above-mentioned condition.24 Production of recombinant IFTase by fed-batch fermentation After cultivating a seed culture to OD600 of 7 in YPD medium at 30 °C, 10% (v/v) of the culture was inoculated into a 5 L fermenter (LiFlus GM BioTRON, Korea) with 2 L basal salts medium and 4.4 mL L−1 PTM1. The pH of the medium was adjusted and controlled at 6.0 with the addition of 250 mL L−1 ammonium hydroxide and 30 mL L−1 phosphoric acid. Fermentation was operated at 30 °C and the dissolved oxygen (DO) level was maintained above 30% of air saturation. The feeding medium, containing 500 g L−1 glycerol and 12 mL L−1 PTM1 solution, was pumped into the fermenter according to a predetermined protocol.25 During the induction phase, the temperature was reduced to 22 °C and pure methanol containing 12 mL L−1 PTM1 solution was fed to maintain methanol concentration of 15 mL L−1 by the methanol online control station (FC2002, East China University of Science and Technology). During the fed-batch fermentation, the broth was centrifuged at 10,000×g and the supernatant was collected for activity assay.23 SDS-PAGE was performed on a 100 g L−1 polyacrylamide gel, which was visualized with 2.5 g L−1 Coomassie Brilliant Blue (CBB)-R250.21 RESULTS AND DISCUSSIONS Screening of multicopy strains After transformation, expression vectors were integrated into the host genome at varying copy numbers, which maximized the stability of expression strains. As the level of antibiotic G418 resistance was roughly correlated to vector copy number, multicopy transformants
could be screened by plates containing G418.26 Moreover, a strain that contains multiple integrated copies of an expression cassette can sometimes yield more heterologous protein than single-copy strains.15 To investigate the effect of copy number on the recombinant IFTase activity, four GS115-IFTase transformants with different copy number of ift gene, selected using YPD medium containing 0.25, 1, 2 and 4 mg mL−1 G418, were induced by 10 mL L−1 methanol under the same culture conditions in shake flasks. The results showed that IFTase activity was detected in the culture supernatant of all the strains integrated with the mature ift gene. As shown in Fig. 1, the strain with 4 mg mL−1 G418 resistance, which suggested a higher copy number of ift gene, exhibited higher IFTase activity in the induction phase. The activity of the extracellular IFTase expressed by this multicopy strain was 2.3-fold of that secreted by the single copy-strain and was significantly higher than other strains. Therefore, P. pastoris GS115-IFTase with 4 mg mL−1 G418 resistance was selected for use in subsequent studies. Effect of culture conditions on recombinant IFTase production In order to improve the protein productivity, the culture conditions used for P. pastoris expression systems are important factors for consideration.14 The shake flask cultivation of P. pastoris is executed in two stages. Biomass is generated in a repressive media, then the recombinant protein is expressed in an induction media containing methanol. Therefore, the biomass and the methanol concentration are important limiting factors in heterologous protein production. As shown in Fig. 2, methanol concentration had a great effect on the activity of recombinant IFTase. The maximum IFTase activity (8.5 U mL−1) was obtained at 15 mL L−1 methanol and was 3-fold higher than the activity induced by 5 mL L−1 methanol
(Fig. 2A). The optimal induction period studies showed that initial cell concentrations of OD600 = 7 was the best stage for recombinant IFTase induction in BMMY medium. Within a wide range of initial cell concentrations (OD600 from 4 to 9), the fluctuations in the IFTase activity was only 37.9%, which suggested that P. pastoris in the logarithmic growth phase maintained a similar capability for IFTase production while induced by methanol (Fig. 2B). In addition, the pH condition is also an important factor for optimizing the expression of yeast recombinant proteins.27 It was reported that lowering pH during the production stage generally mitigated protease activity and improved the product quality.28 However, Fig. 2C showed that a relatively high pH value (pH 6.5) was the more suitable pH condition for IFTase expression by GS115-IFTase. This result was presumably due to the good IFTase stability at pH 4.5-8.0.29 Moreover, owing to the capacity of performing many eukaryotic post-translational modifications, such as glycosylation, disulphide bond formation and proteolytic processing,30 the P. pastoris expression system is suspected to play a role in the pH tolerance of recombinant IFTase. IFTase production in shake flask According to the screening results, P. pastoris GS115-IFTase with 4 mg mL−1 G418 resistance was confirmed to be the best strain and was therefore further characterized in flask cultivation under the previously mentioned suitable conditions. As shown in Fig. 3, the extracellular IFTase activity increased consistently with cell growth and reached its maximum level (11.9 U mL−1) at 96 h, in the late log phase of cell growth. However, no IFTase activity was detected in the control strain supernatant (data not shown). Moreover, the IFTase recombinant P. pastoris accumulated less biomass than the control strain, which might
be due to the burden of overexpression of IFTase.25 Laboratory-scale production of IFTase in a 5 L fermenter Fermentation growth is important for secreted proteins, as the concentration of product in the medium is roughly proportional to the concentration of cells in culture.15 The recombinant IFTase subunit showed molecular mass of about 43 kDa and the fermentation supernatant exhibited a major protein band of IFTase with little undesired bands on SDS-PAGE (Fig. 5). As shown in Fig. 4, this fed-batch fermentation strategy consisted of glycerol batch, glycerol fed-batch and methanol induction phases. after glycerol phase for 18 h and glycerol fed-batch phase for 10 h, the biomass OD600 was up to 74.5, which was much higher than in shake flask cultivation. Then, the methanol feeding was started to induce the production of IFTase. After the induction of methanol for 60 h, the biomass OD600 and extracellular IFTase activity reached final values of 230.2 and 105.4 U mL−1, respectively. This is the highest extracellular activity of IFTase to date. It was reported by our laboratory that Arthrobacter aurescens SK 8.001 IFTase with a relatively high activity (about 19.6 U mL−1) was thermostable and could be a good candidate for the industrial production of DFA III.29 After expression of this ift gene in E. coli., extracellular IFTase activity reached 81.0 U mL−1, however, this value was just 76.8% of the recombinant IFTase in P. pastoris. The results suggested that secretory IFTase in P. pastoris could be an viable method to resolve low extracellular IFTase activity both in the E. coli system and in the original strain. In fact, Pichia high density fermentation can greatly improve the recombinase activity are common.15,25,31 Moreover, the activity of lipase B (secoreted from Pichia) was reported to increase from 57.9 to 11900 U mL−1 when cultured from flask culture to 50 L fermenter.32 So, this system might be expected to achieve
higher IFTase activity in larger volume fermentation of industrial production. On the other hand, the addition of methanol activated pAOX, which could immediately drive the expression of the downstream genes encoding for AOX and IFTase. However, the intracellular AOX activity decreased dramatically from 57.4 to 3.7 U g−1 within 24 h (Fig. 4), suggesting that IFTase have low or no expression. This fluctuation in the AOX activity in the fed-batch fermentation, was probably due to the feedback inhibition of pAOX by metabolic intermediates generated in the methanol utilization pathway.33 In addition, under the constant agitation speed and aeration rate during the induction phase, the relative DO level was dependent on the cell growth strictly but reversely (Fig. 4). CONCLUSIONS In summary, IFTase achieve high level extracellular expression in P. pastoris system with fed-batch fermentation, which suggested that P. pastoris could be an effective alternative to E. coli for secretory expression of IFTase. The extracellular IFTase activity reached 105.4 U mL−1 in a laboratory-scale fermenter, which was 8.9-fold of the activity in shake flasks, and 5.3-fold of that obtained from wild-type strain. Meanwhile, the recombinant IFTase activity was correlated to the copy number of IFTase gene inserted into the GS115 genome, and was greatly affected by methanol concentration. The extracellular expression of IFTase in this system may provide new opportunities for industrial production of IFTase and DFA III. ACKNOWLEDGEMENTS This work was supported by the NSFC Project (No. 31371788), the 863 Project (No. 2013AA102102), the Fundamental Research Funds for the Central Universities (No. JUSRP51304A), and the Support Project of Jiangsu Province (BK20130001) and Shaoxing
city (No. 2013A23002). REFERENCES 1
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10 Haraguchi K, Cloning of inulin fructotransferase (DFA III-producing) gene from Arthrobacter sp. L68-1. Carbohyd Polym 93:473-477 (2013). 11 Haraguchi K, Mori S and Hayashi K, Cloning of inulin fructotransferase (DFA III-producing) gene from Arthrobacter globiformis C11-1. J Biosci Bioeng 89:590–595 (2000). 12 Jahnz U, Schubert M, Baars-Hibbe H and Vorlop KD, Process for producing the potential food ingredient DFA III from inulin: screening, genetic engineering, fermentation and immobilization of inulase II. Int J Pharm 256:199–206 (2003). 13 Choi JH and Lee SY, Secretory and extracellular production of recombinant protein using Escherichia coli. App Microbiol Biot 64:625–623 (2004). 14 Daly R and Hearn MTW, Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18:119–138 (2005). 15 Cereghino JL and Cregg JM, Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66 (2000).
16 Macauley-Patrick S, Fazenda ML, McNeil FB and Harvey LM, Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270 (2005). 17 Cregg JM, Tolstorukov I, Kusari A, Sunga J, Madden K, and Chappell T, Expression in the yeast Pichia pastoris. Method Enzymol 463:169–189 (2009). 18 Zheng J, Zhao W, Guo N, Lin F, Tian J, Wu L, et al, Development of an industrial medium
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and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation. Bioresour Technol 101:1318–1323 (2010). 25 Ling Z, Ma T, Li J, Du G, Kang Z and Chen J, Functional expression of trypsin from Streptomyces griseus by Pichia pastoris. J Ind Microbiol Biot 39:1651–1662 (2012). 26 Scorer CA, Clare JJ, Mccombie WR, Romanos MA and Sreekrishna K, Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Nat Biotechnol 12:181–184 (1994). 27 Charoenrat T, Khumruaengsri N, Promdonkoy P, Rattanaphan N, Eurwilaichitr L, Tanapongpipat S, et al, Improvement of recombinant endoglucanase produced in Pichia pastoris KM71 through the use of synthetic medium for inoculum and pH control of proteolysis. J Biosci Bioeng 116:193–198 (2013). 28 Zhang H, Wang J, Wu M, Gao S, Li J and Yang Y, Optimized expression, purification and characterization of a family 11 xylanase (AuXyn11A) from Aspergillus usamii E001 in Pichia pastoris. J Sci Food Agric 94:699–706 (2014). 29 Zhao M, Mu W, Jiang B, Zhou L, Zhang T, Lu Z, et al, Purification and characterization of inulin fructotransferase (DFA III-forming) from Arthrobacter aurescens SK 8.001. Bioresource Technol 102:1757–1764 (2011). 30 Gellissen G, Heterologous protein production in methylotrophic yeasts. App Microbiol and Biot 54:741–75 (2000). 31 Wang H, Li J, Liu L, Li X, Jia D, Du G, et al, Increased production of alkaline polygalacturonate lyase in the recombinant Pichia pastoris by controlling cell concentration during continuous culture. Bioresource Technol 124:338–346 (2012).
32 Eom GT, Lee SH, Song BK, Chung KW, Kim YW and Song JK, High-level extracellular production and characterization of Candida Antarctica lipase B in Pichia pastoris. J Biosci Bioeng 116:165–170 (2013). 33 He M, Wu D, Wu J and Chen J, Enhanced expression of endoinulinase from Aspergillus niger by codon optimization in Pichia pastoris and its application in inulooligosaccharide production. J Ind Microbiol Biotechnol 41:105–114 (2014).
FIGURE LEGENDS Fig. 1 Comparison of extracellular IFTase activity expressed by P. pastoris IFTase-GS115
with different ift gene copy number, which were screened by different antibiotic G418. The single-copy strain was screened by YPD solid medium containing 0.25 mg mL−1 G418, while other multicopy strains were obtained by same medium with 1, 2, 4 mg mL−1 G418. Data were compared by ANOVA and Duncan’s tests with SPSS statistics package software (Version 18.0). Data points are means of three replicates with error bars of standard deviation. Significant differences were defined at p
Rongrong Zhana,b, Wanmeng Mua,b, Bo Jianga,b,*, Yungao Lia,b, Liuming Zhoua,c, Tao Zhanga,b * Correspondence to: Bo jiang, State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, 214122 Wuxi, Jiangsu, China. E-mail: [email protected] a State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China b Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China c Roquette America, Keokuk, IA 52632, USA
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.6931
This article is protected by copyright. All rights reserved
Abstract BACKGROUND: Inulin fructotransferase (IFTase) catalyses inulin conversion to difructose anhydride (DFA III), which is a natural low-calorie sweetener. Although heterologous expression of IFTase was achieved in Escherichia coli, the extracellular enzyme activity was very low which limited the commercialization of IFTase. RESULTS: Active IFTase of about 43 kDa molecular mass of subunit was extracellularly expressed by P. pastoris, and was greatly regulated by IFTase gene copy number integrated into the P. pastoris genome and methanol concentration in the induction phase. Under the optimized culture conditions, the multicopy P. pastoris exhibited a maximum extracellular IFTase activity of 105.4 U mL−1 in a 5 L fermenter, which was 8.9-fold higher than in shake flasks, and 5.3-fold of that obtained with wild-type strain. CONCLUSION: IFTase was expressed in eukaryotic Pichia pastoris system for the first time and achieved high level extracellular expression using high cell density fed-batch cultivation strategy. It demonstrated that P. pastoris was a good candidate for potential DFA III production as a novel IFTase expression system. Keywords: Difructose anhydride; Extracellular expression; Fed-batch fermentation; Inulin fructotransferase; Pichia pastoris
This article is protected by copyright. All rights reserved
Introduction Difructose anhydride (DFA III) is a cyclic disaccharide consisting of two fructose units linked by their reducing carbons. DFA III is a natural and low-calorie sweetener with half the sweetness and one fifteenth the calories of sucrose.1 It increases the absorption of calcium at low doses, in both the small and large intestines of rats and humans.2,3 Inulin fructotransferase (IFTase; EC 4.2.2.18), catalysing inulin conversion to DFA III with a β-2,1-fructans depolymerisation reaction,4 is considered to be the most promising enzyme for the production of DFA III. DFA III-forming IFTase was firstly described in Arthrobacter ureafaciens5 and was produced industrially only by Nippon Beet Sugar Mfg. Co., Ltd.6 In order to obtain excellent strains for industrial production, studies have been focused on the IFTase heterologous expression in Escherichia coli since 1997.7 Although the IFTase activity was greatly improved, most of the recombinant IFTase exhibited intracellular enzyme activity.8,9 Till now, only few IFTase achieved extracellular expression in E. coli, and the activity was very low, for example, the activity of recombinant IFTase from A. globiformis C11-1 and Arthrobacter sp L68-1 was just 1.5 U mL−1 and 8 U mL−1, respectively.10,11 It was presumably because that the native signal-peptide of IFTase could not be completely removed in E. coli and the remaining parts had a negative effect on the folding of the following protein.12 Compared with intracellular expression, secretory production of proteins has several advantages, such as few contaminating proteins, no space limitation for the accumulation of the protein, simple downstream processing steps and little harm against host cells.13
Therefore, exploring new expression system for high level extracellular IFTase production was of great significance for the industrial production of IFTase and DFAIII. With the successful α-factor single peptide of Saccharomyces cerevisiae, the methylotrophic Pichia pastoris expression system was able to avoid the intracellular accumulation of recombinant protein and achieve extracellular expression of foreign proteins at high levels.14 Meanwhile, compared with bacterial expression systems, it has provided several advantages, such as accurate execution of post-translational modifications which conserve protein function, extremely high densities cultivation (500 OD600 U mL−1) in simple mineral salts medium, and low levels secretion of unwanted endogenous proteins.15–17 These advantages make recombinant protein production in P. pastoris popular for scientific research and industrial application.18–20 However, the studies on IFTase expression in P. pastoris systems have not been reported. The IFTase gene from Arthrobacter aurescens SK 8.001, encoding a 410-amino acid mature protein was used in this work.21 In our previous study, the P. pastoris expression vector pPIC9K-IFTase was constructed.22 The present study was focused on exploring the feasibility of IFTase extracellular expression in P. pastoris and optimising the growth conditions of P. pastoris to obtain the maximum expression of recombinant IFTase in shake flasks and then scale up further in a lab scale fermenter. MATERIALS AND METHODS Materials, strains and medium DFA III was purchased from Wako Pure Chemical Industries (Osaka, Japan). strain P. pastoris GS115 were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals
were of analytical grade and used as received. MD solid medium:1.34 g L−1 YNB, 4 mg L−1 biotin, 20 g L−1 glucose and 20 g L−1 agar. YPD solid medium: 10 g L−1 yeast extract, 20 g L−1 peptone, 20 g L−1 glucose and 20 g L−1 agar. BMGY medium: 1.34 g L−1 yeast nitrogen base (YNB), 4 mg L−1 biotin, 10 g L−1 yeast extract, 20 g L−1 peptone, 0.1 M potassium phosphate buffer (pH 6.0) and 10 g L−1 glycerol. BMMY medium (pH 6.0): BMGY medium replacing glycerol with 5 mL L−1 methanol. Basal salts medium: 40 g L−1 glycerol, 18 g L−1 K2SO4, 4.13 g L−1 KOH, 14.9 g L−1 MgSO4 · 7H2O, 27 mL L−1 H3PO4, and 0.93 g L−1 CaSO4. PTM1: 6 g L−1 CuSO4 · 5H2O, 0.09 g L−1 KI, 3 g L−1 MnSO4 · H2O, 0.02 g L−1 H3BO3, 0.2 g L−1 MoNa2O4 · 2H2O, 0.5 g L−1 CoCl2, 20 g L−1 ZnCl2, 65 g L−1 FeSO4 · 7H2O, 0.2 g L−1 biotin, and 5.0 mL L−1 H2SO4. Screening of recombinant P. pastoris GS115-IFTase The recombinant expression vector pPIC9K-IFTase was linearised by digestion with the restriction enzyme Sal I and transformed into the yeast P. pastoris GS115 by electroporation. Successful transformations were preliminarily selected by growth on MD solid medium. The MD-positive transformants were further screened by growth on YPD solid medium containing the antibiotic G418 and confirmed by polymerase chain reaction (PCR). The empty vector pPIC9K was used as negative control. Expression of the recombinant IFTase P. pastoris GS115-IFTase transformants were inoculated into 25 mL BMGY medium and incubated at 30 °C with shaking (200 rpm). When an optical density at 600 nm (OD600) of 6 was obtained, the cells were harvested by centrifugation and resuspended to OD600 = 1 in a 1 L shake flask containing 100 ml BMMY medium with 5 mL L−1 methanol and the cells
incubated with shaking. Every 24 h, a 1 mL sample was collected for analysis and methanol was added to the remaining culture to a final concentration of 5 mL L−1. Enzyme assay To measure IFTase activity, the IFTase-containing solution (0.2 mL) at a suitable dilution, distilled water (0.3 mL), 0.1 M citrate buffer (pH 6.0, 0.5 mL) and 20 g L−1 inulin solution (1.0 mL) were mixed. The mixture was incubated at 60 °C for 15 min, then the reaction was halted by incubating in boiling water for 10 min. The concentration of DFA III produced was determined by high-performance liquid chromatography (HPLC) using a water Sugur-PakTM 1 column (6.5 mm × 300 mm, USA) and refractive index detector (Shodex RI101). The HPLC conditions and the definition of IFTase activity used are consistent with Hang et al.23 One unit of IFTase was defined as the amount of enzyme which could produce 1 μmol DFA III from inulin per min at pH 5.5 and 60 °C. Measurement of alcohol oxidase activity The recombinant P. pastoris was constructed under the control of the alcohol oxidase promoter (pAOX) for extracellular expression of IFTase. Alcohol oxidase (AOX) can be induced by methanol, which can further achieve induction of foreign proteins. The intracellular AOX activity was assayed by measuring H2O2 production during the oxidation of methanol. The standard assay mixture contained 100 μmol L−1 sodium phosphate buffer (pH 7.0), 4.3 μmol L−1 phenol, 5 units peroxidase mL−1, 1 μmol L−1 4-aminoantipyrine and 400 μmol L−1 methanol. Samples (100 μL) were immediately mixed with 2.9 mL standard assay mixture at 37 °C for 10 min. The reaction rate was measured by the increase in absorbance at 500 nm using a temperature-controlled UV2450 spectrophotometer. One AOX
activity was defined as formation of 1 μmol L−1 of H2O2 per minute at above-mentioned condition.24 Production of recombinant IFTase by fed-batch fermentation After cultivating a seed culture to OD600 of 7 in YPD medium at 30 °C, 10% (v/v) of the culture was inoculated into a 5 L fermenter (LiFlus GM BioTRON, Korea) with 2 L basal salts medium and 4.4 mL L−1 PTM1. The pH of the medium was adjusted and controlled at 6.0 with the addition of 250 mL L−1 ammonium hydroxide and 30 mL L−1 phosphoric acid. Fermentation was operated at 30 °C and the dissolved oxygen (DO) level was maintained above 30% of air saturation. The feeding medium, containing 500 g L−1 glycerol and 12 mL L−1 PTM1 solution, was pumped into the fermenter according to a predetermined protocol.25 During the induction phase, the temperature was reduced to 22 °C and pure methanol containing 12 mL L−1 PTM1 solution was fed to maintain methanol concentration of 15 mL L−1 by the methanol online control station (FC2002, East China University of Science and Technology). During the fed-batch fermentation, the broth was centrifuged at 10,000×g and the supernatant was collected for activity assay.23 SDS-PAGE was performed on a 100 g L−1 polyacrylamide gel, which was visualized with 2.5 g L−1 Coomassie Brilliant Blue (CBB)-R250.21 RESULTS AND DISCUSSIONS Screening of multicopy strains After transformation, expression vectors were integrated into the host genome at varying copy numbers, which maximized the stability of expression strains. As the level of antibiotic G418 resistance was roughly correlated to vector copy number, multicopy transformants
could be screened by plates containing G418.26 Moreover, a strain that contains multiple integrated copies of an expression cassette can sometimes yield more heterologous protein than single-copy strains.15 To investigate the effect of copy number on the recombinant IFTase activity, four GS115-IFTase transformants with different copy number of ift gene, selected using YPD medium containing 0.25, 1, 2 and 4 mg mL−1 G418, were induced by 10 mL L−1 methanol under the same culture conditions in shake flasks. The results showed that IFTase activity was detected in the culture supernatant of all the strains integrated with the mature ift gene. As shown in Fig. 1, the strain with 4 mg mL−1 G418 resistance, which suggested a higher copy number of ift gene, exhibited higher IFTase activity in the induction phase. The activity of the extracellular IFTase expressed by this multicopy strain was 2.3-fold of that secreted by the single copy-strain and was significantly higher than other strains. Therefore, P. pastoris GS115-IFTase with 4 mg mL−1 G418 resistance was selected for use in subsequent studies. Effect of culture conditions on recombinant IFTase production In order to improve the protein productivity, the culture conditions used for P. pastoris expression systems are important factors for consideration.14 The shake flask cultivation of P. pastoris is executed in two stages. Biomass is generated in a repressive media, then the recombinant protein is expressed in an induction media containing methanol. Therefore, the biomass and the methanol concentration are important limiting factors in heterologous protein production. As shown in Fig. 2, methanol concentration had a great effect on the activity of recombinant IFTase. The maximum IFTase activity (8.5 U mL−1) was obtained at 15 mL L−1 methanol and was 3-fold higher than the activity induced by 5 mL L−1 methanol
(Fig. 2A). The optimal induction period studies showed that initial cell concentrations of OD600 = 7 was the best stage for recombinant IFTase induction in BMMY medium. Within a wide range of initial cell concentrations (OD600 from 4 to 9), the fluctuations in the IFTase activity was only 37.9%, which suggested that P. pastoris in the logarithmic growth phase maintained a similar capability for IFTase production while induced by methanol (Fig. 2B). In addition, the pH condition is also an important factor for optimizing the expression of yeast recombinant proteins.27 It was reported that lowering pH during the production stage generally mitigated protease activity and improved the product quality.28 However, Fig. 2C showed that a relatively high pH value (pH 6.5) was the more suitable pH condition for IFTase expression by GS115-IFTase. This result was presumably due to the good IFTase stability at pH 4.5-8.0.29 Moreover, owing to the capacity of performing many eukaryotic post-translational modifications, such as glycosylation, disulphide bond formation and proteolytic processing,30 the P. pastoris expression system is suspected to play a role in the pH tolerance of recombinant IFTase. IFTase production in shake flask According to the screening results, P. pastoris GS115-IFTase with 4 mg mL−1 G418 resistance was confirmed to be the best strain and was therefore further characterized in flask cultivation under the previously mentioned suitable conditions. As shown in Fig. 3, the extracellular IFTase activity increased consistently with cell growth and reached its maximum level (11.9 U mL−1) at 96 h, in the late log phase of cell growth. However, no IFTase activity was detected in the control strain supernatant (data not shown). Moreover, the IFTase recombinant P. pastoris accumulated less biomass than the control strain, which might
be due to the burden of overexpression of IFTase.25 Laboratory-scale production of IFTase in a 5 L fermenter Fermentation growth is important for secreted proteins, as the concentration of product in the medium is roughly proportional to the concentration of cells in culture.15 The recombinant IFTase subunit showed molecular mass of about 43 kDa and the fermentation supernatant exhibited a major protein band of IFTase with little undesired bands on SDS-PAGE (Fig. 5). As shown in Fig. 4, this fed-batch fermentation strategy consisted of glycerol batch, glycerol fed-batch and methanol induction phases. after glycerol phase for 18 h and glycerol fed-batch phase for 10 h, the biomass OD600 was up to 74.5, which was much higher than in shake flask cultivation. Then, the methanol feeding was started to induce the production of IFTase. After the induction of methanol for 60 h, the biomass OD600 and extracellular IFTase activity reached final values of 230.2 and 105.4 U mL−1, respectively. This is the highest extracellular activity of IFTase to date. It was reported by our laboratory that Arthrobacter aurescens SK 8.001 IFTase with a relatively high activity (about 19.6 U mL−1) was thermostable and could be a good candidate for the industrial production of DFA III.29 After expression of this ift gene in E. coli., extracellular IFTase activity reached 81.0 U mL−1, however, this value was just 76.8% of the recombinant IFTase in P. pastoris. The results suggested that secretory IFTase in P. pastoris could be an viable method to resolve low extracellular IFTase activity both in the E. coli system and in the original strain. In fact, Pichia high density fermentation can greatly improve the recombinase activity are common.15,25,31 Moreover, the activity of lipase B (secoreted from Pichia) was reported to increase from 57.9 to 11900 U mL−1 when cultured from flask culture to 50 L fermenter.32 So, this system might be expected to achieve
higher IFTase activity in larger volume fermentation of industrial production. On the other hand, the addition of methanol activated pAOX, which could immediately drive the expression of the downstream genes encoding for AOX and IFTase. However, the intracellular AOX activity decreased dramatically from 57.4 to 3.7 U g−1 within 24 h (Fig. 4), suggesting that IFTase have low or no expression. This fluctuation in the AOX activity in the fed-batch fermentation, was probably due to the feedback inhibition of pAOX by metabolic intermediates generated in the methanol utilization pathway.33 In addition, under the constant agitation speed and aeration rate during the induction phase, the relative DO level was dependent on the cell growth strictly but reversely (Fig. 4). CONCLUSIONS In summary, IFTase achieve high level extracellular expression in P. pastoris system with fed-batch fermentation, which suggested that P. pastoris could be an effective alternative to E. coli for secretory expression of IFTase. The extracellular IFTase activity reached 105.4 U mL−1 in a laboratory-scale fermenter, which was 8.9-fold of the activity in shake flasks, and 5.3-fold of that obtained from wild-type strain. Meanwhile, the recombinant IFTase activity was correlated to the copy number of IFTase gene inserted into the GS115 genome, and was greatly affected by methanol concentration. The extracellular expression of IFTase in this system may provide new opportunities for industrial production of IFTase and DFA III. ACKNOWLEDGEMENTS This work was supported by the NSFC Project (No. 31371788), the 863 Project (No. 2013AA102102), the Fundamental Research Funds for the Central Universities (No. JUSRP51304A), and the Support Project of Jiangsu Province (BK20130001) and Shaoxing
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FIGURE LEGENDS Fig. 1 Comparison of extracellular IFTase activity expressed by P. pastoris IFTase-GS115
with different ift gene copy number, which were screened by different antibiotic G418. The single-copy strain was screened by YPD solid medium containing 0.25 mg mL−1 G418, while other multicopy strains were obtained by same medium with 1, 2, 4 mg mL−1 G418. Data were compared by ANOVA and Duncan’s tests with SPSS statistics package software (Version 18.0). Data points are means of three replicates with error bars of standard deviation. Significant differences were defined at p
