ISSN 00036838, Applied Biochemistry and Microbiology, 2015, Vol. 51, No. 7, pp. 766–773. © Pleiades Publishing, Inc., 2015. Original Russian Text © K.V. Sidoruk, L.I. Davydova, D.G. Kozlov, I.I. Gubaidullin, A.V. Glazunov, V.G. Bogush, V.G. Debabov, 2014, published in Biotekhnologiya, 2014, No. 6, pp. 27–35.
TECHNOLOGY OF BIOPREPARATIONS
Fermentation Optimization of a Saccharomyces cerevisiae Strain Producing 1F9 Recombinant Spidroin K. V. Sidoruk, L. I. Davydova, D. G. Kozlov, I. I. Gubaidullin, A. V. Glazunov, V. G. Bogush, and V. G. Debabov State Research Institute for Genetics and Selection of Industrial Microorganisms, Moscow, 117545 Russia email:
[email protected] Received December 1, 2014
Abstract—The fermentation of a Saccharomyces cerevisiae strain producing recombinant spidroin IF9 was optimized. A simplified twostage scheme of the process was developed; the effect of sucrose, glucose, fruc tose, and galactose on the efficiency of the process was investigated. The optimal concentration of sucrose in the medium and replenishment was determined. The influence of peptone, tryptone, and casein hydrolysate of various brands on the effectiveness of the fermentation was analyzed. The optimal concentrations of pep tone and yeast extract in the medium and replenishment were determined. As a result, owing to the optimi zation, the process of fermentation was simplified, a new composition of a complex replenishment was designed, and sucrosespecific consumption was reduced by two times, whereas the expenses of peptone and yeast extract were decreased by about 2.5 times, the yield of biomass per unit of the culture broth volume grew by ~ 40%, and that of the protein of interest increased by ~ 60%. Keywords: fermentation, galactose, peptone, replenishment, recombinant spidroin, saccharomyces yeast, strainproducer, sucrose, yeast extract DOI: 10.1134/S0003683815070066
The obtaining and studying of features of recombi nant proteins, which are analogs of spider web pro teins, started more than twenty years ago [1, 2]. Differ ent expression systems were used to produce recombi nant analogs, especially analogs of proteins of the web dragline of ordweb spiders (spidroins): bacteria [3, 4], yeast [5], plants [6], insects [7–9], mammals cell cul ture [10], and transgenic animals [11]. Many substances derived from spider silk proteins have been tested in laboratories for medical and veter inary use [12]. However, development of a commercial product so far has been difficult because of the high original cost of these recombinant proteins. Earlier, we constructed genes coding 1F9 and 2E12 proteins, which are analogs of spider web dragline (spidroins 1 and 2). We used different expression sys tems in their production: Pichia pastoris yeast [13–15] and tobacco [16]. However, the best results were achieved with a developed expression system based on genetic modified strains of Saccharomyces cerevisiae yeast. This system provides high density of the target protein in the insoluble fraction. It is the basis for the Abbreviations: CB—culture broth, PAAG—polyacrylamide gel, EDTA—ethylenediaminetetraacetate, EP—electrophoresis, SDS—sodium dodecyl sulfate.
development of a cost effective technology for obtain ing recombinant spidroin. Saccharomyces cerevisiae SCR702T1F9 [17] is a diploid prototrophic strain. It was obtained as a result of cellular transformation of the D702 recipient strain by expressional multicopy vector pPDX4T1F9, a derivative of 2 μm of yeast plasmid that bears a struc tural gene of recombinant protein of spidroin under GAL1 promoter control (data are not presented). Vectors obtained from 2 μm of plasmid usually have a low segregation stability and often can be lost during cultivation. In this strain the stability of the expression vector is determined by its homozygosis by genome mutation in the phosphoglycerate kinase gene (pgkl/pgkl genotype). This genotype assures a lack of cell growth of strains losing the plasmid on any medium with sugar digestible for S. cerevisiae yeast. PGK1 gene in pPDX4T1F9 vector results in the growth of plasmidcontaining cells of this strain and selective maintenance of the expressive vector in cells during fermentation. The regulation of GAL1 promoter in the D702 strain was also changed. The strain bears a homozygotic mutation in the gal80 gene (gal80::LEU2/gal80::LEU2 genotype) that is responsible for inactivation of a pro tein repressing GAL1 promoter in the case of a lack of galactose. Thereby, expression of target gene is inde
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pendent of galactosis induction. Another mutation is related to a promoter of the GAL4 gene coding an acti vator of the GAL1 promoter. Its own GAL4 promoter was changed to one of the variants of yeast gene pro moter sta2, which was activated in response to the exhausting of fermented sugar in the cultivation medium. It induces GAL4 protein expression because of a decrease of sugar concentration in the medium, which leads to activation of GAL1 promoter–con trolled 1F9 target protein expression in cells. Together with mutation in the gal80 gene, the changed regulation and enhanced expression of GAL4 proteins makeit pos sible to realize simple galactoseindependent activation of the GAL1 promoter controlling 1F9 protein expres sion. Thus, GAL1 promoter activity in S. cerevisiae strain SCR702T1F9 is defined not by presence of galactose in the medium but by the low sugar concen tration. High concentrations of fermented carbohy drates inhibit the activity of GAL1 promoter in the strain. The rate of decrease in promoter activity depends on the composition of carbohydrates and their concentration. GAL1 promoter is usually repressed when the carbohydrate concentration is higher than 0.3% (data are not presented). The laboratory methods for detection and purifica tion that we conducted made it possible to receive pro tein in a highly purified condition and to use it in labo ratory research. Different forms of this protein were determined: hydrogels, microgels, films, matrices, and microspheres. Their high biocompatibility with human and animal cells, lack of toxicity, capacity for low biore sorption in animals, and ability to initiate vascularisa tion, innervation, and tissue regeneration during trans plantation into an organism were shown [18, 19]. The results allow us to consider that materials cre ated on the basis of recombinant spidroins have high potential for their use in a wide range of fields—prima rily in medicine. Thus, it was necessary to develop tech nology to obtain recombinant spidroin for manufac ture. One of the first necessary conditions for a shift from artificial spider silk to commercial manufacturing is to optimize the fermentation of Saccharomyces cere visiae, a producer of recombinant spidroin 1F9 [17]. This can reduce the production cost. The following tasks were implemented to achieve the research objective: (1) selection of the optimal process image (number and duration of stages); (2) selection of optimal medium composition and makeup, including the detection of the optimal min eral and carbohydrate composition of the medium and complex components (yeast extract, peptone, etc.), and detection of the optimal ratio of medium ele ments. APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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EXPERIMENTAL CONDITIONS Strains, medium, and conditions of cell cultivation. Spidroin expression was carried out during cultivation of a laboratory strain of Saccharomyces cerrvisiae, SCR702T1F9 [17]. We used following reagents to grow yeast: J849 pep tone 140 (Amresco), J636 peptone (Amresco), casein peptone CM7 (Costantino), meat peptone CM (Cos tantino), tryptone (Pancreac), yeast extract (Bio springer), sucrose (Panreac), D(+)glucose mono hydrate (Pancreac), D(+)fructose (Applichem), D(+)galactose (Reachim, Ch), bacteriological agar (Pancreac), 85% phosphoric acid (Reahim, ch), 25% ammonium hydroxide (Reahim, Ch), and minimal medium YNB (Sigma). The medium was sterilized by autoclaving at 110° for 30 min. Galactose in the medium was sterilized separately by Millipore filters with a pore diameter of 0.2 μm. Yeast cells were grown in broth in a 750mL flask (with a 50mL volume of broth) at 28° with a Multi tron 2 threelevel shaker incubator, an SU25HSCB (Infors) at 250 rpm, or a 3L Biostat BDCU fer menter (B.Braun Biotech International Sartorius Group). Protein extraction and purification. Cells were col lected by centrifugation at 8000 rpm for 20 min in an Avanti J26 XP HighPerformance Centrifuge (Beck man Coulter) with JLA8 1000 Fixed Angle 20° rotor. Fivetenths of a kilogram of wet biomass was suspended in buffer for destruction (0.05 M of sodium phosphate, pH 7.4 (Amresco); 0.001 M of EDTA (Amresco); 5% glycerin (Merck)). Cells were destroyed by glass balls in an MS3 flow mill (Russia) for 1.5 h. The sus pension was centrifuged at 14000 rpm in an Avanti J26 XP HighPerformance Centrifuge (Beckman Coulter) in JLA14 Fixed Angle 25° rotor; the supernatant was removed. The target protein was extracted from the pre cipitate by a 10% lithium chloride solution (Panreac) in 90% formic acid (Reahim) for 16–18 h with intensive mixing in a magnetic mixer. The sample was then cen trifuged at 14000 rpm in a Avanti J26 XP HighPerfor mance Centrifuge (Beckman Coulter) with JLA14 Fixed Angle 25° rotor. The supernatant was dialyzed against 10 mM of sodium acetate (Panreac), pH 4.0, and clarificated by centrifugation. The final purification proceeded via ionexchange chromatography in a HiPrep 16/10 SP FF column (GE Healthcare) with CTAApirifierTM (GE Healthcare) and a change of the pH (pH 4.0 → pH 7.0 → 4.0). Protein was eluted by the NaCl concentration gradient and dia lyzed against deionized water, frozen at –70°, and lyo philized. Detection of protein production. The protein pro duction rate was detected by weighing of the extracted, purificated, and lyophilized protein during the growth
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of yeast in the fermenter; we used the micromethod of detection and analysis of the protein production level during growth in the flask. Micromethod of protein production detection. Extraction proceeded in 1.5 mL tubes. One hundred microliters of buffer for destruction (0.05 M of sodium phosphate, pH 5.0; 0.001 M of EDTA, 5% of glycerin) and 570 mg of glass balls (d = 0.45–0.65 mm (Sigma)) were added to 100 mg of wet cellular precipitate. The sample was mixed and then shaken by a vortex (Multi vortex V32, BioSan) for 90 s. Then 250 μL of buffer for destruction was added and shaking was repeated for 60 s. We added 500 μL more of buffer for destruction and shook it; it was then centrifuged for 10 min at 16000 g. The supernatant was removed, and the pre cipitate with insoluble proteins was washed by the add ing of 500 mL of buffer for destruction, shaking, and transferral to a new tube and centrifuged for 15 min at 16000 g. The precipitate was resuspended in 400 μL of 6.5G buffer with 6.5 M of guanidine hydrochloride (Applichem) and stored overnight at 4°. The solution was then centrifuged for 15 min at 16000 g, and super natant with the target protein passing into it was dia lyzed against 300 mL of 5 mM of sodium acetate (Rea him) for 1.5 h. The obtained sample was centrifuged for 15 min at 16000 g. Supernatant was used for elec trophoretic analysis of the level of target protein pro duction. Electrophoresis in SDS PAAG. Sample preparation and electrophoresis were conducted with SDS (Sigma) in 10% PAAG by the standard Laemmli method. The protein fraction obtained from 100 mg of cells was spread on one track. RESULTS AND DISCUSSION The Main Lines of Fermentation Optimization The main purpose of fermentation optimization and the obtainment of recombinant IF9 is a reduction in the original cost of the target protein. To achieve this goal, it is necessary to complete two tasks: a decrease in the cost of enzyme exploitation per unit mass of the target product and in the original cost of the medium. To solve the first problem, it is necessary to increase the composition of the target protein in a working vol ume unit of fermenter (or culture broth volume). For example, the number of fermentations necessary to obtain the desired amount of the protein depends on this characteristic. The target protein content per vol ume unit is detected in two ways: the yeast biomass yield from a volume unit of the culture broth and the spidroin density in the biomass. At the same time, these two characteristics can be changed indepen dently from each other in response to a change in cul tivation conditions.
In summary, we can detect the following fermenta tion parameters that are necessary for optimization: biomass yield from 1 L of culture broth, protein den sity in 1 kg of biomass, expenditure of peptone and yeast extract per 1 g of spidroin and sucrose per 1 g. Selection of Optimal Scheme of the Process The growth of Saccharomyces in a fermenter for laboratory purposes, which fully satisfied our research needs for spidroin, traditionally involves three stages. (1) Yeast is grown in a rich, complex medium Y25P25S50 containing 25 g/L of yeast extract, 25 g/L of peptone, and 50 g/L of sucrose without replenishment and with pHstat until the index value exceeds 6.5 (this usually happens after 20–24 h). After this, replenishment and pHstat is included (while pH > 6.0, it is carried out using 12% ammonium). (2) Growth occurs for 1 day with a singlecomponent sucrose replenishment S500 (50 g/L of sucrose), which is given at a constant rate of 1 g of sucrose/L/h. (3) Growth occurs for 1 day with complex replenishment Y70P70S250 (yeast extract is 70 g/L, peptone is 70 g/L, and sucrose is 250 g/L), which is given at a constant rate of 1 g of sucrose/L/h. Fermentation lasts 68–72 h and makes it possible to obtain 95 ± 5 g of wet yeast biomass from 1 L of liquid broth with and a spidroin density of 2.6 ± 0.2 g/kg of biomass. These data were obtained via analysis of the results of 20 cultivation experiments. It is clear that a process including three stages and the use of two different replenishments contains many more parameters that require optimization than a pro cess limited to two stages and only one replenishment. This is why the opportunity to proceed to a twostage process and to move from two different replenish ments to one without a decrease in fermentation effi ciency was studied for the first time. The amount of sucrose, peptone, and yeast extract, together with replenishments during the entire pro cess, was thus detected. Based on the obtained data, the average composition of the complex replenish ment Y60P60S400 composed of 60 g/L of yeast extract, 60 g/L of peptone, and 400 g/L of sucrose was calcu lated. This replenishment makes it possible to preserve the previous expenditure level for all components and combines the second and third stages together into one phase. The designed twostage process included the fol lowing phases: (1) yeast growth on a rich, complex medium Y25P25S50 without replenishment until the pH exceeds 6.5 (usually in 20–24 h), followed by replen ishment, and pHstat proceeded (6.0 < pH < 7.0) by means of 12% ammonium solution and 20% ophos phoric acid; and (2) growth for 2 days with the addi
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tion of complex replenishment Y60P60S400 (see above) with 1 g of sucrose/L medium/h speed. The fermentation duration was 68–72 h. The pHstat parameters were changed because the new process has had a constant tendency toward a pH increase after 40–44 h of cultivation. Both processes give the same yield of biomass 90 ± 5 g/L and the same spidroin density 2.6 ± 0.2 g/kg) (the data was obtained in accordance with the analysis of five experiments). We then provided optimization of only the two stage process. Selection of Optimal Carbohydrate Composition of Medium and Replenishments In the original process, sucrose was used as the car bohydrate component of the medium and replenish ments. However, Saccharomyces can assimilate a wide spectrum of carbohydrates, including glucose and fructose, which are also cheap and accessible as sucrose. The gene encoding recombinant spidroin IF9 is under GAL1 promoter control, the activity of which, as was mentioned above, is regulated in a given strain by the concentration of available carbohydrates in the medium; this regulation depends on the variet ies of sugar. Thus, it is appropriate to examine the role of sucrose, glucose, and fructose as a carbohydrate components absorbed in fermentation conditions. A comparison showed that a change from sucrose to glucose does not affect the biomass yield but leads to a decrease in celluar spidroin density to 2.2 ± 0.2 g/kg of the biomass. This favors sucrose as a fermentation medium component. A change from sucrose to fructose leads to a small increase in the biomass yield from 95 ± 5 g/L to 110 ± 10 g/L, but the cellular spidroin density decreased to 2.2 ± 0.2 g/kg (the data was based on an analysis of three experiments) in the presence of fructose, as with glucose. Both of the processes with sucrose and fructose have approximately the same protein yield from the working volume of the fer menter 0.25 ± 0.2 g/L of culture broth. However, spidroin extraction and purification is better for pro duction from a smaller number of cells under other wise equal conditions. This is another reason why sucrose is superior to fructose. As was mentioned before, the strainproducing gene encoding spidroin is under GAL1 promoter con trol. Its activity increases when the concentration of available carbohydrates in the medium decreases. Based on this, we decided to determine the possibility of using catabolite repression effect for a decrease in the “apparent” carbohydrate concentration. Thus, one of the two carbohydrates cannot be used before the APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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other in the medium is exhausted. As a result of adding a mixture of carbohydrates to the medium, the con centration of each of them used consequently at any one time is lower than the total concentration. At the same time, the promoter activity should increase. Since it was shown earlier that the density of spidroin grown on glucose or fructose in yeast cells is lower than that grown on sucrose, we decided to use a mixture of sucrose and another monosaccharide—galactose. Due to the high price of galactose, the first experi ments on the induction of spidroin expression were in flasks. We used the following media, g/L: Y20P20S30 (yeast extract—20, peptone—20, and sucrose—30); Y20P20S15G15 (yeast extract—20, peptone—20, sucrose—15, and galactose—15); Y20P20S20G10 (yeast extract—20, peptone—20, sucrose—20, and galac tose—10); Y20P20S25G5 (yeast extract—20, pep tone—20, sucrose—25, and galactose—5), and Y20P20G30 (yeast extract—20, peptone—20, and galactose—30). The cellular spidroin density was estimated qualitatively by PAAG SDS EP. The best results for spidroin density (70 ± 2 g/L) were obtained when acombination of sucrose and galac tose in Y20P20S15G15, Y20P20S20G10, and Y20P20S25G5 medium was used (Fig. 1) (the sucrose/galactose ratio was 1 : 1, 2 : 1, and 5 : 1, respectively). The high est biomass entry values were obtained on these media 70 ± 5 g/L. Media with pure sucrose or galac tose (Y20P20S30 and Y20P20G30) provided a biomass yield that equaled 50 ± 3 g/L (the data was based on three experiments). After confirming in experiments with flasks the supposition about the advantages of using a combina tion of carbohydrates, we conducted cultivation with a 3liter fermenter. The process with sucrose was com pared with a process in which the medium and replen ishment have a mixture of sucrose/galactose in a 2 : 1 ratio. A change from sucrose to a mixture of sucrose and galactose in the fermenter conditions did not affect biomass growth or spidroin density in yeast cells, as was the case for the experiments with flasks. This probably occurred because of the fractional delivery of replenishment in fermenter and the concentration of carbohydrates, which can suppress promoter activity only at the beginning of the process and has no serious effect on fermentation at all. Based on these data, we can conclude that sucrose is the optimal carbohydrate for cultivation of Saccha romyces cerevisiae, a producer of 1F9 spidroin. Selection of Optimal Sucrose Concentration in Medium Besides the quality of the composition of the medium and replenishments, the concentration of
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1
2
3
4
5
6
Fig. 1. Effect of galactose on density level of recombinant spidroin (electrophoresis in 12% of SDS PAAG). 1⎯markers of molec ular mass (PageRulerTM Prestained Protein Ladder, Fermentas); 2⎯Y20P20S30 medium; 3⎯Y20P20S15G15; 4⎯Y20P20S20G10; 5⎯Y20P20S25G5; 6⎯Y20P20G30 (ref. to text).
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Yield, g/L
50 40 30 20 10 0 0
5
10 15 20 25 30 35 40 45 50 Sucrose concentration, g/L
Fig. 2. Dependence of biomass yield on the sucrose concentration in the medium.
carbohydrates in the main cultivation medium should have a large effect on the process. In experiments with flasks, we checked the effect of the sucrose concentration on yeast growth. With the same 25 g/L concentration of yeast extract and 25 g/L concentration of peptone, we tested the following concentrations of sucrose, g/L: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50. Growth was provided in 750 mL flasks and with 50 mL of medium at 2° with a mixer at 250 rpm for 48 h. The dependence of biomass yield on the sucrose concentration in the medium is shown in Figure 2. It would seem that the curve of dependence can be split into three parts. At a sucrose concentration of 0–
15 g/L, a direct proportion is observed: each added gram of sucrose adds 3 g of cells in 1 L of culture broth. At a concentration of 15–40 g/L, the biomass continues to increase in response to an increase in sucrose, but the effect is not as great. A further increase in the sucrose concentration up to 40 g/L leads to a decrease in the biomass concentration. Thus, the maximum sucrose concentration with the maximum effect on biomass yield is 15 g/L. The effect of the sucrose concentration observed during the growth of the spidroin producer in flasks was also studied during growth in fermenter. At the same time, media with 50 g/L and 15 g/L of sucrose were compared. The peptone and yeast extract con tents in both media were the same: 25 g/L. Fermen
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tation proceeded as was described earlier, for 72 h in two stages. Comparison of the results shows that a decrease of the sucrose concentration in the medium from 50 g/L to 15 g/L leads to an increase in the bio mass yield from 95 ± 5 g/L to 135 ± 5 g/L and an increase in spidroin density from 2.6 ± 0.2 g/kg to 3.0 ± 0.3 g/kg of biomass. Since a change in the sucrose concentration results in a large effect on the effectiveness of the process, this factor was fully studied with the use of media containing the following sucrose, g/L: 10, 12, 15, and 20. The best results were obtained with concentrations from 10 g/L to 15 g/L; the biomass yield was 135 ± 5 g/kg, and the spidroin density was 3.0 ± 0.3 g/kg of wet biomass. At a concentration of 20 g/L, a small decrease in the biomass yield to 125 ± 5 g/L was observed without a decrease in spidroin density in yeast cells. Selection of Optimal Concentration of Peptone and Yeast Extract in the Medium After the optimal sucrose concentration was deter mined, we studied the effect of the peptone and yeast extract compositions in the medium during growth in the fermenter. Media with peptone and yeast extract concentrations of 25 g/L and media in which the pep tone and yeast extract concentrations were 20, 15, 10, and 5 g/L were analyzed. A decrease in the peptone and yeast extract composition from 25 to 10 g/L was not negative for the process. A further decrease in the peptone and yeast extract concentration to 5 g/L of each leads to the termination of strain growth. When we brought the data for the optimization of peptone and yeast extract concentrations together with the results obtained during optimization of the sucrose concentration, we developed a new composi tion of the main medium Y12P12S12 that was used in our subsequent experiments, g/L: peptone—12, yeast extract—12, and sucrose—12. Selection of Optimal Concentration of Peptone and Yeast Extract in Replenishment After the optimal concentrations of sucrose, pep tone, and yeast extract in the main medium were deter mined, it was necessary to determine the optimal con centrations of these components in the replenishment. The average composition of the complex replenish ment Y60P60S400 was calculated based on the use of peptone, yeast extract, and sucrose during a nonopti mized process that included three stages and used two different replenishments (see above). At the same time, the sucrose concentration was determined as the highest, in which replenishment retains an acceptable viscosity. A decrease in the sucrose concentration in the replenishment to 300 g/L leads to a strong dilution APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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of the medium and an increase in the culture broth volume during fermentation. An increase in the sucrose concentration up to 500 g/L depends on the increase in viscosity when it became difficult to deliver replenishment by peristaltic pump. Thus, the sucrose concentration in the replenishment has been main tained (400 g/L). The fact that a twofold decrease in the peptone and yeast extract concentrations in the main medium was not negative for results of fermentation made it possi ble to decrease the peptone and yeast extract concen trations in the replenishment. We compared the effectiveness of Y60P60S400 replen ishment (g/L: yeast extract—60, peptone—60, and sucrose–400) and Y40P40S400 (g/L: yeast extract—40, peptone—40, and sucrose—400). A decrease of pep tone and yeast extract concentrations by half had a large negative effect on the process: the biomass yield decreased to 80–90 g/L, and the spidroin density was reduced by 2 times. We then tested the possibility of partial replace ments of peptone and yeast extract for a complex of macroelements, microelements, and vitamins. For this purpose we added to Y12P12S12 a single standard minimal medium for yeast growth, YNB (6.7 g/L, V/OAMINO ACIDS), which has a balanced complex of all of the necessary elements and vitamins. We added triple YNB medium to the replenishment. However, there was no large effect from either of them. This result shows that a decrease in the peptone and yeast extract concentrations in replenishment is impossible without compromising the process. The supposition regarding an increase in the pep tone and yeast extract concentrations in the replenish ment was also verified. The original replenishment, Y60P60S400, was compared with Y90P90S400 replenish ment (g/L: yeast extract—90, peptone—90, and sucrose—400). An increase of the peptone and yeast extract concentrations by half leads to a small (
