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Process Biochemistry, Vol. 31, No. 5, pp. 499-506, 1996 Couvrieht 0 1996 Elsevier Science Ltd Printed-i; &eat Britain. All rights resewed 0032-9592/96 $15.00 +O.OO 0032-9592(95)00093-j :LSEVIER Determination of Optimal Glucose Concentrations for Glucoamylase Production from PlasmidHarboring and Chromosome-Integrated Recombinant Yeasts using a WC2 Promoter Hyung Joon Cha & Young Je Yoo* Department of Chemical Seoul 151-742, Korea (Received 4 September Engineering and The Institute 1995; accepted 21 December for Molecular Biology and Genetics, Seoul National University, 1995) During foreign protein production using a promoter such as SUC2, which is controllable by the glucose concentration in the culture broth, it is important to determine the optimal glucose concentration for maximum protein production. The optimal glucose concentrations for maximal production of glucoamylase in plasmid-harboring recombinant yeast MMY2SUCSTA culture and in chromosome-integrated recombinant yeast MMY2SUCSTA-I culture were 0.13 and 0.09 g litre - ‘, respectively. Using glucose feeding to maintain these optimal glucose concentrations, the extracellular glucoamylase productivities of fedbatch culture were increased (12-fold in MMy2SUCSTA and 23-fold in MMY2SUCSTA-I) over simulation results from batch cultures. The yeast Saccharomyces cerevisiae is widely used as a recombinant host that expresses foreign genes and secretes proteins.’ Glucoamylase (EC 3.2.1.3) which is used to saccharify starchy feed stocks in commercial processes for glucose and ethanol productions, is not produced by S. cerevisiae. With this in mind, the STA gene (glucoamylase gene of Saccharomyces diastaticus) was chosen as a glucoamylase gene source for this research.2 The STA gene used was cloned from a wild type S. diastaticus DS101.3 When a heterologous gene is cloned into yeast, a promoter of the heterologous gene may usefully be replaced by a yeast promoter. Regulated promoters which control gene expression INTRODUCTION One of the principal tools in new biotechnology is the recombinant DNA technique which allows us to manipulate directly the genetic material of individual cells. By inserting forgein genetic information into fast-growing microorganisms, foreign gene products such as proteins may be produced at higher rates and yields than possible with other cellular systems. Recombinant microorganisms may be divided into plasmid-harboring recombinant cells and chromosome-integrated recombinant cells. *To whom correspondence should be addressed. 499 500 Hyung Joon Cha, Young Je Yoo by changes of medium composition, addition of chemicals, or change of cultural conditions are commonly used for foreign protein production.4’5 In this research, the SUC2 promoter regulated by glucose in the culture broth was used6,7 and has the following advantages. Firstly, the problems of the host’s growth inhibition and plasmid instability due to the expression of the plasmid gene can be reduced since the SUC2 promoter is a regulatable promoter. Secondly, since the SUC2 promoter is affected only by glucose, the addition of an inducer is unnecessary in comparison with other inducible promoters. Also, the problem of medium substitution does not exist in comparison with other repressible promoters. When using a promoter controllable by the glucose concentration in the culture broth (such as SUC2 promoter), the close control of glucose concentration is essential and it is very importthe optimal glucose ant to determine concentration for maximum protein production, In this research, optimal glucose concentrations for higher production of foreign glucoamylase from plasmid-harboring and chromosome-integrated recombinant yeasts were determined with special consideration for expression and secretion. MATERIALS AND METHODS Strains and culture medium Saccharomyces cerevisiae MMY2 (a, ura3-52, sta’, stal0) strain was used as the host, Recombinant plasmids YEpSUCSTA and YIpSUCSTA containing the glucoamylase coding STA gene fused with the SUC2 promoter and original STA signal sequence were transformed into the host. The transformants were named MMY2SUCSTA (plasmid-harboring recombiand MMY2SUCSTA-I nant yeast)’ (chromosome-integrated recombinant yeast).’ Yeast was grown at 30°C in semi-synthetic minimal medium containing 0.2% MgS04 - 7H20, 0.2% (NH4)$04, 0.1% KH2P04, 0.025% CaC&. 2H20, 0.2% yeast extract, 0.3% bactopeptone, 1% glucose, 1% starch, and 50 mM succinate. Starch was used as a carbon source for glucoamylase biosynthesis.8 Measurements of cell mass and glucoamylase activity Estimations of cell mass as absorbance were carried out with a spectrophotometer (Kontron; UVICON930) at 600 nm. To measure glucoamylase activity, O-7 ml culture supernatant was incubated in 0.1 ml 1 M sodium acetate buffer (pH 5.0) and 0.2 ml 8% soluble starch (Junsei) at 50°C for 30 min and boiled at 100°C for 5 min to inactivate glucoamylase. Glucose produced by the action of glucoamylase on soluble starch was assayed using a glucose-diagnostic kit (Sigma, No. 510). Units of glucoamylase activity are defined as 1 ymol of glucose released per 1 min. Cell fractionation Cells were harvested, washed with 10 mM sodium azide, suspended in lysis buffer (0.1 M sodium acetate (pH 5.0) 10 mM sodium azide, 1 mM EDTA and 0.1% (v/v) Triton X-100) and mechanically lysed by vortexing with glass beads (Sigma, 425-600 microns). After intermitent vortexing and cooling on ice, the suspension was harvested by centrifugation at 12 000 rpm at 4°C for 15 min, and the supernatant used for enzyme assay. Simulation of foreign glucoamylse production in recombinant yeast culture The parameters of model equations were estimated from experimental data of batch culture using the least-squares regression method. Determination of optimal glucose concentration and simulation of recombinant yeast culture were performed using MATLAB software (version 4.0, The MathWorks, Inc.). Ordinary differential equations of model equations were solved using the 2nd and 3rd order RungeKutta method. RESULTS AND DISCUSSION Foreign glucoamylase production from plasmidharboring and chromosome-integrated recombinant yeasts In order to compare the characteristics of cultures of chromosome-integrated recombinant yeast (MMY2SUCSTA-I) and plasmid-harboring recombinant yeast (MMY2SUCSTA), a Glucoamylase production in recombinant yeasts number of cultivations were carried out. Since r,hanges of pH were very small and expression of glucoamylase occurred normally in this pH range of the culture, control of pH was not liecessary in the fermentation experiments (see 1.6 - 6.6 h ‘: 1.2 22 - 6.4 = -7 ob d- 6.2 i X S - 6.0 a .4 6 0 - 5.8 ?? 0.; .03 J 5.6 r c 1 VI .z J .02 .e .g Y s .a 75 0 70 z e 65 ; .O' 0 E 60 gj a 55 50 + Extra activi d E” 8 0.00 a 0 ;; 2 ” 4 6 8 10 Culture time (h) (A) 10 1 1.6 , 6.6 7 1.2 t 2 8’1 s gq .a z3 4- 6.4 6.2 X 6.0 & 0 .4 s 2?? ^ 0+ .020 1 4 ,015 i 0 I 5.6 0.0 100 3 95 k E 12 .OlO 3 g s 90 g .$ jj ,005 a5 E" g 0.000 0 s B 5.8 2 4 6 8 d 80 + 10 Culture time (h) (6) Fig. 1. Batch profiles of plasmid-harboring recombinant yeast MMY2SUCSTA culture (A) and chromosome-integratred recombinant yeast MMY2SUCSTA-I culture (B). 501 Fig. 1). The patterns of cell growth and glucose consumption were similar in both types of recombinant yeast culture. The profiles of pH were similar to the pattern of glucose consumption in both recombinant yeasts. While the pH in the culture broth decreased depending on the cell growth, the decrease of pH was small because succinate added to the medium played a role in protecting the decrease of pH. In the case of MMY2SUCSTA, plasmid stability depended on glucoamylase expression. As the expression of glucoamylase increased then plasmid stability decreased. In contrast, the genetic stability of the chromosome-integrated recombinant yeast MMY2SUCSTA-I remained good. Glucoamylase was produced late in the culture in both recombinant yeast cultures since the SUC2 promoter was regulated by glucose concentration in the culture broth as previously reported.‘.‘. I0 In the case of MMY2SUCSTA, glucoamylase synthesis began below 7 g litre-’ glucose concentration and secretion occurred below 5 g litre ~ ’ (Fig. l(A)). Expression of glucoamylase then increased rapidly but secretion increased slowly as glucose concentration in the culture broth decreased. In the case of MMY2SUCSTA-I, glucoamylase synthesis began below 4 g litre- ’ glucose concentration and secretion below 2 g litree’ (Fig. l(B)). Expression of glucoamylase then rapidly increased and secretion of glucoamylase increased slowly as with MMY2SUCSTA. The behaviour of the two yeasts with respect to glucose concentration clearly differed. These might be due to a gene dosage (the number of copies of a given gene present in a cell) effect.’ ’ Since MMY2SUCSTA had a high plasmid copy number (20-40) transcription regulation of the SUC2 promoter by glucose in the recombinant plasmid was not tight. In contrast, MMY2SUCSTA-I had three copy numbers of the desired gene, expression of glucoamylase was tightly regulated by glucose and the optimal glucose concentration for maximum expression of glucoamylase was lower from that in the plasmid-harboring recombinant yeast culture. Determination of optimal glucose concentration for glucoamylase production The determination of optimal glucose concentration for protein production is important from a biotechnological point of view. In this research, we considered glucoamylase secretion 502 Hyung Joon Cha, Young Je Yoo as well as glucoamylase expression in the determination of optimal glucose concentration. The optimal glucose concentration for maximum production of foreign glucoamylase, the specific expression rate and the specific secretion rate of each recombinant yeast were calculated as functions of glucose concentration in the culture broth. The mass balance for total glucoamylase protein can be written asi2,13 d(PTX) x =F dt ” d(f’d) dt Fern G (kp + G + G2/kpi) ’ (3) where K= specific secretion rate (h-l), PMX=extracellular glucoamylase activity (units ml-‘). The specific secretion rate K was related to cell growth condition (data not shown).‘3,‘4 (1) where Fp=specific expression rate (units mg ~ ’ cell h-l), PTX=total glucoamylase activity (units mll’), t=operation time (h), X=cell mass (g litre-‘). The SUC2 promoter used in this work, is regulated by glucose concentration in the culture broth. When the glucose concentration is high, the expression of SUC2 promoter is repressed. However, when glucose is decreased to a threshold concentration then expression is not repressed. Thus, to consider glucose repression on the expression of SK2 promoter, a glucose repression term (G’/kpi) is introduced into the specific expression rate Fp’2,13 Fp= =K(P-&-PM@, 0.0 (2) where FP,,,=maximum specific expression rate (units mgg’ cell hh’), G=glucose concentration in the culture broth (g litre-‘), kp=constant of specific expression rate (g litre - ’), kpi = repression constant of specific expression rate (g litre-‘). The specific glucoamylase expression rate FP of MMY2SUCSTA is indicated in Fig. 2(A) and its maximum value calculated to be O-054 units mg-’ cell h- ’. Maximum expression of plasmid-harboring yeast occurs at a glucose concentration of 0.17 g litre-’ in the culture broth. Similarly, the specific glucoamylase expression rate of chromosome-integrated recombinant yeast MMY2SUCSTA-I is indicated in Fig. 2(B) and its maximum value calculated as O-026 units mg-’ cell hh’. In this case, maximum expression occurs at a glucose concentration of 0.13 g litre-‘. The mass balance for secreted glucoamylase13 is *- .025 I .5 1.0 1.5 2.0 2.5 3.0 , .4 Fig. 2. Optimal glucose concentrations for maximum specific expression rate and specific secretion rate in plasmid-harboring recombinant yeast MMY2SUCSTA (A) and chromosome-integrated recombinant yeast MMY2SUCSTA-I (B). SER: specific expression rate; SSR: specific secretion rate; SOR: specific optimal rate. 503 Glucoamylase production in recombinant yeasts Since specific growth rate was a function of glucose concentration, the specific secretion rate may also be described as a function of glucose concentration.‘” described below and the optimization was performed in fed-batch culture as shown in Fig. 3. PI =mF K= GIG a+pG ’ {Productivity} =mp {‘$V,!)” 1, (4) where K,=maximum specific secretion rate I h-r), m=constant of specific secretion rate (g litreel), /?=constant of specific scretion rate. The dependence of specific secretion rates on glucose concentration in both recombinant yeasts is indicated in Fig. 2. In both recombihant yeasts, the specific secretion rate increased with increase in glucose and finally reached s,aturation. The optimal glucose concentration might exist between the specific expression rate and rhe specific secretion rate. The optimal glucose concentration for maximum extracellular gluco<imylase activity was calculated through two equations of specific expression rate (eqn 2) tend specific secretion rate (eqn 4). In the case ,)f MMY2SUCSTA, the optimal glucose con::entration for specific expression rate was 0.17 g litre-’ (Fig. 2(A)) but, the optimal glucose conzentration for both specific expression rate and specific secretion rate was 0.76 g litree’. In the zase of MMY2SUCSTA-I, the optimal glucose concentration for specific expression rate was 9.13 g litreel and the optimal glucose concentration for both specific expression rate and specific secretion rate was O-25 g litre-’ (Fig. 2(B)). In this case, the difference between two optimal glucose concentrations was smaller than that of MMY2SUCSTA since the slope of the specific secretion rate of MMY2SUCSTA-I was larger in the region of low glucose concentration. This calculation was performed using only eqn (2) for specific expression rate and eqn (4) for specific secretion rate. Values calculated in this manner can be used in simulation or real experiment for fed-batch culture. However, in the real culture, many other factors that affect final extracellular product concentration exist including, expression level, secretion capacity, genetic stability, final operation time, final vessel volume, and culture type. Therefore, in order to determine real optimal glucose concentration, glucoamylase productivity (unit h-‘) was used using a performance index (PI) as (5) where V,=final vessel volume (litre). A fixed final vessel was used as a constraint condition, the initial vessel volume being set as 0.5 litre and final vessel volume set as 2 litre. In the case of MMY2SUCSTA, the optimal glucose concentration for the desired extracellular glucoamylase productivity was 0.16 g 1itre-l and its value was close to the optimal Glucose concentration (g litre-‘) (4 I I I Glucose concentration I I (g litre-‘) W of optimal glucose concentrations Fig. 3. Determination for maximum glucoamylase productivity in fed-batch culture of plasmid-harboring recombinant yeast MMY2SUCSTA (A) and chromosome-integrated recombinant yeast MMY2SUCSTA-I (B). Initial and final conditions; X+=0.1 g litre-‘, X- =O*Og litre-‘, X=0*1 g litre-‘, G=S g litre-‘, Gr=20 g litre- , V,,--05 lure, and l/f=2 litre. X + : plasmid-harboring cells, X - : plasmid-free cells, X: chromosome-integrated cells, P,W/t: extracellular glucoamylase productivity and P,XV/t: total glucoamylase productivity. 504 Hyung Joon Cha, Young Je Yoo value for specific expression rate (O-17 g litre-‘) (Fig. 3(A)), while that for total glucoamylase productivity was 0.13 g litre-‘. In the case of MMY2SUCSTA-I, the optimal glucose concentrations for the desired extracellular glucoamylase productivity and for total glucoamylase productivity were equal and their value was 0.09 g litre-’ (Fig. 3(B)). These optimal glucose concentrations (O-16 and 0.09 g litre-‘) were closer to the glucose concentrations for maximum specific expression rate (O-17 and 0.13 g litre-‘) than those for maximum specific expression rate and specific secretion rate (0.76 and 0.25 g litre-‘). The reason for this may be that expression was rate-limiting for overall foreign glucoamylase production compared to secretion in both recombinant yeasts. Simulation of foreign glucoamylase production using optimal glucose concentration Simulations of foreign glucoamylase production in fed-batch cultures of plasmid-harboring and chromosome-integrated recombinant yeasts were performed using optimal glucose concentrations for 30 h operation times as shown in Fig. 4. Glucose feeding rate was calculated using growth conditions to maintain the optimal glucose concentration in the culture broth as shown below.‘” !g-G& 1 P -++m Y f= Cultu~ time(b) (4 xv’ (6) where Gf=glucose concentration in the feed (g litre-‘), GOPT=optimal glucose concentration for glucoamylase production (g litre-‘), (O-16 g litre-’ for MMY2SUCSTA and 0.09 g litre-’ MMY2SUCSTA-I), m =maintenance coeffivolume (litre), YXG=yield cient, V=vessel coefficient, p=specific growth rate (h-l). This equation includes state variables (cell mass and vessel volume) and feed rate was calculated using these state variables. As shown in Fig. 4(A), cell mass reached a maximum at 20 h in the MMY2SUCSTA culture. A larger fraction of total cells was occupied by plasmid-free cells in the final culture period and the internal glucoamylase expression was therefore maximal at 25 h and then decreased. The final extracellular glucoamylase activity was O-215 units ml-’ and the productivity was 13.0 units h-‘. 5 10 15 20 Cnlbtime(h) 2s 30 1 (61 Fig. 4. Simulation results for fed-batch culture of plasmid-harboring recombinant yeast MMY2SUCSTA (A) and chromosome-integrated recombinant yeast MMY2SUCSTA-I (B). Initial conditions; X+ =O.l g litre-‘, X-=O.Og litre-‘, X=0*1 g litre-‘, G=S g litre-‘, Gf=20 g litreP’, V,=O5 lit re, and t,=30 h. X+: plasmidharboring cells, X - : plasmid-free cells, XT: total cells, X: chromosome-integrated cells, G: glucose concentration, P,X: intracellular glucoamylase activity, PMX: extracellular glucoamylase activity and PTX: total glucoamylase activity. 505 Glucoamylase production in recombinant yeasts Table 1. Summary of simulation results for glucoamylase chromosome-integrated recombinant yeasts production in fed-batch culture Chromosome-integrated MMy2SlJCSTA-I Plasmid-harboring yeast MMYZSUCSTA Batch culture Fed-batch culture (30 h culture time) Fed-batch culture (2 litre final vessel volume) using plasmid-harboring and yeast Productivity (units h ‘) Relative increase Productivity (units h _ ‘) Relative increase 1.2 13.0 1.0 10.8 0.8 1I.9 1.0 14.9 14.0 11.7 18.4 23.0 This pattern became more marked with an increase of plasmid-free cell fraction in the initial culture. In contrast, cell mass steadily increased in the case of MMY2SUCSTA-I (Fig. 4(B)). The glucoamylase activity continued to increase and the final extracellular glucoamylase activity was 0.274 units ml-‘; while the extracellular glucoamylase productivity reached 11.9 units hh ‘. When using final operation time as a constraint condition, the extracellular glucoamylase productivities of both recombinant yeasts were similar. This resulted from a final small vessel volume of the chromosomeintegrated yeast culture. In the case of using final vessel volume as a constraint condition, the final extracellular glucoamylase productivity of the chromosome-integrated yeast culture was 31.4% larger (18.4 units hh’) than that of plasmid-harboring yeast culture (14.0 units hh’). This resulted from higher genetic stability and higher secretion efficiency (68.1% compared to 48.7% of MMY2SUCSTA). These simulation results of foreign glucoamylase production using fed-batch culture of the plasmid-harboring recombinant yeast MMY2SUCSTA and the chromosome-integrated recombinant yeast MMY2SUCSTA-I are summarized in Table 1. By using glucose feeding to maintain optimal glucose concentrations (0.16 g litre - ’ in MMY2SUCSTA and 0.09 g litre - ’ in MMY2SUCSTA-I), the final glucoamylase productivities of fed-batch cultures were increased (11*7-fold in MMY2SUCSTA and 23-O-fold in MMY2SUCSTA-I) compared to those of the batch culture when using final vessel volume as a constraint condition. ACKNOWLEDGEMENT This research was supported by the Genetic Engineering Research Fund from the Ministry of Education, Korea. REFERENCES 1. Kingsman, production S. M., Kingsman, A. J. & Mellor, J., The of mammalian proteins in Saccharomyces cerevisiae. Trends Biotechnol., 5 (1987) 53-7. 2. Yamashita, I., Suzuki, K. & Fukui, S., Nucleotide sequence of the extracellular glucoamylase gene STA 1 in the yeast Saccharomyces diastaticus. J. Bactetiol., 161 (1985) 567-73. 3. Ahn, J. H., Regulation of glucoamylase gene expression in Saccharomyces cerevisiae var. diastaticus. 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