bioreactor studies of heterologous protein production by ...

More documents

Recommendations

Info

$uWaterloo LaTeX Thesis Template - UWSpace - University of Waterloo$

the aqueous fermentation broth. b such systerns, Iiquid-solid mass W e r could be a limiting step due to the low relative velocity between the immobilized ceil particIes and the fennentation broth. Thus, novel mimobilized-cdl bioreactors are needed to be developed for successful application with recombinant œüs. 1.5. Objectives In thk research. a recombinant S. cerevisiae C468/pGAC9 (ATCC 20690). which expresses Aspergillus mcullo~ glucoamylase gene under the control of the yeast enoiase 1 (ENOI) promoter and secretes glucoamylase into the emaoeilular medium. is used as a mode1 system to investigate an immobibd recombinant yeast bioprocess. The C468 suain of S. cerevisiae was Wormed with the plasmid vector pGAC9 (2 p-based) which contains a section of the ghcoamyiase gene h m A. awmnori. The 2pm-based shuttle plasmid has characteNncs common to many yeast vectors and therefore serves as a good mode1 system. The objectives of this study are to develop an efficient immobilid ceii bioreactor for cultivation of the recombinant yeast œik, to investigate and compare the genetic stability and recombinant protein productivity of fiex and immobilized yeast ceU systerns. to examine the mechanhm which facilitate the high degree of plasmid stabüity in the immobilization aystem and to &velop a mathematicai mode! for describing ce0 growth. substrate consumption. ethanol metabolism, genetic stabiiity, and recombinant protein production. In order to achieve these objectives, the foiiowing tasks were carried out:
1. Study the gmwth characteristics, the plasmid stability and recombinant protein pmductivity of this soain in batch and continuous suspuision cultures and gain knowledge about the importance of metabolic pathways with regard to the recombinant protein formation. 2. Develop a novel immobilized-Cen-hlm airlift bionactor for the recombinant yeast culture. 3. Compare genetic stability and protein productivïty be~een the fiee and immobiüzed recombinant yeast systems. 4. Mode1 the fiee and irnmobilizcd yeast cell systems based on metabolic pathways. plasmid segregation and stabWg mechanisns. 5. Seek a general explanation for the irnproved stability in the imrnobilllcd ce0 systems.
Page 1 and 2: BIOREACTOR STUDIES OF HETEROLOGOUS
Page 3 and 4: nie University of Waterloo requin%
Page 5 and 6: ate depended on activities of the p
Page 7 and 8: Acknowiedgments Fmt of aü, 1 am ve
Page 9 and 10: 2.6. Concluding Rernarks ..........
Page 11 and 12: 5.3.3. Simlilated Results for Prese
Page 13 and 14: Table 1.1. Table 1.2. Table 1.3. Ta
Page 15 and 16: Figure 2.1. Figure 3.1. Figure 3.2.
Page 17 and 18: during Continuous Culture in Airlif
Page 19 and 20: Figure 5.3. Comparison between Mode
Page 21 and 22: Cell Systern at Glucose Feed Concen
Page 23 and 24: a .......... growth ratio (dimensio
Page 25 and 26: Y, ... ethanol yield for fermentati
Page 27 and 28: foreign plasmids. One major chalien
Page 29 and 30: than the denanired, inclusion body
Page 31 and 32: Table 1.2. Strategies for Enhancing
Page 33 and 34: Table 1.3. Host Cek, Pfasmids, Gene
Page 35: 1.4. Immo bilized-Cell Bioreactors
Page 39 and 40: plasmid are used; or chromaromal DN
Page 41 and 42: 2.2.1. Genetic Factors 2.2.1.1. Pid
Page 43 and 44: 1- fkquently than a plasmid with Io
Page 45 and 46: Da Silva and Bailey (1991) used the
Page 47 and 48: LEU2 or TRPI is cloned into the aux
Page 49 and 50: eported reduced stabiliity in the s
Page 51 and 52: on metabolic pathways. In the ferme
Page 53 and 54: OC), oniy about 35% of the populati
Page 55 and 56: loss @) is dehned as the probabilit
Page 57 and 58: + s Dilution Rate = p' = CI,- - K*+
Page 59 and 60: concentration couid Iead to coexist
Page 61 and 62: that factors other than immobiiizat
Page 63 and 64: earing and plasmid-tke =Ils. Gel be
Page 65 and 66: productivity irrespective of the mo
Page 67 and 68: inactive and useless. The problern
Page 69 and 70: Aerobic fermentation osing iamnobih
Page 71 and 72: (ranghg h m 1 to 7) on citric acid
Page 73 and 74: celi fraction decreases monotonicai
Page 75 and 76: (1987) model aliows for plasmid ins
Page 77 and 78: affect plasmid stability have ben o
Page 79 and 80: CEtAPTER 3 MATERIALS AND METHODS 3.
Page 81 and 82: stiU deleted hm the plasmid. Such p
Page 83 and 84: 1) Reagent solution preparation was
Page 85 and 86: nonse1ective agar plates, but only
Page 87 and 88:
working Liquid volume. The aeration
Page 89 and 90:
3.5. UNnobilized Ceil Culture 35.1.
Page 91 and 92:
3.52 Cd Lmmobiiization Yeast attach
Page 93 and 94:
3.6. Experimental Error and Reprodu
Page 95 and 96:
-0-Cell(1) +Total CeII (2) 4- Ethan
Page 97 and 98:
* . I I a O 2 4 6 8 10 12 14 Batche
Page 99 and 100:
Cell Maso, Glucose and EthanoI(g1L)
Page 101 and 102:
4.2.2. Batch CuIture in Stirreà-Ta
Page 103 and 104:
[t ~lucoamylase (ALR) -e Glucoamyla
Page 105 and 106:
The finai giucoamyiase concentratio
Page 107 and 108:
Bentley and Kompala, 1989; Satyagal
Page 109 and 110:
160 First ExponenUaI Phase 1 -c ALR
Page 111 and 112:
Figure 4hl. Pathways of Yeast Intem
Page 113 and 114:
fermentation (using nitrogen spargi
Page 115 and 116:
43. Continuous Suspension Culture C
Page 117 and 118:
Figure 4.9. Continuous Suspension C
Page 119 and 120:
1 +Enzyme +Fradlan of PBC +CeIl Mas
Page 121 and 122:
Figures 4.12 and 4.13 show that by
Page 123 and 124:
Fi- 4.13. Cornparison of Glu~iase C
Page 125 and 126:
where 6 = p- - p' + pp*, which is a
Page 127 and 128:
Figure 4-14. Effeds of Dilution Rat
Page 129 and 130:
Impoolsup et ai. (1989a) and Hardji
Page 131 and 132:
Figure 4.16. dB/dt versus B Plots a
Page 133 and 134:
Figure 4.17. Totai Cell Mas at Diff
Page 135 and 136:
another recombinant yeast (impoolsu
Page 137 and 138:
other recombinant yeast under nonse
Page 139 and 140:
4.3.4. Effixt of Dilution Rate On R
Page 141 and 142:
Figure 4.18- Etlects of Mluiioa Rat
Page 143 and 144:
Number of Generations - - - - - - *
Page 145 and 146:
B Table 4.5. Metabolic Pathway Anal
Page 147 and 148:
concentration. But this may be more
Page 149 and 150:
[+ Enzyme (ALR) + Enzyme (STR) +Fra
Page 151 and 152:
necessary for host ce& to grow in t
Page 153 and 154:
Glucoamylase ConcentraUon (unltsll)
Page 155 and 156:
[a Selective Medium A Nonseiective
Page 157 and 158:
5.1 Introduction CHAPTER 5 MODELING
Page 159 and 160:
handle. excess glucose is femented
Page 161 and 162:
The carbon fluxes h m the three met
Page 163 and 164:
53. Modei Simulation in Batch CuItu
Page 165 and 166:
Table 5.1. Summary of Parameters fo
Page 167 and 168:
1 n Glucose o Cell Mass O Ethano1 A
Page 169 and 170:
detennined in the cunent study, the
Page 171 and 172:
Figure 5.4. Cornparison between Mod
Page 173 and 174:
equations are üsted in Appendix G.
Page 175 and 176:
a dilution rate at which the giucoa
Page 177 and 178:
[ o Expetfmental data -Simuiated mt
Page 179 and 180:
The effixts of dilution rate on the
Page 181 and 182:
Ttme (hrs) 1 a Enzyme A Phsrnid-Bea
Page 183 and 184:
It may take several generations for
Page 185 and 186:
understanding of the nature of the
Page 187 and 188:
On the other hand. the net accumula
Page 189 and 190:
Differentiating Equation (6.20) res
Page 191 and 192:
Enyme collcentration m the t>ioreac
Page 193 and 194:
Glucoamylase Concentration (units/L
Page 195 and 196:
50 100 150 200 Time (hrs) 1 O Free
Page 197 and 198:
F i 6.6. Cornparison of Fnx Cd Mars
Page 199 and 200:
. - O 50 100 150 200 250 nme (hrs)
Page 201 and 202:
Nasi et ai.. 1988) and recombinant
Page 203 and 204:
estimated by using Equation (6.24)
Page 205 and 206:
[a lmmobilked System O Free System
Page 207 and 208:
oxidation pathway. W~th increasing
Page 209 and 210:
O 50 100 150 200 250 300 Tirne (hrs
Page 211 and 212:
epeated batch fermentations, the fe
Page 213 and 214:
Glucoamylase Concentration (unitslL
Page 215 and 216:
6) Suitable for repeated-batch and
Page 217 and 218:
ecause glucose fermentation dominat
Page 219 and 220:
cells. Tests of the proposed model
Page 221 and 222:
7.2. Recommendations The foUowing f
Page 223 and 224:
APPENDIX A: PLASMID MAP FOR pGAC9 l
Page 225 and 226:
where Bi (Biot number) = - ks De @
Page 227 and 228:
APPENDK C. EFFECTS OF INITIAL GLUCO
Page 229 and 230:
Figure C.2. Effect of Initial Gluco
Page 231 and 232:
Figure C.4 Effect of Initial Glucos
Page 233 and 234:
APPENDLX D. COMPARISON OF ORIGINAL
Page 235 and 236:
(continue)
Page 237:
(continue)
Page 240 and 241:
(continue)
Page 242 and 243:
E.2. Batch Fermentation with Recomb
Page 244 and 245:
(continue)
Page 246 and 247:
(continue) tfhJ 0.5 10.5 12.75 12.5
Page 248 and 249:
(continue)
Page 250 and 251:
E.3.2. Experimental Data (Parker an
Page 252 and 253:
Run No: CALE (Selected Strain. D =
Page 254 and 255:
Run No: CALR 5 (Original Suain, D =
Page 256 and 257:
Run No: CSTR3 (Selected Suain, D =
Page 258 and 259:
(continue)
Page 260 and 261:
Run No: CICR2 (Selected Suain, D =
Page 262 and 263:
Run No: CiCR4 (Selected Suain, D =
Page 264 and 265:
Eh. Repeated Batch Culture in hmobi
Page 266 and 267:
APPENDIX F. C CODE FOR THE PROPOSED
Page 268 and 269:
* the initial data */ /* gening the
Page 270 and 271:
* getting the founh term in R-K met
Page 272 and 273:
I*Plasrnid-bearing ceU concentratio
Page 274 and 275:
* Func tion C 1 */ îloat C l(float
Page 276 and 277:
APPEMIM G. EXAMPLES OF MAPLE V CODE
Page 278 and 279:
REFERENCES Ataai. MM. and Shuler. M
Page 280 and 281:
Caunt, P.. hpoolsup, A.. and Greenf
Page 282 and 283:
cuitanes of E. coli BZ18@TG 201) wi
Page 284 and 285:
Gabrieisen. O.S. et aL (1990), Effi
Page 286 and 287:
Huang, C-T., Peretti, S.W., and Bry
Page 288 and 289:
Kim, B.G. and Shuler, M.L. (1990a),
Page 290 and 291:
Lee, S.B.. Ryu, D.D.Y., seigel, R,
Page 292 and 293:
Nasri, M., Sayadi. S.. Barbotin, J.
Page 294 and 295:
Pin, S.J. and Kmwski. W.M. (1970),
Page 296 and 297:
Seo, I-H. and Bailey. J.E. (1985).
Page 298 and 299:
Tumer. B.G., Avgerinos G-C.? Melnic
Page 300:
Yu, J. and Tang, X (Editor, 1991),
show all

bioreactor studies of heterologous protein production by ...

Create successful ePaper yourself

Delete template?

Save as template?