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Table 1.1. Examples of Extracellular Recombinant Proteins Expressed in S. cerevisiue. 1 Recombinant Protein Recombinant Yeast Strain Referenœ t- Hwnan Epidennal Growth Factor &endorphin Hunan Atrial Nabnuretic 1 Peotide Hunan Nerve Growth 1 Factor S. cerevisiue Coppella et al, 1989 (AB lO3.l/pY CCEGF-2s) S. cerevrcioe (20B 12/pYE/aE) Zsebo et 1,1986 S. cerevisiae Zsebo et & 1986 (2OB 12fpWacaic) S. cerevisiue (W30 1 - Vlasuk et al., 1986 18AhJCl-5) S. cerevisiae 1 Mullenbach, 1986 (AB 103.11paEFG-24) S. cerevtsiae (C468/pGAC9) Nunberg et ai. 1988 S. cerevisiue 1 Lin. 1987 S. cerevisiue (CGY 189 1) 1 ~Ürner et aÏ., 1990 S. cerevkiae (MC 16fpMA.230) Wak and Gainer, 1989 I S. cerevisiae GabrieIsen, 1990 Wûû/paUo S. cerevisiue Barthel et aï.. 1993 (F5rWL292/PS7041) S. cerevisiue (m345fpNB6 1) Uozumi et al.. 1993 S. cereviriae (20B-lUpTKi0) Nishuawa et al, 1992 S.cerevkiae(FY178IpW1) SodeetaL,1988a S. cerevkiae (BJ1991/p336/1) Sode et al., 1988b l - -
than the denanired, inclusion body types wially produced by recombinant baneria (Qiisti and Moo-Young, 1994; Rouf et al., 1996). Due to all these advantages, it is not surprishg that use of the yeaa S. cerevisiae, as a host for expression of recombinant proteins has rapidiy accelerated in recent years. S. cmevisiae is now an important workhorse in indusW biotechnology and many recombinant proteins. including commercial products, are produced by recombinant S. cerdiae. Table 1.1. provides an ove~ew of some recombinant proteins expnssed in S. cerdiae which are secmed into the extracellular medium. Therefore. investigation and development of efficient recombinant yeast bioprocesses are very important 12. Instability of Recombinant Yeast in commercial production with recombinant microorgaMsms, a critical problem is the instability of the recombinant organisms harboring unstable plas& where instability is defined as the tendency of the ttansformed ceils to Iose their engineered properties due to the los andlor modification of fonign plasmids. This problem is especially signifiant with recombinant yeasts because almost al yeast plasmid vectors are hybrid plasrnids, or so-caiied "shuttie vectors", which are nlatively unstable. Plasmid instability cm be divided into two categories: stnictud instability and segregational instability (Ryu et ai., 1988). Structural instabiiity is ofwi caused by the DNA's deletion, insertion, recombination, or other events, whïie segngationai instability is caused by uneven partition of plasrnids during ceil division et ai., 1981; Ryu and Lee, 1988). The genetic instabiîities in combination with effm of environmentaï hctors iead recombinant
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: foreign plasmids. One major chalien
Page 31 and 32: Table 1.2. Strategies for Enhancing
Page 33 and 34: Table 1.3. Host Cek, Pfasmids, Gene
Page 35 and 36: 1.4. Immo bilized-Cell Bioreactors
Page 37 and 38: 1. Study the gmwth characteristics,
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
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CEtAPTER 3 MATERIALS AND METHODS 3.
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stiU deleted hm the plasmid. Such p
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1) Reagent solution preparation was
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nonse1ective agar plates, but only
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working Liquid volume. The aeration
Page 89 and 90:
3.5. UNnobilized Ceil Culture 35.1.
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3.52 Cd Lmmobiiization Yeast attach
Page 93 and 94:
3.6. Experimental Error and Reprodu
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-0-Cell(1) +Total CeII (2) 4- Ethan
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* . I I a O 2 4 6 8 10 12 14 Batche
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Cell Maso, Glucose and EthanoI(g1L)
Page 101 and 102:
4.2.2. Batch CuIture in Stirreà-Ta
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[t ~lucoamylase (ALR) -e Glucoamyla
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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
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fermentation (using nitrogen spargi
Page 115 and 116:
43. Continuous Suspension Culture C
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Figure 4.9. Continuous Suspension C
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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
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Impoolsup et ai. (1989a) and Hardji
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Figure 4.16. dB/dt versus B Plots a
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Figure 4.17. Totai Cell Mas at Diff
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another recombinant yeast (impoolsu
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other recombinant yeast under nonse
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4.3.4. Effixt of Dilution Rate On R
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Figure 4.18- Etlects of Mluiioa Rat
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Number of Generations - - - - - - *
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B Table 4.5. Metabolic Pathway Anal
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concentration. But this may be more
Page 149 and 150:
[+ Enzyme (ALR) + Enzyme (STR) +Fra
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necessary for host ce& to grow in t
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Glucoamylase ConcentraUon (unltsll)
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[a Selective Medium A Nonseiective
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5.1 Introduction CHAPTER 5 MODELING
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handle. excess glucose is femented
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The carbon fluxes h m the three met
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53. Modei Simulation in Batch CuItu
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Table 5.1. Summary of Parameters fo
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1 n Glucose o Cell Mass O Ethano1 A
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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.
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a dilution rate at which the giucoa
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[ 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
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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
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Differentiating Equation (6.20) res
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Enyme collcentration m the t>ioreac
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Glucoamylase Concentration (units/L
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50 100 150 200 Time (hrs) 1 O Free
Page 197 and 198:
F i 6.6. Cornparison of Fnx Cd Mars
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. - O 50 100 150 200 250 nme (hrs)
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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
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(continue)
Page 237:
(continue)
Page 240 and 241:
(continue)
Page 242 and 243:
E.2. Batch Fermentation with Recomb
Page 244 and 245:
(continue)
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(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
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* the initial data */ /* gening the
Page 270 and 271:
* getting the founh term in R-K met
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I*Plasrnid-bearing ceU concentratio
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* 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),
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