发酵过程优化控制4

1、1第四节第四节基于代谢网络模型的发酵过程控制基于代谢网络模型的发酵过程控制发酵过程优化和控制发酵过程优化和控制2磷酸戊糖磷酸戊糖(PP)途径途径解糖解糖(EMP)途径途径TCAMetabolic network of lysine Production by Corynebacterium glutamicum发酵过程优化和控制发酵过程优化和控制3GlucosePYRATPEtOHO2NADH2CO2TCABiomassNH3发酵过程优化和控制发酵过程优化和控制40 0 0 0 1 0 0 0 0-1 0 0 0 -0.68 0 0 0 00 1 0 0 0 -1 0 0 00 0 0 -1

2、0 0 -1 0 00 1 3 0 0 0 0 -1 00 0 0 0 -1 0 0 0 -12 -1 -1 0 -0.2 0 0 0 02 0 1 4P/O 0 0 0 0 02 -1 4.7 -2 -14.7 0 0 0 0r1r2r3r4r5rerO2rCO2rNH3= 0mmccccmmrAArrArAAr10A：n k阶的代谢反应总行列矩阵；阶的代谢反应总行列矩阵；r是是k 1阶的反应速度的向量。阶的反应速度的向量。n：表示代谢网络的总的（节点）个数（：表示代谢网络的总的（节点）个数（9）；）；k：则表示反应速度的总个数（：则表示反应速度的总个数（11），它是代谢网），它是代谢网络模

3、型的反应式个数（络模型的反应式个数（5）与着眼物质速度式的个数（）与着眼物质速度式的个数（6）之和。）之和。如果如果k个总反应速度中有个总反应速度中有m个是在线可测的，对应的可测速度向量为个是在线可测的，对应的可测速度向量为rm，其余不可测速度向量为，其余不可测速度向量为rc，那么那么Ac和和Am就分别是就分别是n (k-m)阶和阶和n m阶的，对应于不可测速度向量阶的，对应于不可测速度向量rc和可测速度向量和可测速度向量rm的，对的，对代谢反应总行列矩阵代谢反应总行列矩阵A进行分割以后形成的小矩阵。进行分割以后形成的小矩阵。 只要只要Ac满秩，其逆矩阵存在，不可测定向量满秩，其逆矩阵存在，不

4、可测定向量 便可以唯一求解。这里，共有便可以唯一求解。这里，共有9个个“节点节点”（n=9），），即即葡萄糖葡萄糖，丙酮酸，丙酮酸， ATP，NADH2，酒精，酒精，CO2，O2， NH3，菌体菌体。有两个速度变量可测，。有两个速度变量可测，m=2。方程组。方程组中的中的 和和 以及方程组可以表示成：以及方程组可以表示成：发酵过程优化和控制发酵过程优化和控制50 0r1r2r3r4r5rerO2rCO2rNH30 0 0 0 1 0 0 0 0-1 0 0 0 -0.68 0 0 0 00 1 0 0 0 -1 0 0 00 0 0 -1 0 0 -1 0 00 1 3 0 0 0 0 -1

5、00 0 0 0 -1 0 0 0 -12 -1 -1 0 -0.2 0 0 0 02 0 1 8 0 0 0 0 02 -1 4.7 -2 -14.7 0 0 0 0rxrs+0.68 -1 0 0 0 0 0 0 0 -2.8 -2.16 0 0 0 0 -0.84 -0.08 -0.161.27 0.16 0 0 0 0 -0.16 0.08 0.163.7 0.46 0 0 0 0 -0.04 0.23 -0.04 1 0 0 0 0 0 0 0 0-2.8 -2.16 -1 0 0 0 -0.84 -0.08 -0.16-3.7 -0.45 0 -1 0 0 -0.04 -0.23

6、0.041 -1.67 0 0 -1 0 -1.32 0.16 0.32-1 0 0 0 0 -1 0 0 0r1r2r3r4r5rerO2rCO2rNH3= -rxrs= -0.68 -1-2.8 -2.161.27 0.163.7 0.461 0-2.8 -2.16-3.7 -1.671 -1.67-1 0rxrs发酵过程优化和控制发酵过程优化和控制6发酵过程优化和控制发酵过程优化和控制7N. Takiguchi et. al., Biotechnol.Bioeng. 55, 170, 1997发酵过程优化和控制发酵过程优化和控制8葡萄糖（胞内）6-磷酸葡萄糖6-磷酸果糖磷酸烯醇丙酮酸3-

7、磷酸甘油醛丙酮酸r1葡萄糖（胞外）赖氨酸TCA细胞NADH + O2NAD + ATPATPADPr2r3r4r5r6r7r8r9r10r11 发酵过程优化和控制发酵过程优化和控制9TCOOsxmTLncmcccmmrrrrrrrrrrrrrrrrrrrrrrrArAAr),(),(),(0221110987654321发酵过程优化和控制发酵过程优化和控制10发酵过程优化和控制发酵过程优化和控制110501001502000102030Time (h)CER & OUR (mol/m3 h)OURCER0306090010203040Time (h)Glutamate concentr

8、ation (g/L) &DO (%)0102030Cell & lactate concentrations(g/L) 发酵过程优化和控制发酵过程优化和控制12EMP Glycolysis Pathway r1: Glucose + ATP = Glu6P + ADP r2: Glu6P = Fru6P r3: Fru6P + ATP = 2 G3P + ADP r5: G3P + NAD +ADP = PEP + ATP + NADH r6: PEP + ADP = PYR + ATP PP Pentose Phosphate Pathway r4: 3 Glu6P + 6

9、 NADP = 2 Fru6P + G3P + 6 NADPH + 3 CO2 TCA Cycle r7: PEP + CO2 + ATP = OaA + ADP r9: PYR + NAD = Ac-CoA + NADH + CO2 r10: Ac-CoA + OaA = Isocit r11: Isocit + NAD = a-KG + NADH + CO2 r13: a-KG + NAD + ADP = Suc + NADH + ATP +CO2 r14: Suc + FAD = Mal + 2/3 NADH r15: Mal + NAD = OaA + NADH Glyoxylate

10、Shunt r16: Isocit = Suc + Glyoxy r17: Ac-CoA + Glyoxy =Mal Metabolic Products Formation r8: PYR + NADH = Lactate + NAD r12: a-KG + NH3 + NADPH = Glutamate + NADP Respiratory Chain & Oxidative Phosphorylation r18: O2 + 2 NADH + 2(P/O) ADP = 2(P/O) ATP + 2 NAD + 2 H2O r19: ATP = ADP + Pi r20: O2 +

11、 2(1+) NADPH + 2 NAD = 2 NADH + 2(1+) NADP + 2H2O GlucoseATPADPGlu6PFru6Pr1r2G3PATPADPADPATPNADNADHr3r5PYRRespiratory Chain &Oxidative PhosphorylationADPATPO2NADHr18Pir19ADPLactateNADHNADNADPNADPHr4r9EMPPP PathwayCO2NADNADHCO2Ac-CoACO2r6PEPATPNADPHNADHIsocit KGSucMalOaANADCO2NH3NADPHGlutamateADP

12、ATPNADNADHCO2FAD2/3NADHNADNADHGlyoxyr10r11r13r14r16r17r17r15r7r8r12r20TCAADPADPATPO2NADH发酵过程优化和控制发酵过程优化和控制13), 1(0)(&:267. 0)(201514131195mqiirrArAconditionstConstrainrrrrrrrMinrateproductionNADHtotalMincmmcc012)0()() 1()(PPTkrMkPkPWP08)0()() 1()(LLTkrMkLkLWL发酵过程优化和控制发酵过程优化和控制1402040608010001020

13、3040Time (h)Glutamate Conccentration (g/L)0306090DO (%)(b) 020406080100010203040Time (h)Glutamate Concentration (g/L)DO=10%DO=30%(a) 实线：在线推定结果发酵过程优化和控制发酵过程优化和控制15GlucoseGlu6PPYRLactateGlutamate100331/282/333921/1282/1023121/882/373571/392/633221/02/230204060010203040Time (h)Glutamate & NH4+Conce

14、ntrations (g/L, mg/L)051015KG Concentration(g/L)(b) 020406005101520Time (h)Glutamate & LactateConcentration (g/L)03060DO (%)(a) 01020304050010203040Time (h)Lactate Concentration (g/L)DO=10%DO=50% 实线：在线推定结果发酵过程优化和控制发酵过程优化和控制160204060801000102030Time (h)Glutamate Concentration (g/L)020406080Lactat

15、e Concentration (g/L)(a) 0120102030Time (h)GDH, LDH activities (U/mg-Protein)(b) 发酵过程优化和控制发酵过程优化和控制17GlucoseGlu6PPYRLactateGlutamate-KGNADHNADPH NH3LDHGDHTCAEMPPP10083115322952CO2 ,NADHCO2 ,NADPHGlucoseGlu6PPYRLactateGlutamate-KGNADHNADPH NH3LDHGDHTCAEMPPP10093105123853CO2 ,NADHCO2 ,NADPHGlucoseGlu6

16、PPYRLactateGlutamate-KGNADHNADPH NH3LDHGDHTCAEMPPP100128139109531CO2 ,NADHCO2 ,NADPHMetabolic flux distribution at 24h, DO=10% Metabolic flux distribution at 24h, DO=30% Metabolic flux distribution at 24h, DO=50% 0204060801000102030Time (h)Glucose Consumption rate (mol/m3 h)050331-DO10%050407-DO50%

17、发酵过程优化和控制发酵过程优化和控制1800.51010203040Time (h)RQ (-)0306090r13 (mol/m3h)RQ, DO=50%RQ, DO=10%r13, DO=50%r13, DO=10% 发酵过程优化和控制发酵过程优化和控制19GlucoseGlu6PPYRLactateGlutamate-KGNADHNADPH NH3LDHGDHTCAEMPPPr1r3r2r5r4r6CO2, NADHATPCO2, NADPHNADHGlucoseGlu6PPYRLactateGlutamate-KGNADHNADPH NH3LDHGDHTCAEMPPPr1r3r2r5r

18、4r6CO2, NADHATPCO2, NADPHNADHHigh DO LevelLow DO LevelGlucoseGlu6PPYRLactateGlutamate-KGNADHNADPH NH3LDHGDHTCAEMPPPr1r3r2r5r4r6CO2, NADHATPCO2, NADPHNADHGlucoseGlu6PPYRLactateGlutamate-KGNADHNADPH NH3LDHGDHTCAEMPPPr1r3r2r5r4r6CO2, NADHATPCO2, NADPHNADHGlucoseGlu6PPYRLactateGlutamate-KGNADHNADPH NH3L

19、DHGDHTCAEMPPPr1r3r2r5r4r6CO2, NADHATPCO2, NADPHNADHHigh DO LevelGlucoseGlu6PPYRLactateGlutamate-KGNADHNADPH NH3LDHGDHTCAEMPPPr1r3r2r5r4r6CO2, NADHATPCO2, NADPHNADHHigh DO LevelLow DO LevelGlucoseGlu6PPYRLactateGlutamate-KGNADHNADPH NH3LDHGDHTCAEMPPPr1r3r2r5r4r6CO2, NADHATPCO2, NADPHNADHBalanced Meta

20、bolic Control by RQGlucoseGlu6PPYRLactateGlutamate-KGNADHNADPH NH3LDHGDHTCAEMPPPr1r3r2r5r4r6CO2, NADHATPCO2, NADPHNADHGlucoseGlu6PPYRLactateGlutamate-KGNADHNADPH NH3LDHGDHTCAEMPPPr1r3r2r5r4r6CO2, NADHATPCO2, NADPHNADHBalanced Metabolic Control by RQ 发酵过程优化和控制发酵过程优化和控制2000.40.81.28.599.51010.511Time

21、(h)RQ (-)0500100015002000Agitation Rate (RPM)RQAgitation Rate 发酵过程优化和控制发酵过程优化和控制2101020300510152025020406080100120 Cell &Lactate Concentrations(g-DWC/L, g/L)Time (h) Glutamate Concentration (g/L) 01020300.00.20.40.60.81.0020406080100120140020040060080010001200RQ (-)Time (h) DO (%) Agitation Rate

22、 (rpm)DO RQ Agitation Rate 01020300510152025020406080100120Cell & Lactate Concentrations(g-DWC/L, g/L)Time (h) Glutamate Concentration (g/L) 01020300200400600800100012000204060801001201400.00.20.40.60.81.0Agitation Rate (rpm)Time (h) DO (%) RQ (-) DO Agitation Rate RQ 发酵过程优化和控制发酵过程优化和控制220306001

23、02030Time (h)r13 (mol/m3 h)MBC by RQDO=50%DO=10% 发酵过程优化和控制发酵过程优化和控制23发酵过程优化和控制发酵过程优化和控制24IA：细胞浓度；B：葡萄糖浓度；C：L-精氨酸浓度；D：L-精氨酸生成速度。供氧模式供氧模式：高供氧（HOS）；：中度供氧（MOS）；：低供氧（LOS）。II不同供氧方式下的溶解氧（DO）的变化模式供氧模式供氧模式1：高供氧（HOS）；2：中度供氧（MOS）；3：低供氧（LOS）。发酵过程优化和控制发酵过程优化和控制25钝齿棒杆菌C.crenatum SYPA5-5发酵的代谢网络图发酵过程优化和控制发酵过程优化和控制26高供氧（高供氧（HOS）和中度供氧（）和中度供氧（MOS）条件下，精氨酸发酵各个阶