基于代谢分析和计算流体力学的头孢菌素C发酵过程优化控制研究
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摘要
头孢菌素C（Cephalosporin C，CPC）是头孢类药物的中间体，由顶头孢霉菌通过发酵法生产制得。采用化学法或酶法对CPC分子进行修饰，可以得到一些高效、低毒、广谱的β-内酰胺类抗生素药物，在临床抗感染药物中占有重要地位。CPC发酵在我国起步较早，但目前CPC发酵生产工艺仍存在一些问题。比如，生产性能严重依赖专家和熟练工人的知识与经验，主要杂质去乙酰氧头孢菌素（Deacetoxycephalosporin C, DAOC）积累量高，发酵过程耗氧剧烈、溶解氧浓度（DO）不易控制，原料和操作成本居高不下，等等。本论文对利用顶头孢霉菌（Cephalosporins acremonium HC-3）发酵生产CPC过程的最优底物流加工艺、发酵罐最优桨型组合以及混合碳源发酵生产CPC工艺进行了研究，探讨和比较了不同碳源、氮源自动流加方式和不同桨型组合对CPC发酵性能的影响，并对不同的豆油和混合碳源自动流加方式下的CPC发酵进行了代谢分析，分析得到了最优碳源自动流加条件下CPC发酵得以高效进行的原因。论文主要结果如下：
(1)建立了一种新型、硫铵-豆油耦联型的硫铵自动补加策略。结果表明，通过这种新型硫铵流加策略，可将发酵液中的氨态氮(NH4+-N)浓度控制在3-5g/L的范围，既满足了细胞生长与CPC合成对氮源和硫源的需求，又促进了顶头孢霉菌的菌丝分化，为主酵期CPC的高效生产奠定了前期基础。
(2)在新型硫铵-豆油耦联型的硫铵流加策略的基础上，比较了CPC主酵期内使用间歇、匀速和DO-Stat法进行豆油自动流加条件下的CPC发酵性能。采用硫铵-豆油耦联型硫铵流加策略+后期通富氧空气的DO-Stat法进行硫铵和豆油补料，可使CPC发酵以高浓度和低副产物积累的方式进行，最终CPC浓度和得率分别达到35.77g/L和13.3%。主代谢副产物DAOC的积累量和DAOC/CPC比分别仅有0.178g/L和0.5%。
(3)利用CFD计算技术对适合于CPC生产的发酵罐的桨叶个数和桨型组合进行了模拟计算和优化。顶头孢霉菌发酵生产CPC是高耗氧的过程，搅拌发酵罐的桨叶个数和桨型组合直接影响发酵液中的O2传质和混合效果。与此同时，顶头孢霉菌对剪切力反应敏感，剪切力过大可造成细胞不可逆转的损伤、导致菌丝体提前断裂，对细胞生长和CPC合成产生负面影响。通过模拟计算寻找得到了适合于CPC合成的最优桨型组合、桨叶个数和安装尺寸：桨叶个数2，下层6直叶圆盘涡轮桨/上层4折叶桨，底层桨叶距罐底距离57mm，桨叶间距97mm。对不同桨叶个数/组合下的发酵性能进行了实验验证，结果表明，采用上述最优桨型组合进行CPC发酵，其整体发酵性能得到提高。与使用标准桨型组合相比，CPC浓度提高了52%、代谢副产物DAOC积累量降低了71%。
(4)利用DO-Stat法同时自动流加葡萄糖和豆油进行CPC生产时，葡萄糖浓度可以控制在很低的水平，因葡萄糖过量所引起的CPC合成抑制不会发生。使用葡萄糖作为辅助碳源，葡萄糖可以被细胞快速利用、维持或提高细胞的代谢活性；可以间接地改善发酵液主体内的O2传质速度、消除了单独流加豆油时DO控制的“平台”效应、提高了豆油的流加速度，使得CPC合成速度得到进一步改善。在采用该新型混合碳源流加策略、标准搅拌桨型、全程通空气供氧的条件下，CPC浓度最大可以达到36.99g/L，与单独流加豆油相比、CPC对相对昂贵的底物-豆油的得率从11.39%提高到25.14%，代谢副产物去乙酰氧头孢菌素C (DAOC)的积累量少、DAOC/CPC比只有0.28%，达到工业生产要求。使用新型混合碳源流加策略，可以调节碳源物质的流向、提高走向CPC合成途径的碳通量，在维持较高CPC生产强度的前提下，大幅提高CPC得率，抑制代谢副产物的积累、改善CPC发酵产品的质量。
(5)对CPC主酵期不同豆油流加方式下的CPC发酵进行了代谢分析。结果发现，采用DO-Stat法补加豆油，可以使更多的碳源物质流向CPC的合成前体，菌体可高效利用初始培养基中的蛋氨酸，TCA循环的代谢通量适度弱化。在此条件下，CO2的释放量减少，CPC得率得到提高。
(6)在50L发酵罐上验证了一种新型匀速+DO-Stat结合补油法，该方法同时汲取了匀速补油和DO-Stat法补油的优点，既提高了豆油流加速度和CPC合成速度，又可以将DO稳定地控制在20%-40%之间。与石药集团中诺药业有限公司原有补料工艺相比，豆油转化率得到提高，主要代谢副产物积累量明显下降、DAOC/CPC比仅为0.46%，达到工业生产要求。
Cephalosporin C (CPC) is an important intermediate for producing the widely used β-lactamantibiotics, and Cephalosporins are fermentatively produced by several Streptomyces spp. andfilamentous fungi. The semi-synthetic derivatives of CPC are less toxic, high stability onβ-lactamase and broad-spectrum antibiotics. Even though the history of CPC fermentativeproduction in China is relatively long, a lot of practical problems still remained unsolved inindustrial CPC productions, such as fermentation performance/stability dependence onknowledge/experience of expert/skilled operators, high accumulation of the majorby-metabolite of deacetoxycephalosporin (DAOC), difficulty in controlling dissolved oxygenconcentration (DO) control due to the extensively high oxygen consumption, high costs ofraw materials and operations, etc. Focusing on CPC fermentations by Cephalosporinsacremonium HC-3, this thesis investigated the optimal substrates feeding strategies, theoptimal impeller combinations in a stirred bioreactor for CPC production, and CPCfermentation techniques of co-feeding mixed carbon sources. The effects of various automaticfeeding strategies of carbon/nitrogen sources and impeller combinations on CPC fermentationperformance were explored and compared. Metabolic analysis under different feedingmanners of soybean and mixed carbon sources was also conducted to find out the reasons ofenhanced CPC fermentation performance achieved under the optimal feeding conditions. Themajor results of this dissertation were summarized as follows:
(1) A novel and automatic (NH_4)_2SO_4feeding strategy which coupled (NH_4)_2SO_4supplementwith soybean oil addition was proposed. The results indicated that the (NH_4)_2SO_4-soybeanoil coupled feeding strategy (mass ratio of soybean oil versus (NH_4)_2SO_4about1:0.17)could control NH4+-N concentration in a range of3-5g/L throughout the fermentations,which satisfied the requirements of cell growth/CPC synthesis on N-/S-sources andaccelerated mycelia differentiation. This strategy laid a platform or prerequisite forefficient CPC production during its major production phase.
(2) On the basis of the proposed (NH_4)_2SO_4-soybean oil coupled feeding strategy, CPCfermentations during the major CPC production phase using different soybean oil feedingmethods including intermittent, constant rate and DO-Stat based feeding were conductedand their performance was compared. The method of adopting DO-Stat for soybean oilfeeding with oxygen-enriched-air for aeration could conduct CPC fermentations in a wayof higher CPC but lower DAOC accumulation. In this case, final CPC concentration andCPC yield reached the levels of35.77g/L and13.3%, respectively. DAOC accumulationand DAOC/CPC ratio ended at lower levels of0.178g/L and0.5%, respectively.
(3) CFD calculation technique was used for simulation and optimization of the suitableimpeller numbers and combinations for CPC production. CPC fermentation is anextremely high oxygen consuming process, the impeller numbers/combinations directlyaffects oxygen mass transfer/mixng effects in the broth. C.acremonium is sensitive toshear rate. Extrassively high shear rate causes irreservable damage on cells and mycelia breakage at improper/early stage, and severely deteriorates cell growth/CPC synthesis.The optimal impeller combination/numbers/mounting positions for CPC fermentationwere determined on the basis of CFD simulation/calculation results, which were impellernumber of2; impeller combination of a bottem six-bladed turbine plus a upperfour-pitched-blade turbine; mounting distance of the bottom turbine and tank bottom of57mm; and mounting distance of the two turbines of97mm. The optimal impellers wereapplied for CPC fermentation experiments for verification. The experimental resultsindicated that the entire fermentation performance was largely enhanced when adoptingthe optimized impellers, CPC concentration increased by52%and DAOC accumulationdecreased by71%as compared with those of using the standard impellers.
(4) When automatically feeding mixed carbon sources consisting of soybean oil and glucose,glucose concentration could be controlled at very low level and CPC synthesis repressiondue to excessive glucosed did not occur. When using glucose as an assistant carbon source,cellular activity could be maintained or even enhanced as glucose was quickly utilized bythe cells; soybean oil feeding and CPC synthesis rates were increased as O2mass transferrate in broth was indirectly improved and “platform” effect in DO control was eliminated.Under the conditions of adopting the novel mixed carbon sources feeding strategy andstandard impellers, aerating air for oxygen supply throughout the fermentation, maximalCPC concentration reached a higher level of36.99g/L; CPC yield on expensive soybeanoil increased up to25.14%; DAOC/CPC ratio stayed at very low level of0.28%qualifying industrial standard. With the new mixed carbon sources feeding strategy,carbon fluxes towards to CPC synthesis route was improved; CPC yield was significantlyenhanced; accumulation of by-metabolite was largely relieved; and quality of the CPCproduct was ensured, while CPC productivity was maintained at compabably high level.
(5) During CPC major production phase, metabolic analysis under various soybean oilfeeding conditions was conducted. The results revealed that, when using DO-Stat basedfeeding method for supplementing soybean oil, more carbon fluxes could be directed intothe formation of CPC synthesis precursors; extracellular methionine could be directlyutilized to run the entire CPC biosynthesis in a more efficient way; carbon fluxesdistribution in TCA could be slightly weakened. Under this condition, CO2releasedecreased and CPC yield increased.
(6) A novel soybean oil feeding strategy, namely, a combined strategy of constant rate feeding+DO-Stat feeding which absorbed the advantages of both was proposed and conducted in50L scaled-up fermentors. This strategy increased rates of soybean oil feeding and CPCsynthesis, and stably controlled DO in a range of20%-40%. In this case, CPC yieldsignificantly increased, DAOC accumulation decreased, and DAOC/CPC ratio reduced toa lower level of0.46%qualifying the industrial standard, as compared with the results ofusing the original feeding method.
(1)建立了一种新型、硫铵-豆油耦联型的硫铵自动补加策略。结果表明，通过这种新型硫铵流加策略，可将发酵液中的氨态氮(NH4+-N)浓度控制在3-5g/L的范围，既满足了细胞生长与CPC合成对氮源和硫源的需求，又促进了顶头孢霉菌的菌丝分化，为主酵期CPC的高效生产奠定了前期基础。
(2)在新型硫铵-豆油耦联型的硫铵流加策略的基础上，比较了CPC主酵期内使用间歇、匀速和DO-Stat法进行豆油自动流加条件下的CPC发酵性能。采用硫铵-豆油耦联型硫铵流加策略+后期通富氧空气的DO-Stat法进行硫铵和豆油补料，可使CPC发酵以高浓度和低副产物积累的方式进行，最终CPC浓度和得率分别达到35.77g/L和13.3%。主代谢副产物DAOC的积累量和DAOC/CPC比分别仅有0.178g/L和0.5%。
(3)利用CFD计算技术对适合于CPC生产的发酵罐的桨叶个数和桨型组合进行了模拟计算和优化。顶头孢霉菌发酵生产CPC是高耗氧的过程，搅拌发酵罐的桨叶个数和桨型组合直接影响发酵液中的O2传质和混合效果。与此同时，顶头孢霉菌对剪切力反应敏感，剪切力过大可造成细胞不可逆转的损伤、导致菌丝体提前断裂，对细胞生长和CPC合成产生负面影响。通过模拟计算寻找得到了适合于CPC合成的最优桨型组合、桨叶个数和安装尺寸：桨叶个数2，下层6直叶圆盘涡轮桨/上层4折叶桨，底层桨叶距罐底距离57mm，桨叶间距97mm。对不同桨叶个数/组合下的发酵性能进行了实验验证，结果表明，采用上述最优桨型组合进行CPC发酵，其整体发酵性能得到提高。与使用标准桨型组合相比，CPC浓度提高了52%、代谢副产物DAOC积累量降低了71%。
(4)利用DO-Stat法同时自动流加葡萄糖和豆油进行CPC生产时，葡萄糖浓度可以控制在很低的水平，因葡萄糖过量所引起的CPC合成抑制不会发生。使用葡萄糖作为辅助碳源，葡萄糖可以被细胞快速利用、维持或提高细胞的代谢活性；可以间接地改善发酵液主体内的O2传质速度、消除了单独流加豆油时DO控制的“平台”效应、提高了豆油的流加速度，使得CPC合成速度得到进一步改善。在采用该新型混合碳源流加策略、标准搅拌桨型、全程通空气供氧的条件下，CPC浓度最大可以达到36.99g/L，与单独流加豆油相比、CPC对相对昂贵的底物-豆油的得率从11.39%提高到25.14%，代谢副产物去乙酰氧头孢菌素C (DAOC)的积累量少、DAOC/CPC比只有0.28%，达到工业生产要求。使用新型混合碳源流加策略，可以调节碳源物质的流向、提高走向CPC合成途径的碳通量，在维持较高CPC生产强度的前提下，大幅提高CPC得率，抑制代谢副产物的积累、改善CPC发酵产品的质量。
(5)对CPC主酵期不同豆油流加方式下的CPC发酵进行了代谢分析。结果发现，采用DO-Stat法补加豆油，可以使更多的碳源物质流向CPC的合成前体，菌体可高效利用初始培养基中的蛋氨酸，TCA循环的代谢通量适度弱化。在此条件下，CO2的释放量减少，CPC得率得到提高。
(6)在50L发酵罐上验证了一种新型匀速+DO-Stat结合补油法，该方法同时汲取了匀速补油和DO-Stat法补油的优点，既提高了豆油流加速度和CPC合成速度，又可以将DO稳定地控制在20%-40%之间。与石药集团中诺药业有限公司原有补料工艺相比，豆油转化率得到提高，主要代谢副产物积累量明显下降、DAOC/CPC比仅为0.46%，达到工业生产要求。
Cephalosporin C (CPC) is an important intermediate for producing the widely used β-lactamantibiotics, and Cephalosporins are fermentatively produced by several Streptomyces spp. andfilamentous fungi. The semi-synthetic derivatives of CPC are less toxic, high stability onβ-lactamase and broad-spectrum antibiotics. Even though the history of CPC fermentativeproduction in China is relatively long, a lot of practical problems still remained unsolved inindustrial CPC productions, such as fermentation performance/stability dependence onknowledge/experience of expert/skilled operators, high accumulation of the majorby-metabolite of deacetoxycephalosporin (DAOC), difficulty in controlling dissolved oxygenconcentration (DO) control due to the extensively high oxygen consumption, high costs ofraw materials and operations, etc. Focusing on CPC fermentations by Cephalosporinsacremonium HC-3, this thesis investigated the optimal substrates feeding strategies, theoptimal impeller combinations in a stirred bioreactor for CPC production, and CPCfermentation techniques of co-feeding mixed carbon sources. The effects of various automaticfeeding strategies of carbon/nitrogen sources and impeller combinations on CPC fermentationperformance were explored and compared. Metabolic analysis under different feedingmanners of soybean and mixed carbon sources was also conducted to find out the reasons ofenhanced CPC fermentation performance achieved under the optimal feeding conditions. Themajor results of this dissertation were summarized as follows:
(1) A novel and automatic (NH_4)_2SO_4feeding strategy which coupled (NH_4)_2SO_4supplementwith soybean oil addition was proposed. The results indicated that the (NH_4)_2SO_4-soybeanoil coupled feeding strategy (mass ratio of soybean oil versus (NH_4)_2SO_4about1:0.17)could control NH4+-N concentration in a range of3-5g/L throughout the fermentations,which satisfied the requirements of cell growth/CPC synthesis on N-/S-sources andaccelerated mycelia differentiation. This strategy laid a platform or prerequisite forefficient CPC production during its major production phase.
(2) On the basis of the proposed (NH_4)_2SO_4-soybean oil coupled feeding strategy, CPCfermentations during the major CPC production phase using different soybean oil feedingmethods including intermittent, constant rate and DO-Stat based feeding were conductedand their performance was compared. The method of adopting DO-Stat for soybean oilfeeding with oxygen-enriched-air for aeration could conduct CPC fermentations in a wayof higher CPC but lower DAOC accumulation. In this case, final CPC concentration andCPC yield reached the levels of35.77g/L and13.3%, respectively. DAOC accumulationand DAOC/CPC ratio ended at lower levels of0.178g/L and0.5%, respectively.
(3) CFD calculation technique was used for simulation and optimization of the suitableimpeller numbers and combinations for CPC production. CPC fermentation is anextremely high oxygen consuming process, the impeller numbers/combinations directlyaffects oxygen mass transfer/mixng effects in the broth. C.acremonium is sensitive toshear rate. Extrassively high shear rate causes irreservable damage on cells and mycelia breakage at improper/early stage, and severely deteriorates cell growth/CPC synthesis.The optimal impeller combination/numbers/mounting positions for CPC fermentationwere determined on the basis of CFD simulation/calculation results, which were impellernumber of2; impeller combination of a bottem six-bladed turbine plus a upperfour-pitched-blade turbine; mounting distance of the bottom turbine and tank bottom of57mm; and mounting distance of the two turbines of97mm. The optimal impellers wereapplied for CPC fermentation experiments for verification. The experimental resultsindicated that the entire fermentation performance was largely enhanced when adoptingthe optimized impellers, CPC concentration increased by52%and DAOC accumulationdecreased by71%as compared with those of using the standard impellers.
(4) When automatically feeding mixed carbon sources consisting of soybean oil and glucose,glucose concentration could be controlled at very low level and CPC synthesis repressiondue to excessive glucosed did not occur. When using glucose as an assistant carbon source,cellular activity could be maintained or even enhanced as glucose was quickly utilized bythe cells; soybean oil feeding and CPC synthesis rates were increased as O2mass transferrate in broth was indirectly improved and “platform” effect in DO control was eliminated.Under the conditions of adopting the novel mixed carbon sources feeding strategy andstandard impellers, aerating air for oxygen supply throughout the fermentation, maximalCPC concentration reached a higher level of36.99g/L; CPC yield on expensive soybeanoil increased up to25.14%; DAOC/CPC ratio stayed at very low level of0.28%qualifying industrial standard. With the new mixed carbon sources feeding strategy,carbon fluxes towards to CPC synthesis route was improved; CPC yield was significantlyenhanced; accumulation of by-metabolite was largely relieved; and quality of the CPCproduct was ensured, while CPC productivity was maintained at compabably high level.
(5) During CPC major production phase, metabolic analysis under various soybean oilfeeding conditions was conducted. The results revealed that, when using DO-Stat basedfeeding method for supplementing soybean oil, more carbon fluxes could be directed intothe formation of CPC synthesis precursors; extracellular methionine could be directlyutilized to run the entire CPC biosynthesis in a more efficient way; carbon fluxesdistribution in TCA could be slightly weakened. Under this condition, CO2releasedecreased and CPC yield increased.
(6) A novel soybean oil feeding strategy, namely, a combined strategy of constant rate feeding+DO-Stat feeding which absorbed the advantages of both was proposed and conducted in50L scaled-up fermentors. This strategy increased rates of soybean oil feeding and CPCsynthesis, and stably controlled DO in a range of20%-40%. In this case, CPC yieldsignificantly increased, DAOC accumulation decreased, and DAOC/CPC ratio reduced toa lower level of0.46%qualifying the industrial standard, as compared with the results ofusing the original feeding method.
引文
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