1，3-丙二醇高产菌株的选育及其发酵研究
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摘要
聚对苯二甲酸丙二醇酯(PTT)是一种新型的合成聚酯,具有许多优良特性和很好的应用前景,而1,3-丙二醇(1,3-PDO)是其合成的关键性材料。近年来,1,3-丙二醇的研究受到了全球众多知名企业的重视,与化学合成法相比,微生物发酵法生产1,3-丙二醇具有显著的优点,成为当前的研究热点。
产物抑制和副产物多造成微生物发酵法产1,3-丙二醇的产量低,另外,副产物多也是甘油转化率低和产物分离困难的原因之一,选育既有1,3-丙二醇高浓度耐受性,同时又具有副产物代谢途径突变的菌株,是提高1,3-丙二醇产量和甘油转化率并简化后期提取工艺的一种尝试。
通过向培养基中添加外源性产物的方法,考察了1,3-丙二醇和其它有机酸如乙酸、乳酸、琥珀酸对肺炎克雷伯氏杆菌(K.pneumonia)发酵过程的影响。结果表明90g/L的1,3-丙二醇完全抑制了K.pneumoniae的生长。乙酸虽有利于菌体生长却降低了1,3-丙二醇的产量,而乳酸则对菌体生长有抑制。琥珀酸有利于1,3-丙二醇和乳酸形成。
用mini-Tn5随机转座和亚硝基胍分别诱变,在含100g/L 1,3-丙二醇和0.17M溴化钠-溴酸钠(质子自杀方法)的培养基上筛选既有1,3-丙二醇高浓度耐受性,同时又有产酸代谢途径突变的菌株,传代后获得44个稳定突变株,摇瓶培养后作产物分析,经初筛和复筛获得一个1,3-丙二醇高产菌株,其乳酸脱氢酶酶活为162U,仅为亲株的50%。
对突变株在自动发酵罐上作发酵研究,考察了溶氧对其产1,3-丙二醇的影响,最终选定适时调整搅拌转速和通气量做流加补料批式发酵,获得59.85g/L的1,3-丙二醇最高产量,此时其生产强度为0.88g/L/h,甘油的摩尔转化率为53.94%。突变株在初始甘油浓度为43.9g/L的批式发酵中,1,3-丙二醇终浓度为21.2g/L,较对照提高了80%以上,1,3-丙二醇的生产强度为0.96g/L/h,较对照提高了65%,且发酵液中只含1,3-丙二醇和乙酸两种产物。
Poly-Trimethylene Terephthalate (PTT), a newfashioned polyester, has numerous promising properties and applications. 1,3-propanediol (1,3-PDO) as a pivotal monomer to the synthesis of PTT, it has attracted particular attention of many famous corporation in the world in these years. Compared with chemical synthesis, microbial fermentation processes to 1,3-PDO have many obvious advantages, the environmental friendship and price advantage make it more promising, and become the focuses of research.The inhibition of products on microorganism cells will limit the product titer, the yield of 1,3-PDO on glycerol, and lead the product recovery and purification to a troublesome task. These problems may be effectively solved if mutants with tolerance to high concentration of 1,3-PDO and less byproducts were obtained by selective breeding.1,3-PDO、lactate、 acetate and succinate were added to fermention media, and their effects were investigated on the aerobic fermentation of Klebsiella pneumoniae. As a result, 1,3-PDO at a concentration of 90 g/L would inhibit the growth of cells completely. Acetate was favorable to cells growth, but repressive to 1,3-PDO accumulation. Cells grow was repressive by lactate. At a certainty concentration, succinate would increase the producion of 1,3-PDO and lactate.Forty-four mutants were obtained after the mini-Tn5 transposon insertion or NTG mutagenesis of K. pneumoniae and selected on seed medium containing 100 g/L 1,3-PDO and 0.17M NaBr-NaBrO_3 (proton suicide method). Mutants were investigated by shaking flasks, and a mutant named No.9 with high 1,3-propanediol productivity was selected. Its lactate dehydrogenase (LDH) activity was only 162U (about half of the parent strain).In the fed-batch cultures of No.9, a 1,3-propanediol titers of 59.85 g/L was obtained (at the moment, the productivity of 1,3-PDO is 0.88 g/L/h), the mole conversion efficiency of glycerol was 53.94%. In the batch fermentation with a initial glycerol concentration at 43.9 g/L, No.9 finally accumulated 21.2 g/L of 1,3-PDO (80% higher than wild type strain), its productivity of 1,3-PDO was 0.96 g/L/h (65% higher than wild type strain). Furthermore, only two products, 1,3-PDO and acetate, were found in the culture.
产物抑制和副产物多造成微生物发酵法产1,3-丙二醇的产量低,另外,副产物多也是甘油转化率低和产物分离困难的原因之一,选育既有1,3-丙二醇高浓度耐受性,同时又具有副产物代谢途径突变的菌株,是提高1,3-丙二醇产量和甘油转化率并简化后期提取工艺的一种尝试。
通过向培养基中添加外源性产物的方法,考察了1,3-丙二醇和其它有机酸如乙酸、乳酸、琥珀酸对肺炎克雷伯氏杆菌(K.pneumonia)发酵过程的影响。结果表明90g/L的1,3-丙二醇完全抑制了K.pneumoniae的生长。乙酸虽有利于菌体生长却降低了1,3-丙二醇的产量,而乳酸则对菌体生长有抑制。琥珀酸有利于1,3-丙二醇和乳酸形成。
用mini-Tn5随机转座和亚硝基胍分别诱变,在含100g/L 1,3-丙二醇和0.17M溴化钠-溴酸钠(质子自杀方法)的培养基上筛选既有1,3-丙二醇高浓度耐受性,同时又有产酸代谢途径突变的菌株,传代后获得44个稳定突变株,摇瓶培养后作产物分析,经初筛和复筛获得一个1,3-丙二醇高产菌株,其乳酸脱氢酶酶活为162U,仅为亲株的50%。
对突变株在自动发酵罐上作发酵研究,考察了溶氧对其产1,3-丙二醇的影响,最终选定适时调整搅拌转速和通气量做流加补料批式发酵,获得59.85g/L的1,3-丙二醇最高产量,此时其生产强度为0.88g/L/h,甘油的摩尔转化率为53.94%。突变株在初始甘油浓度为43.9g/L的批式发酵中,1,3-丙二醇终浓度为21.2g/L,较对照提高了80%以上,1,3-丙二醇的生产强度为0.96g/L/h,较对照提高了65%,且发酵液中只含1,3-丙二醇和乙酸两种产物。
Poly-Trimethylene Terephthalate (PTT), a newfashioned polyester, has numerous promising properties and applications. 1,3-propanediol (1,3-PDO) as a pivotal monomer to the synthesis of PTT, it has attracted particular attention of many famous corporation in the world in these years. Compared with chemical synthesis, microbial fermentation processes to 1,3-PDO have many obvious advantages, the environmental friendship and price advantage make it more promising, and become the focuses of research.The inhibition of products on microorganism cells will limit the product titer, the yield of 1,3-PDO on glycerol, and lead the product recovery and purification to a troublesome task. These problems may be effectively solved if mutants with tolerance to high concentration of 1,3-PDO and less byproducts were obtained by selective breeding.1,3-PDO、lactate、 acetate and succinate were added to fermention media, and their effects were investigated on the aerobic fermentation of Klebsiella pneumoniae. As a result, 1,3-PDO at a concentration of 90 g/L would inhibit the growth of cells completely. Acetate was favorable to cells growth, but repressive to 1,3-PDO accumulation. Cells grow was repressive by lactate. At a certainty concentration, succinate would increase the producion of 1,3-PDO and lactate.Forty-four mutants were obtained after the mini-Tn5 transposon insertion or NTG mutagenesis of K. pneumoniae and selected on seed medium containing 100 g/L 1,3-PDO and 0.17M NaBr-NaBrO_3 (proton suicide method). Mutants were investigated by shaking flasks, and a mutant named No.9 with high 1,3-propanediol productivity was selected. Its lactate dehydrogenase (LDH) activity was only 162U (about half of the parent strain).In the fed-batch cultures of No.9, a 1,3-propanediol titers of 59.85 g/L was obtained (at the moment, the productivity of 1,3-PDO is 0.88 g/L/h), the mole conversion efficiency of glycerol was 53.94%. In the batch fermentation with a initial glycerol concentration at 43.9 g/L, No.9 finally accumulated 21.2 g/L of 1,3-PDO (80% higher than wild type strain), its productivity of 1,3-PDO was 0.96 g/L/h (65% higher than wild type strain). Furthermore, only two products, 1,3-PDO and acetate, were found in the culture.
引文
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