L-苏氨酸基因工程菌的改造及发酵过程的优化

发布时间：2019-05-23 19:14

【摘要】：L-苏氨酸是人体所必须的八种氨基酸之一,具有独特的生理功能,在食品、医药、化妆品、饲料等多个行业有较广泛的应用。本论文首先对菌种进行了改造,探究了磷酸烯醇式丙酮酸羧化酶(PEPC)过表达对L-苏氨酸发酵的影响。然后对大肠杆菌产生L-苏氨酸的发酵过程进行了优化,探究了甜菜碱、B族维生素等不同发酵促进剂对L-苏氨酸发酵的影响。同时分析了添加甜菜碱对L-苏氨酸发酵的代谢流分布的影响。最后探究了细胞循环发酵对L-苏氨酸发酵的影响。本论文所进行的发酵实验是在实验室5L发酵罐中完成的。主要研究内容和结果如下:(1)将过表达pep C基因的质粒p JL225-9导入到E.coli JLTHR菌株,并进行发酵。发酵30h,产量达到111.2g/L,比E.coli JLTHR产量提高2.8%。代谢流分析结果表明通过过表达pep C基因,对菌种的代谢途径的代谢流量进行重新分配。其中在GLC6P节点处,流向HMP途径代谢流量提高了22%,流向EMP途径代谢流量降低了7.5%。在PEP节点处,E.coli JLTHR p JL225-9的代谢流量流向草酰乙酸回补途径提高了4%。(2)通过添加sigma无水甜菜碱、国产无水甜菜碱以及去除了硬脂酸钙的国产无水甜菜碱等三种不同类型的无水甜菜碱探究了其对L-苏氨酸发酵的影响。结果表明,去除了硬脂酸钙的国产无水甜菜碱对L-苏氨酸发酵的提升效果最显著,产量达到119.3g/L,与对照组比较提高7.3%。此外,通过添加不同浓度的甜菜碱盐酸盐和甜菜碱磷酸盐探究了甜菜碱盐类类型和浓度对L-苏氨酸发酵的影响,发酵30h结果表明:甜菜碱盐酸盐在浓度为2g/L的添加量下,L-苏氨酸的产量最高,达到了127.3g/L,与对照组比较提高14.5%;甜菜碱磷酸盐在添加浓度为2g/L的条件下,大肠杆菌的OD_(600nm)最高,达到了63.4,与对照组比较提高了15.3%。甜菜碱含有三个活性甲基,氯化胆碱和甲硫氨酸同样含有活性甲基,在L-苏氨酸合成方面有促进作用。实验表明添加氯化胆碱L-苏氨酸产量为119.3g/L,与对照组比较相等。添加甲硫氨酸进行L-苏氨酸发酵,产量为118.4g/L,与无水甜菜碱效果相似。(3)探究甜菜碱对L-苏氨酸菌种代谢流分布的影响。构建L-苏氨酸菌株的代谢网络,根据代谢流分析理论,使用MATLAB软件线性规划得到L-苏氨酸菌株在发酵中后期的代谢流量的分配。通过代谢流分析,得出葡萄糖在EMP、HMP以及TCA循环中代谢流量分配情况,并确定关键节点GLC6P、PEP和-KG。实验结果表明在节点GLC6P处,添加甜菜碱发酵液后,细胞内葡萄糖流向HMP途径的代谢流量比不添加甜菜碱提高了57.3%。在节点PEP处,添加甜菜碱中细胞中的草酰乙酸(OAA)代谢流比未添加甜菜碱菌体中的OAA的代谢流,提高了10.1%,流向TCA途径的代谢流降低了6.9%。a(4)通过添加不同发酵维生素B族探究其对L-苏氨酸发酵的影响。本论文使用的维生素B有氯化胆碱(VB_4)、烟酰胺(VB_3)、泛酸钙(VB_5)以及钴胺素(VB_(12)),实验表明:添加氯化胆碱对L-苏氨酸的促进效果最显著,L-苏氨酸产量达到133.4g/L。VB_3对L-苏氨酸发酵促进效果较显著,产量达到130.6g/L。添加甜菜碱盐酸盐、VB_4和VB_3的混合发酵促进剂,L-苏氨酸产量达到138.4g/L。(5)细胞循环发酵试验结果表明:离心分离发酵L-苏氨酸产量(达到141.4g/L)较不进行循环发酵提高了7.9%,效果优于陶瓷膜分离发酵。对细胞循环周期和循环发酵策略进行初步探究,发现在菌体循环周期16h,循环策略为V(上清液):V(浓缩液)=1:2时,L-苏氨酸产量最高,达到148.4g/L,较不进行循环发酵提高了13.1%。
[Abstract]:L-threonine is one of the eight amino acids necessary for human body, has a unique physiological function, and has a wide application in many industries such as food, medicine, cosmetics and feed. The influence of the over-expression of phosphoenolase (PEPC) on L-threonine fermentation was studied. The fermentation process of L-threonine produced by E. coli was optimized, and the effects of different fermentation promoters such as betaine and B vitamins on the fermentation of L-threonine were investigated. The effect of betaine on the distribution of L-threonine fermentation was also analyzed. The effect of cell cycle fermentation on L-threonine fermentation was studied. The fermentation experiments carried out in this paper were completed in a 5 L fermentor in the laboratory. The main contents and results are as follows: (1) The plasmid p JL225-9, which overexpresses the pep C gene, is introduced into the E. coli JLHR strain and is fermented. The yield reached 111.2 g/ L, and the yield of E. coli JLHR was increased by 2.8%. The analysis of the metabolic flow showed that the metabolic flux of the metabolic pathway of the strain was reallocated by overexpressing the pep C gene. At the GLC6P node, the metabolic flux to the HMP pathway increased by 22%, and the flow rate to the EMP pathway was reduced by 7.5%. The metabolic rate of E. coli JLHR p JL225-9 was increased by 4% at the PEP node. (2) The effect of three different types of anhydrous betaines on L-threonine fermentation was investigated by addition of three different types of anhydrous betaines, such as sima anhydrous betaines, domestic anhydrous betaines, and domestic anhydrous betaines, other than calcium stearate. The results showed that the effect of the domestic anhydrous betaine on L-threonine fermentation with the addition of calcium stearate was the most significant, the yield reached 119.3 g/ L, and the comparison with the control group was increased by 7.3%. In addition, the effect of betaine salt type and concentration on L-threonine fermentation was investigated by adding betaine hydrochloride and betaine phosphate at different concentrations. The results showed that the yield of L-threonine was the highest under the addition of 2 g/ L of betaine hydrochloride. The result showed that the OD _ (600 nm) of E. coli was the highest in the condition of the addition of 2 g/ L, the OD _ (600 nm) of E. coli reached 63.4, and the comparison with the control group increased by 15.3%. The betaine contains three active methyl groups, the choline chloride and the methionine also contain the active methyl groups, and the betaine has the effect of promoting the synthesis of L-threonine. The results showed that the yield of L-threonine with choline chloride was 119.3 g/ L, which was equal to that of the control group. The L-threonine fermentation was carried out with methionine and the yield was 118.4 g/ L, which was similar to that of the anhydrous betaine. (3) To investigate the effect of betaine on the distribution of L-threonine species. The metabolic network of L-threonine strain was constructed. According to the metabolic flow analysis theory, the distribution of the metabolic flux of L-threonine strain in the middle and late stage of the fermentation was obtained by using the MATLAB software linear programming. The distribution of glucose in the EMP, HMP, and TCA cycle was obtained by metabolic flow analysis and the key nodes GLC6P, PEP and-KG were determined. The experimental results show that, at the node GLC6P, the metabolic flux of glucose in the cells to the HMP pathway after addition of the betaine fermentation broth is 57.3% higher than that of the non-addition of betaine. At the node PEP, the metabolic flux of the OAA in the cells added to the betaine was increased by 10.1%, and the metabolic flow to the TCA pathway was decreased by 6.9%. a (4) the effect on L-threonine fermentation was investigated by the addition of the different fermentation vitamin B groups. The vitamin B used in this paper has choline chloride (VB _ 4), bichromine (VB _ 3), calcium pantothenate (VB _ 5) and cobalamin (VB _ (12)). The results show that the effect of adding choline chloride to L-threonine is the most significant. The yield of L-threonine reached 133.4 g/ L. VB _ 3 had a significant effect on L-threonine fermentation, and the yield reached 130.6 g/ L. The yield of L-threonine was 138.4 g/ L by adding betaine hydrochloride, VB _ 4 and VB _ 3. (5) The results of cell cycle fermentation showed that the yield of L-threonine (up to 141.4 g/ L) was increased by 7.9%, and the effect was better than that of the ceramic membrane. The cycle of cycle and the strategy of circulating fermentation were studied, and it was found that the yield of L-threonine was up to 148.4 g/ L when the cycle time was 16 h and the circulation strategy was V (supernatant): V (concentrated solution) = 1:2, and 13.1% higher than that of the non-cyclic fermentation.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ922

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