L-蘇氨酸基因工程菌的改造及發(fā)酵過程的優(yōu)化
發(fā)布時(shí)間：2019-05-23 19:14
【摘要】：L-蘇氨酸是人體所必須的八種氨基酸之一,具有獨(dú)特的生理功能,在食品、醫(yī)藥、化妝品、飼料等多個(gè)行業(yè)有較廣泛的應(yīng)用。本論文首先對菌種進(jìn)行了改造,探究了磷酸烯醇式丙酮酸羧化酶(PEPC)過表達(dá)對L-蘇氨酸發(fā)酵的影響。然后對大腸桿菌產(chǎn)生L-蘇氨酸的發(fā)酵過程進(jìn)行了優(yōu)化,探究了甜菜堿、B族維生素等不同發(fā)酵促進(jìn)劑對L-蘇氨酸發(fā)酵的影響。同時(shí)分析了添加甜菜堿對L-蘇氨酸發(fā)酵的代謝流分布的影響。最后探究了細(xì)胞循環(huán)發(fā)酵對L-蘇氨酸發(fā)酵的影響。本論文所進(jìn)行的發(fā)酵實(shí)驗(yàn)是在實(shí)驗(yàn)室5L發(fā)酵罐中完成的。主要研究內(nèi)容和結(jié)果如下:(1)將過表達(dá)pep C基因的質(zhì)粒p JL225-9導(dǎo)入到E.coli JLTHR菌株,并進(jìn)行發(fā)酵。發(fā)酵30h,產(chǎn)量達(dá)到111.2g/L,比E.coli JLTHR產(chǎn)量提高2.8%。代謝流分析結(jié)果表明通過過表達(dá)pep C基因,對菌種的代謝途徑的代謝流量進(jìn)行重新分配。其中在GLC6P節(jié)點(diǎn)處,流向HMP途徑代謝流量提高了22%,流向EMP途徑代謝流量降低了7.5%。在PEP節(jié)點(diǎn)處,E.coli JLTHR p JL225-9的代謝流量流向草酰乙酸回補(bǔ)途徑提高了4%。(2)通過添加sigma無水甜菜堿、國產(chǎn)無水甜菜堿以及去除了硬脂酸鈣的國產(chǎn)無水甜菜堿等三種不同類型的無水甜菜堿探究了其對L-蘇氨酸發(fā)酵的影響。結(jié)果表明,去除了硬脂酸鈣的國產(chǎn)無水甜菜堿對L-蘇氨酸發(fā)酵的提升效果最顯著,產(chǎn)量達(dá)到119.3g/L,與對照組比較提高7.3%。此外,通過添加不同濃度的甜菜堿鹽酸鹽和甜菜堿磷酸鹽探究了甜菜堿鹽類類型和濃度對L-蘇氨酸發(fā)酵的影響,發(fā)酵30h結(jié)果表明:甜菜堿鹽酸鹽在濃度為2g/L的添加量下,L-蘇氨酸的產(chǎn)量最高,達(dá)到了127.3g/L,與對照組比較提高14.5%;甜菜堿磷酸鹽在添加濃度為2g/L的條件下,大腸桿菌的OD_(600nm)最高,達(dá)到了63.4,與對照組比較提高了15.3%。甜菜堿含有三個(gè)活性甲基,氯化膽堿和甲硫氨酸同樣含有活性甲基,在L-蘇氨酸合成方面有促進(jìn)作用。實(shí)驗(yàn)表明添加氯化膽堿L-蘇氨酸產(chǎn)量為119.3g/L,與對照組比較相等。添加甲硫氨酸進(jìn)行L-蘇氨酸發(fā)酵,產(chǎn)量為118.4g/L,與無水甜菜堿效果相似。(3)探究甜菜堿對L-蘇氨酸菌種代謝流分布的影響。構(gòu)建L-蘇氨酸菌株的代謝網(wǎng)絡(luò),根據(jù)代謝流分析理論,使用MATLAB軟件線性規(guī)劃得到L-蘇氨酸菌株在發(fā)酵中后期的代謝流量的分配。通過代謝流分析,得出葡萄糖在EMP、HMP以及TCA循環(huán)中代謝流量分配情況,并確定關(guān)鍵節(jié)點(diǎn)GLC6P、PEP和-KG。實(shí)驗(yàn)結(jié)果表明在節(jié)點(diǎn)GLC6P處,添加甜菜堿發(fā)酵液后,細(xì)胞內(nèi)葡萄糖流向HMP途徑的代謝流量比不添加甜菜堿提高了57.3%。在節(jié)點(diǎn)PEP處,添加甜菜堿中細(xì)胞中的草酰乙酸(OAA)代謝流比未添加甜菜堿菌體中的OAA的代謝流,提高了10.1%,流向TCA途徑的代謝流降低了6.9%。a(4)通過添加不同發(fā)酵維生素B族探究其對L-蘇氨酸發(fā)酵的影響。本論文使用的維生素B有氯化膽堿(VB_4)、煙酰胺(VB_3)、泛酸鈣(VB_5)以及鈷胺素(VB_(12)),實(shí)驗(yàn)表明:添加氯化膽堿對L-蘇氨酸的促進(jìn)效果最顯著,L-蘇氨酸產(chǎn)量達(dá)到133.4g/L。VB_3對L-蘇氨酸發(fā)酵促進(jìn)效果較顯著,產(chǎn)量達(dá)到130.6g/L。添加甜菜堿鹽酸鹽、VB_4和VB_3的混合發(fā)酵促進(jìn)劑,L-蘇氨酸產(chǎn)量達(dá)到138.4g/L。(5)細(xì)胞循環(huán)發(fā)酵試驗(yàn)結(jié)果表明:離心分離發(fā)酵L-蘇氨酸產(chǎn)量(達(dá)到141.4g/L)較不進(jìn)行循環(huán)發(fā)酵提高了7.9%,效果優(yōu)于陶瓷膜分離發(fā)酵。對細(xì)胞循環(huán)周期和循環(huán)發(fā)酵策略進(jìn)行初步探究,發(fā)現(xiàn)在菌體循環(huán)周期16h,循環(huán)策略為V(上清液):V(濃縮液)=1:2時(shí),L-蘇氨酸產(chǎn)量最高,達(dá)到148.4g/L,較不進(jìn)行循環(huán)發(fā)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：TQ922
本文編號：2484153
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：TQ922
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