產(chǎn)N乙酰神經(jīng)氨酸重組大腸桿菌的構(gòu)建及其生物轉(zhuǎn)化合成
發(fā)布時(shí)間：2018-07-17 06:04
【摘要】：唾液酸是一類含有9個(gè)碳原子并具有吡喃糖結(jié)構(gòu)的酸性氨基糖,又稱神經(jīng)氨酸;唾液酸在自然界中分布廣泛,且種類繁多,迄今已發(fā)現(xiàn)超過60種唾液酸。N-乙酰神經(jīng)氨酸(Neu5Ac)是最重要的一種唾液酸,是其他唾液酸合成的前體物質(zhì),與人類的健康關(guān)系最為密切;Neu5Ac可以用于合成抗病毒藥物,治療H1N1和H5N1流感等疾病。此外,Neu5Ac也有很大的營養(yǎng)價(jià)值,它能促進(jìn)嬰兒的大腦發(fā)育,同時(shí)對(duì)維持早產(chǎn)兒腦功能和健康也有積極作用。目前制備和生產(chǎn)Neu5Ac常用的方法是酶催化法,該方法產(chǎn)量雖高,但需要添加過量的丙酮酸導(dǎo)致原料浪費(fèi)、產(chǎn)品純化困難以及環(huán)境壓力增大。本論文嘗試建立一個(gè)不依賴丙酮酸的Neu5Ac合成方法:以N-乙酰葡萄糖胺(Glc NAc)為底物,在N-乙酰葡萄糖胺異構(gòu)酶(AGE)的催化下異構(gòu)化為N-乙酰甘露糖胺(Man NAc);然后,在Neu5Ac合成酶(Neu B)的催化下,與磷酸烯醇式丙酮酸(PEP)發(fā)生不可逆反應(yīng)合成Neu5Ac。在大腸桿菌中構(gòu)建上述Neu5Ac合成途徑,并利用代謝工程和發(fā)酵工程方法強(qiáng)化Neu5Ac的合成。主要結(jié)論如下:(1)分別考察不同來源的AGE和Neu B的活性及酶學(xué)性質(zhì),對(duì)比得到在大腸桿菌中表達(dá)活性最高的酶。結(jié)果表明,豬腎臟中p AGE和項(xiàng)圈藻中b AGE的穩(wěn)定性、溫度偏好和p H偏好性相似,其最適p H均為7.0,最適溫度均為50°C,并且在35-55°C范圍內(nèi)保持較高活性,達(dá)到其在50°C時(shí)活性的90%以上。在37°C Tris-HCl(p H 7.0)條件下,p AGE和b AGE活性分別為392.1和2411.5 U?mg-1總蛋白。此外,e Neu B和c Neu B的最適p H均為中性,且最適溫度均為45°C,在30-47.5°C范圍內(nèi)保留超過60%的活性。在37°C Tris-HCl(p H 7.0)條件下,e Neu B和c Neu B活性分別為0.037和6.380 U?mg-1總蛋白。而c Neu B的穩(wěn)定性不佳,2 h后僅有50%的催化活性保留。當(dāng)與分子伴侶質(zhì)粒p Gro7共表達(dá)時(shí),測(cè)得的c Neu B活性比對(duì)照組提高17.8%;其誘導(dǎo)表達(dá)的Gro EL/Gro ES復(fù)合體,能阻止蛋白質(zhì)的錯(cuò)誤聚集并提高蛋白質(zhì)的折疊速度,提高大腸桿菌可溶性表達(dá)c Neu B的能力。因此考慮基于b AGE和c Neu B兩種酶構(gòu)建Neu5Ac合成途徑。(2)基于b AGE和c Neu B,構(gòu)建Neu5Ac合成途徑;分別敲除宿主菌的Neu5Ac分解途徑和Glc NAc跨膜轉(zhuǎn)運(yùn)途徑的相關(guān)基因,促進(jìn)Neu5Ac的合成。由于c Neu B活性遠(yuǎn)小于b AGE,因此重點(diǎn)關(guān)注體系中的c Neu B催化活性。對(duì)調(diào)兩個(gè)基因的次序,分別得到共表達(dá)載體p DTrc-AB和p DTrc-BA,發(fā)現(xiàn)p DTrc-AB中cneu B基因位于下游其表達(dá)活性更高。宿主大腸桿菌中的Neu5Ac分解途徑保留時(shí),無法實(shí)現(xiàn)Neu5Ac的積累;利用Red重組系統(tǒng)敲除nan ATEK基因簇后,得到E.coli SA-01,其Neu5Ac分解途徑被阻斷;將共表達(dá)載體p DTrc-AB和p DTrc-BA分別轉(zhuǎn)化E.coli SA-01,經(jīng)誘導(dǎo)后的菌體成功實(shí)現(xiàn)Neu5Ac的合成,Neu5Ac合成量分別為2.61和2.03 g?L-1。改造Glc NAc跨膜轉(zhuǎn)運(yùn)途徑,避免底物Glc NAc與PEP在旁路上的無效消耗,增加胞內(nèi)底物Glc NAc濃度和PEP供給,強(qiáng)化了Neu5Ac的合成,Neu5Ac合成量提高38.3%,達(dá)到3.61 g?L-1。(3)將大腸桿菌中PEP合成相關(guān)基因單獨(dú)或共同過表達(dá),提高胞內(nèi)PEP的供給,進(jìn)一步促進(jìn)了Neu5Ac的合成。從E.coli MG1655基因組中克隆得到PEP合成相關(guān)基因pck和pps A,利用Duet系列載體構(gòu)建兩個(gè)基因的單獨(dú)過表達(dá)和共過表達(dá)載體,并導(dǎo)入DE3溶源化的E.coli。當(dāng)pck和pps A單獨(dú)過表達(dá)時(shí),Neu5Ac合成量分別為5.80和7.09 g?L-1。當(dāng)pck和pps A共同過表達(dá)時(shí),兩個(gè)基因的轉(zhuǎn)錄水平同步提高,并正相關(guān)于載體的拷貝數(shù)。最優(yōu)的PEP強(qiáng)化供給系統(tǒng)由中拷貝載體p CDF-pck-pps A構(gòu)成,此時(shí)Neu5Ac合成量達(dá)到8.63 g?L-1,比出發(fā)菌株提高139%。(4)考察生物轉(zhuǎn)化階段合成Neu5Ac的營養(yǎng)條件以及表面活性劑對(duì)Neu5Ac合成的影響,優(yōu)化Neu5Ac合成過程,并在發(fā)酵罐中實(shí)現(xiàn)Neu5Ac的高效合成。在含有氮源的培養(yǎng)基中,宿主大腸桿菌大量生長,而Neu5Ac的合成受到抑制;在無氮葡萄糖培養(yǎng)基中,宿主大腸桿菌生長受到限制,而Neu5Ac的合成抑制解除,合成量達(dá)到8.61 g?L-1。當(dāng)宿主大腸桿菌生長旺盛時(shí)會(huì)消耗PEP,與Neu5Ac合成競(jìng)爭(zhēng)胞內(nèi)的PEP,反而不利于Neu5Ac的合成。當(dāng)甘油為碳源時(shí),其跨膜轉(zhuǎn)運(yùn)不依賴于PTS,節(jié)省胞內(nèi)PEP從而使其更多流向Neu5Ac合成方向,Neu5Ac合成量也隨之提高到10.43 g?L-1。轉(zhuǎn)化培養(yǎng)基中添加Triton X-100,對(duì)Neu5Ac合成有一定的促進(jìn)作用,但僅為6.1%。最后,在發(fā)酵罐中進(jìn)行生物轉(zhuǎn)化合成Neu5Ac過程,培養(yǎng)基中磷酸鹽濃度大幅降低,減小了反應(yīng)體系滲透壓力;同時(shí)發(fā)酵罐中更好的傳質(zhì)狀況,有利于Neu5Ac的合成。在發(fā)酵罐中65 h合成Neu5Ac達(dá)到16.02 g?L-1,比同條件搖瓶水平顯著增加53.6%,實(shí)現(xiàn)了Neu5Ac的高效合成。
[Abstract]:Sialic acid is a class of acid aminosaccharides containing 9 carbon atoms and with the structure of Piran sugar, also known as neuraminic acid; sialic acid is widely distributed in nature and has a wide variety. Up to now, more than 60 sialic acid.N- acetyl neuroammonia acid (Neu5Ac) is the most important sialic acid, the precursor of other sialic acid synthesis, and human The health relationship is most closely related; Neu5Ac can be used to synthesize antiviral drugs and to treat diseases such as H1N1 and H5N1 influenza. In addition, Neu5Ac also has great nutritional value. It can promote the development of the brain in infants, and also have a positive effect on the maintenance of brain function and health in premature infants. At present, the method of preparing and producing Neu5Ac is enzyme catalysis, which is a common method. Although the yield of the method is high, it needs to add excess pyruvic acid to lead to waste of raw materials, difficult product purification and environmental pressure. This paper attempts to establish a Neu5Ac synthesis method without pyruvic acid: N- acetyl glucosamine (Glc NAc) as the substrate and the N- acetyl glucosamine isomerase (AGE) catalyzed isomerization to N- acetyl mannose Amines (Man NAc); then, under the catalysis of Neu5Ac synthetase (Neu B), an irreversible reaction with phosphoenolpyruvic acid (PEP) is used to synthesize Neu5Ac. in Escherichia coli to construct the above-mentioned Neu5Ac synthesis pathway, and to strengthen the aggregation of Neu5Ac by metabolic engineering and fermentation engineering. The main conclusions are as follows: (1) examine AGE and Neu B from different sources, respectively. The results showed that the stability of B AGE in P AGE and colipora colipora was similar in the pig kidney, and the temperature preference and P H preference were similar. The optimum P H was 7, the optimum temperature was 50 degrees C, and the high activity was maintained in the 35-55 degree C peri, which reached 90 of its activity at 50 degree C. Under the condition of 37 C Tris-HCl (P H 7), P AGE and B AGE activity are 392.1 and 2411.5 U mg-1 total protein respectively. And the total protein of 6.380 U? Mg-1. But the stability of C Neu B was not good, only 50% of the catalytic activity was retained after 2 h. When co expression with the molecular chaperone P Gro7, the B activity of C Neu was 17.8% higher than that of the control group. Enterobacteriaceae expressed the ability to express C Neu B. Therefore, we consider the construction of Neu5Ac synthesis pathway based on the two enzymes of B AGE and C Neu B. (2) the synthesis pathway is constructed based on B AGE and C. Therefore, we focus on the catalytic activity of C Neu B in the system. A co expression vector p DTrc-AB and P DTrc-BA are obtained in order to adjust the two genes. It is found that cneu B gene in the P DTrc-AB is more active downstream, and the accumulation of the Neu5Ac decomposition pathway in the host Escherichia coli can not be realized. After the Nan ATEK gene cluster, E.coli SA-01 was obtained, and its Neu5Ac decomposition pathway was blocked; the co expression vector p DTrc-AB and P DTrc-BA were converted to E.coli SA-01. The induced organisms were successfully synthesized, and the synthesis amount was 2.61 and 2.03 respectively. Ineffective consumption, increasing the concentration of intracellular substrate Glc NAc and PEP supply, enhanced the synthesis of Neu5Ac, increased the synthesis of Neu5Ac by 38.3%, reached 3.61 G? L-1. (3), by which PEP synthesis related genes in Escherichia coli were expressed individually or together, improving the supply of intracellular PEP and further promoting the synthesis of Neu5Ac. Related genes PCK and PPS A, using Duet series vectors to construct a single overexpression and co expression vector of two genes, and import DE3 soluble E.coli. when PCK and PPS A are expressed separately, the Neu5Ac synthesis is 5.80 and 7.09 g, and the transcriptional level of the two genes increases synchronously and is positively correlated. The number of copies of the carrier. The optimal PEP fortified supply system is composed of medium copy carrier P CDF-pck-pps A. At this time, the Neu5Ac synthesis amount to 8.63 G? L-1, increase 139%. (4) compared with the starting strain, investigate the nutritional conditions of the synthesis of Neu5Ac and the effect of surfactant on Neu5Ac, optimize the Neu5Ac synthesis process, and in the fermenting tank. To achieve high efficiency synthesis of Neu5Ac. In the medium containing nitrogen sources, the host Escherichia coli grew in large quantities, and the synthesis of Neu5Ac was inhibited. In the nitrogen free glucose medium, the growth of Escherichia coli in the host was restricted, and the synthesis of Neu5Ac was inhibited and the synthesis reached 8.61 G? L-1. when the host Escherichia coli grew exuberant PEP and N The synthesis of PEP in the competitive cell of eu5Ac is not conducive to the synthesis of Neu5Ac. When glycerol is a carbon source, its transmembrane transport is not dependent on PTS, which saves the intracellular PEP and makes it more flow to the direction of Neu5Ac synthesis. The Neu5Ac synthesis also increases to the 10.43 G? L-1. conversion medium, adding Triton X-100, but has a certain promotion effect on the Neu5Ac synthesis. In the end of 6.1%., the process of biosynthesis of Neu5Ac in the fermenting tank was carried out. The phosphate concentration in the medium was greatly reduced and the permeation pressure of the reaction system was reduced. At the same time, the better mass transfer in the fermenting tank was beneficial to the synthesis of Neu5Ac. In the fermenting tank, the 65 h synthesis Neu5Ac reached 16.02 G? L-1, which was significantly increased by 53.6% than that of the same condition shake flask. The efficient synthesis of Neu5Ac is realized.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：TQ920.1
本文編號(hào)：2129334
[Abstract]:Sialic acid is a class of acid aminosaccharides containing 9 carbon atoms and with the structure of Piran sugar, also known as neuraminic acid; sialic acid is widely distributed in nature and has a wide variety. Up to now, more than 60 sialic acid.N- acetyl neuroammonia acid (Neu5Ac) is the most important sialic acid, the precursor of other sialic acid synthesis, and human The health relationship is most closely related; Neu5Ac can be used to synthesize antiviral drugs and to treat diseases such as H1N1 and H5N1 influenza. In addition, Neu5Ac also has great nutritional value. It can promote the development of the brain in infants, and also have a positive effect on the maintenance of brain function and health in premature infants. At present, the method of preparing and producing Neu5Ac is enzyme catalysis, which is a common method. Although the yield of the method is high, it needs to add excess pyruvic acid to lead to waste of raw materials, difficult product purification and environmental pressure. This paper attempts to establish a Neu5Ac synthesis method without pyruvic acid: N- acetyl glucosamine (Glc NAc) as the substrate and the N- acetyl glucosamine isomerase (AGE) catalyzed isomerization to N- acetyl mannose Amines (Man NAc); then, under the catalysis of Neu5Ac synthetase (Neu B), an irreversible reaction with phosphoenolpyruvic acid (PEP) is used to synthesize Neu5Ac. in Escherichia coli to construct the above-mentioned Neu5Ac synthesis pathway, and to strengthen the aggregation of Neu5Ac by metabolic engineering and fermentation engineering. The main conclusions are as follows: (1) examine AGE and Neu B from different sources, respectively. The results showed that the stability of B AGE in P AGE and colipora colipora was similar in the pig kidney, and the temperature preference and P H preference were similar. The optimum P H was 7, the optimum temperature was 50 degrees C, and the high activity was maintained in the 35-55 degree C peri, which reached 90 of its activity at 50 degree C. Under the condition of 37 C Tris-HCl (P H 7), P AGE and B AGE activity are 392.1 and 2411.5 U mg-1 total protein respectively. And the total protein of 6.380 U? Mg-1. But the stability of C Neu B was not good, only 50% of the catalytic activity was retained after 2 h. When co expression with the molecular chaperone P Gro7, the B activity of C Neu was 17.8% higher than that of the control group. Enterobacteriaceae expressed the ability to express C Neu B. Therefore, we consider the construction of Neu5Ac synthesis pathway based on the two enzymes of B AGE and C Neu B. (2) the synthesis pathway is constructed based on B AGE and C. Therefore, we focus on the catalytic activity of C Neu B in the system. A co expression vector p DTrc-AB and P DTrc-BA are obtained in order to adjust the two genes. It is found that cneu B gene in the P DTrc-AB is more active downstream, and the accumulation of the Neu5Ac decomposition pathway in the host Escherichia coli can not be realized. After the Nan ATEK gene cluster, E.coli SA-01 was obtained, and its Neu5Ac decomposition pathway was blocked; the co expression vector p DTrc-AB and P DTrc-BA were converted to E.coli SA-01. The induced organisms were successfully synthesized, and the synthesis amount was 2.61 and 2.03 respectively. Ineffective consumption, increasing the concentration of intracellular substrate Glc NAc and PEP supply, enhanced the synthesis of Neu5Ac, increased the synthesis of Neu5Ac by 38.3%, reached 3.61 G? L-1. (3), by which PEP synthesis related genes in Escherichia coli were expressed individually or together, improving the supply of intracellular PEP and further promoting the synthesis of Neu5Ac. Related genes PCK and PPS A, using Duet series vectors to construct a single overexpression and co expression vector of two genes, and import DE3 soluble E.coli. when PCK and PPS A are expressed separately, the Neu5Ac synthesis is 5.80 and 7.09 g, and the transcriptional level of the two genes increases synchronously and is positively correlated. The number of copies of the carrier. The optimal PEP fortified supply system is composed of medium copy carrier P CDF-pck-pps A. At this time, the Neu5Ac synthesis amount to 8.63 G? L-1, increase 139%. (4) compared with the starting strain, investigate the nutritional conditions of the synthesis of Neu5Ac and the effect of surfactant on Neu5Ac, optimize the Neu5Ac synthesis process, and in the fermenting tank. To achieve high efficiency synthesis of Neu5Ac. In the medium containing nitrogen sources, the host Escherichia coli grew in large quantities, and the synthesis of Neu5Ac was inhibited. In the nitrogen free glucose medium, the growth of Escherichia coli in the host was restricted, and the synthesis of Neu5Ac was inhibited and the synthesis reached 8.61 G? L-1. when the host Escherichia coli grew exuberant PEP and N The synthesis of PEP in the competitive cell of eu5Ac is not conducive to the synthesis of Neu5Ac. When glycerol is a carbon source, its transmembrane transport is not dependent on PTS, which saves the intracellular PEP and makes it more flow to the direction of Neu5Ac synthesis. The Neu5Ac synthesis also increases to the 10.43 G? L-1. conversion medium, adding Triton X-100, but has a certain promotion effect on the Neu5Ac synthesis. In the end of 6.1%., the process of biosynthesis of Neu5Ac in the fermenting tank was carried out. The phosphate concentration in the medium was greatly reduced and the permeation pressure of the reaction system was reduced. At the same time, the better mass transfer in the fermenting tank was beneficial to the synthesis of Neu5Ac. In the fermenting tank, the 65 h synthesis Neu5Ac reached 16.02 G? L-1, which was significantly increased by 53.6% than that of the same condition shake flask. The efficient synthesis of Neu5Ac is realized.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：TQ920.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 ;Co-expression of five genes in E coli for L-phenylalanine in Brevibacterium flavum[J];World Journal of Gastroenterology;2003年02期
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