【作者】 朱德强；

【导师】 詹晓北；

【作者基本信息】 江南大学 ， 发酵工程， 2017， 博士

【摘要】 唾液酸是一类含有9个碳原子并具有吡喃糖结构的酸性氨基糖,又称神经氨酸;唾液酸在自然界中分布广泛,且种类繁多,迄今已发现超过60种唾液酸。N-乙酰神经氨酸(Neu5Ac)是最重要的一种唾液酸,是其他唾液酸合成的前体物质,与人类的健康关系最为密切;Neu5Ac可以用于合成抗病毒药物,治疗H1N1和H5N1流感等疾病。此外,Neu5Ac也有很大的营养价值,它能促进婴儿的大脑发育,同时对维持早产儿脑功能和健康也有积极作用。目前制备和生产Neu5Ac常用的方法是酶催化法,该方法产量虽高,但需要添加过量的丙酮酸导致原料浪费、产品纯化困难以及环境压力增大。本论文尝试建立一个不依赖丙酮酸的Neu5Ac合成方法:以N-乙酰葡萄糖胺(Glc NAc)为底物,在N-乙酰葡萄糖胺异构酶(AGE)的催化下异构化为N-乙酰甘露糖胺(Man NAc);然后,在Neu5Ac合成酶(Neu B)的催化下,与磷酸烯醇式丙酮酸(PEP)发生不可逆反应合成Neu5Ac。在大肠杆菌中构建上述Neu5Ac合成途径,并利用代谢工程和发酵工程方法强化Neu5Ac的合成。主要结论如下:(1)分别考察不同来源的AGE和Neu B的活性及酶学性质,对比得到在大肠杆菌中表达活性最高的酶。结果表明,猪肾脏中p AGE和项圈藻中b AGE的稳定性、温度偏好和p H偏好性相似,其最适p H均为7.0,最适温度均为50°C,并且在35-55°C范围内保持较高活性,达到其在50°C时活性的90%以上。在37°C Tris-HCl(p H 7.0)条件下,p AGE和b AGE活性分别为392.1和2411.5 U?mg-1总蛋白。此外,e Neu B和c Neu B的最适p H均为中性,且最适温度均为45°C,在30-47.5°C范围内保留超过60%的活性。在37°C Tris-HCl(p H 7.0)条件下,e Neu B和c Neu B活性分别为0.037和6.380 U?mg-1总蛋白。而c Neu B的稳定性不佳,2 h后仅有50%的催化活性保留。当与分子伴侣质粒p Gro7共表达时,测得的c Neu B活性比对照组提高17.8%;其诱导表达的Gro EL/Gro ES复合体,能阻止蛋白质的错误聚集并提高蛋白质的折叠速度,提高大肠杆菌可溶性表达c Neu B的能力。因此考虑基于b AGE和c Neu B两种酶构建Neu5Ac合成途径。(2)基于b AGE和c Neu B,构建Neu5Ac合成途径;分别敲除宿主菌的Neu5Ac分解途径和Glc NAc跨膜转运途径的相关基因,促进Neu5Ac的合成。由于c Neu B活性远小于b AGE,因此重点关注体系中的c Neu B催化活性。对调两个基因的次序,分别得到共表达载体p DTrc-AB和p DTrc-BA,发现p DTrc-AB中cneu B基因位于下游其表达活性更高。宿主大肠杆菌中的Neu5Ac分解途径保留时,无法实现Neu5Ac的积累;利用Red重组系统敲除nan ATEK基因簇后,得到E.coli SA-01,其Neu5Ac分解途径被阻断;将共表达载体p DTrc-AB和p DTrc-BA分别转化E.coli SA-01,经诱导后的菌体成功实现Neu5Ac的合成,Neu5Ac合成量分别为2.61和2.03 g?L-1。改造Glc NAc跨膜转运途径,避免底物Glc NAc与PEP在旁路上的无效消耗,增加胞内底物Glc NAc浓度和PEP供给,强化了Neu5Ac的合成,Neu5Ac合成量提高38.3%,达到3.61 g?L-1。(3)将大肠杆菌中PEP合成相关基因单独或共同过表达,提高胞内PEP的供给,进一步促进了Neu5Ac的合成。从E.coli MG1655基因组中克隆得到PEP合成相关基因pck和pps A,利用Duet系列载体构建两个基因的单独过表达和共过表达载体,并导入DE3溶源化的E.coli。当pck和pps A单独过表达时,Neu5Ac合成量分别为5.80和7.09 g?L-1。当pck和pps A共同过表达时,两个基因的转录水平同步提高,并正相关于载体的拷贝数。最优的PEP强化供给系统由中拷贝载体p CDF-pck-pps A构成,此时Neu5Ac合成量达到8.63 g?L-1,比出发菌株提高139%。(4)考察生物转化阶段合成Neu5Ac的营养条件以及表面活性剂对Neu5Ac合成的影响,优化Neu5Ac合成过程,并在发酵罐中实现Neu5Ac的高效合成。在含有氮源的培养基中,宿主大肠杆菌大量生长,而Neu5Ac的合成受到抑制;在无氮葡萄糖培养基中,宿主大肠杆菌生长受到限制,而Neu5Ac的合成抑制解除,合成量达到8.61 g?L-1。当宿主大肠杆菌生长旺盛时会消耗PEP,与Neu5Ac合成竞争胞内的PEP,反而不利于Neu5Ac的合成。当甘油为碳源时,其跨膜转运不依赖于PTS,节省胞内PEP从而使其更多流向Neu5Ac合成方向,Neu5Ac合成量也随之提高到10.43 g?L-1。转化培养基中添加Triton X-100,对Neu5Ac合成有一定的促进作用,但仅为6.1%。最后,在发酵罐中进行生物转化合成Neu5Ac过程,培养基中磷酸盐浓度大幅降低,减小了反应体系渗透压力;同时发酵罐中更好的传质状况,有利于Neu5Ac的合成。在发酵罐中65 h合成Neu5Ac达到16.02 g?L-1,比同条件摇瓶水平显著增加53.6%,实现了Neu5Ac的高效合成。更多还原

【Abstract】 Sialic acid, also known as neuraminic acid, is an amino sugar with nine carbons and pyranose structures. It is ubiquitous in nature, and more than 60 categories of sialic acids have been identified. Among these categories, N-acetylneuraminic acid(Neu5Ac) is the most important because it is the precursor for the synthesis of other sialic acids and has a close relationship with human health. Neu5 Ac is used to synthesize several antiviral drugs and cure H1N1 and H5N1. In addition, Neu5 Ac has great nutritional value and can promote brain development in infants. Neu5 Ac is commonly produced through enzyme catalysis. Although enzyme catalysis has high output of Neu5 Ac, adding excessive pyruvate to drive the reaction equilibrium to Neu5 Ac synthesis, but it also leads to substrate waste, high costs, and considerable environmental pressure. This study attempts to develop a Neu5 Ac synthesis strategy without extracellular pyruvic acid. This strategy utilizes N-Acetyl glucosamine(GlcNAc) as the substrate to synthesize N-acetylmannosamine(ManNAc) by GlcNAc 2-epimerase(AGE). ManNAc and intracellular PEP then undergo an irreversible reaction catalyzed by N-acetylneuraminic acid synthetase(NeuB) to synthesize Neu5 Ac. The synthesis pathway was constructed in Escherichia coli and strengthened by metabolic and fermentation engineering. The developed synthesis strategy is summarized below:(1) First, the activity and enzymatic property of AGE and NeuB from different sources were investigated. Results demonstrated that pAGE and bAGE had similar stability, temperature preference, and pH preference. Their optimal pH and temperature were 7.0 and 50°C, respectively. These enzymes maintained high activity at 35–55°C, and higher than 90% of activity at 50°C. At 37°C in Tris-HCl(pH 7.0), pAGE and bAGE activities were 392.1 U?mg-1 and 2411.5 U?mg-1, respectively. The optimal pH of eNeu B and cNeuB was neutral and their optimal temperature was 45°C. They maintained more than 60% activity at 30–47.5°C. At 37°C in Tris-HCl(pH 7.0) eNeuB and cNeuB activities were 0.037 U?mg-1 and 6.380 U?mg-1 total proteins, respectively. However, cNeuB had poor stability and retained only 50% catalytic activity after 2 h. When coexpressed with pGro7, cNeuB activity was 17.8% higher compared with the control group. Inducing the expression of the GroEL/Gro ES composite inhibited the erratic agglomeration of proteins and accelerated protein folding, thus increasing the soluble expression of cNeuB in E. coli. Subsequent experiments constructed the Neu5 Ac synthesis pathway based on bAGE and cNeuB.(2) The Neu5 Ac synthesis pathway was constructed based on bAGE and cNeuB. The eliminating of host bacterial genes related to the Neu5 Ac degradation pathway and GlcNAc transmembrane transport pathway promoted Neu5 Ac synthesis. Given that cNeuB had relatively lower activity than bAGE, we explored strategies to increase cNeuB activity. The exchange sequence of the two genes and pDTrc-AB and pDTrc-BA coexpression carriers was investigated. The results showed that cNeuB in pDTrc-AB was highly active, and that Neu5 Ac did not accumulate when the Neu5 Ac degradation pathway in E. coli MG1655 was retained. E. coli SA-01 was constructed by eliminating the nanATEK gene clusters of the related enzymes in the Neu5 Ac degradation pathway via the ? Red recombinant system. pDTrc-AB and pDTrc-BA were transformed into E. coli SA-01, and the transformed bacteria was used to synthesize Neu5 Ac. The Neu5 Ac outputs were 2.61 and 2.03 g?L-1, respectively. The transformation of the GlcNAc transmembrane transport system not only prevented GlcNAc phosphorylation and saved intracellular PEP, but also increased intracellular GlcNAc concentration and enhanced Neu5 Ac synthesis. Therefore, Neu5 Ac production increased by 38.3% to 3.61 g?L-1.(3) In E. coli, Neu5 Ac synthesis was further improved as the intracellular PEP supply increases via the independent overexpression or co-overexpression of genes related to PEP synthesis. Genes related to PEP synthesis(pck and ppsA) were cloned from the E. coli MG1655 genome. The Duet series carrier induced the independent overexpression or co-overexpression of these genes, and the host E. coli was DE3 lysogenized. Neu5 Ac yield reached 5.80 g?L-1 and 7.09 g?L-1 for independent pck and ppsA overexpression, respectively. However, the transcriptional level of another gene was inhibited. When pck and ppsA were co-overexpressed, their transcriptional levels increased simultaneously and were positively correlated with the copy number of carriers. The optimal Neu5 Ac output was contributed by the PEP supply system, which was composed of the copy carrier, pCDF-pck-ppsA. The Neu5 Ac yield reached 8.63 g?L-1, which was higher than that of the original strain by 139%. The intracellular PEP supply was increased by optimizing pck and ppsA overexpression levels in E. coli, resulting in high Neu5 Ac production.(4) The nutrition for Neu5 Ac synthesis in the biotransformation stage, as well as the effect of surfactants on Neu5 Ac synthesis were investigated. In a culture medium with nitrogen sources, the host E. coli cells grow exponentially, but Neu5 Ac synthesis is inhibited. In nitrogen-free glucose culture medium, host bacterial biomass was slightly lower than the initial value, and the inhibition of Neu5 Ac synthesis was disrupted. Neu5 Ac yield was 8.61 g?L-1. The exponential growth of the host E. coli consumed PEP and competed with Neu5 Ac synthesis for intracellular PEP, and was therefore disadvantageous to Neu5 Ac synthesis. When glycerol was used as the carbon source, transmembrane transport was no longer dependent on PTS and thereby more PEP molecules were available for Neu5 Ac synthesis, Neu5 Ac yield increased to 10.43 g?L-1. Adding Triton X-100 to the transformation culture medium promotes Neu5 Ac synthesis by approximately 6.1%. Finally, Neu5 Ac biotransformation was implemented in a bioreactor. Neu5 Ac biotransformation was carried out under low phosphate concentration and the osmotic pressure of the reaction system. Meanwhile, the fermenter facilitated mass transfer condition for Neu5 Ac synthesis. At 65 h, the Neu5 Ac yield in the fermenter reached 16.02 g?L-1, which was 53.6% higher than that in a shake flask under same conditions. Therefore, the constructed Neu5 Ac synthesis pathway attained highly efficient Neu5 Ac synthesis.更多还原