赖氨酸脱羧酶高效表达、分子定向进化及其催化合成戊二胺的反应过程特性

发布时间：2018-05-30 21:25

本文选题：产酸克雷伯氏菌 + 赖氨酸脱羧酶　；参考：《江南大学》2016年博士论文

【摘要】：戊二胺是一种重要的平台化合物,可以合成高附加值、性能优异的新型聚氨酯、新型聚酰胺等材料。用微生物发酵法生成戊二胺代谢途径复杂、产率低难以工业化。酶法制备戊二胺过程简单、不需要复杂的代谢调控、没有副产物的累积、避免了戊二胺对微生物细胞的毒害作用,但是赖氨酸脱羧酶的酶活低、碱性环境下p H稳定性差,是工业化开发过程中存在的技术瓶颈。目前大多研究者对多物种赖氨酸脱羧酶的生物信息学分析仍不够充分,仅对目的基因表达的局部环节进行考察和研究。本研究选择在自然界可以过量积累戊二胺的产酸克雷伯氏菌赖氨酸脱羧酶作为研究对象,运用分子生物学手段对目的基因表达的转录过程和翻译过程进行系统调控,并对酶分子的结构基因进行定向改造:包括顺式作用元件对基因表达的影响,特别是对基因转录起始的调控、翻译过程对表达的影响如密码子偏爱性、SD序列等调控元件的优化,通过分子定向进化构造新的酶蛋白。通过这些手段提高赖氨酸脱羧酶的表达量、活性以及在碱性环境下的稳定性,并对构造的新型赖氨酸脱羧酶催化合成戊二胺的过程进行研究。大幅度提高了赖氨酸脱羧酶催化合成戊二胺的效率,为生物法戊二胺的产业化奠定基础,主要研究内容和结果如下:(1)首次对产酸克雷伯氏菌赖氨酸脱羧酶进行了异源表达,并对其基因的密码子和启动子进行了优化,显著提高了赖氨酸脱羧酶的表达量。对产酸克雷伯氏菌赖氨酸脱羧酶基因ldc进行扩增,构建了p UC18-KOldc质粒,转入Escherichia coli K12 MG1655构建了重组菌E.coli LN18,实现了产酸克雷伯氏菌赖氨酸脱羧酶的异源表达。密码子优化将产酸克雷伯氏菌赖氨酸脱羧酶基因的GC含量从54%降低到49%,基因序列与野生型的赖氨酸脱羧酶基因序列相比有81.9%的相似性。密码子优化的菌株E.coli LN20发酵液中戊二胺的含量达到6.6 g/L发酵液,比出发菌株E.coli LN18提高了50%,比过表达大肠杆菌赖氨酸脱羧酶的E.coli LN35相比提高了16%,这说明密码子优化显著提高了产酸克雷伯氏菌赖氨酸脱羧酶在大肠杆菌的表达效率,也说明产酸克雷伯氏菌赖氨酸脱羧酶的活力比大肠杆菌赖氨酸脱羧酶活力高。构建了不同启动子的重组菌株,其中E.coli LN24合成的戊二胺浓度最高达到7.6 g/L发酵液,说明Ptac启动子更有利于产酸克雷伯氏菌赖氨酸脱羧酶的表达。(2)基于高通量筛选的产酸克雷伯氏菌赖氨酸脱羧酶基因的RBS(核糖体结合位点)序列优化、分子定向进化和相关生物信息学统计分析。建立了突变株高通量筛选方法,利用quickchange PCR对RBS的序列进行突变,并对1000株突变株进行筛选得到合成戊二胺水平最高的菌株E.coli LN1103,是E.coli LN18合成戊二胺水平的2.3倍,通过RBS强度计算器分析和可溶性蛋白表达量分析,说明核糖体结合位点序列优化可以增强赖氨酸脱羧酶m RNA的翻译效率。通过易错PCR对产酸克雷伯氏菌赖氨酸脱羧酶进行随机突变、进行分子定向进化。从1000株突变株中筛选得到5株合成戊二胺水平明显高于对照的重组菌。最优菌株E.coli LN3014合成的戊二胺水平达到11.2 g/L发酵液,是E.coli LN18合成戊二胺水平的2.5倍。(3)经过分子定向进化的赖氨酸脱羧酶具备了独特的酶学特性,在碱性条件下保持高效催化性和稳定性。构造的赖氨酸脱羧酶(E.coli LN3014)在p H值8.0下保持36h,相对酶活力保留85%,在碱性环境下表现出很高的稳定性。而大肠杆菌的赖氨酸脱羧酶Cad A在p H 8.0没有活力。当p H值从6.0提高到8.0,野生型的赖氨酸脱羧酶(E.coli LN18)的最大反应速度从1.67 mmol/min下降到0.65 mmol/min,下降幅度为60%;而经过分子定向进化的赖氨酸脱羧酶(E.coli LN3014)在p H 8.0时的最大反应速度和p H6.0时相比,仅下降了7.0%。从酶的米氏常数来看,赖氨酸脱羧酶的点突变并不影响蛋白与底物L-赖氨酸的结合。说明分子定向进化改造后的E.coli LN18赖氨酸脱羧酶在碱性条件下具备高效催化性和稳定性,可建立酶法合成戊二胺的新工艺,使之具备工业实施可行性。(4)对重组菌E.coli LN3014在摇瓶和10 L罐规模进行了发酵产酶特性研究。重组菌产酶过程属于生长关联型,随着微生物的生长,酶活不断提高。通过正交实验优化了摇瓶发酵条件,E.coli LN3014发酵液的赖氨酸脱羧酶活为128 U/g发酵液,比未优化时提高了16%。进一步考察了重组菌E.coli LN3014在10 L罐的分批发酵和补料分批发酵工艺。补料分批发酵工艺采用p H-Stat的控制策略,流加葡萄糖和氨水,E.coli LN3014的菌体密度OD600可以达到82,酶活力达到240 U/g发酵液,是摇瓶发酵结果的1.9倍,实现了E.coli LN3014的高密度培养,并显著高于文献报道的酶活。(5)对重组菌细胞催化生成戊二胺的过程参数进行了考察和优化,建立了不调节p H值就能高效合成戊二胺的反应体系。产物戊二胺积累过程中导致的p H上升使酶失活,是酶法制备戊二胺的技术瓶颈之一。对反应体系中的底物浓度、细胞浓度、辅酶添加量等影响因素进行了系统考察,经过优化,戊二胺生成速率为887mmol/(g-cell·h)。所用的催化剂和催化时间大大节约,催化效率有了显著提高,而且整个反应过程中不调节p H,减少了反应体系的无机盐含量,对后续的纯化和产品质量都有很好的帮助,克服了酶法合成戊二胺催化效率低、反应过程中p H升高导致赖氨酸脱羧酶易失活的技术瓶颈。选用聚乙烯醇包埋法固定化E.coli LN3014细胞,固定化细胞可以重复利用30次进行催化反应,底物到产物的转化率没有明显下降,固定化细胞赖氨酸脱羧酶最适p H向碱性区域发生了迁移,在4℃下,固定化细胞赖氨酸脱羧酶活半衰期为30天,是游离细胞的2倍。
[Abstract]:Amyl two amine is an important platform compound, which can synthesize high added value, new polyurethane with excellent performance and a new type of polyamides. The metabolic pathway of amyl two amine is complicated by microbial fermentation, and the yield is low and difficult to industrialize. The process of preparing amyl two amine by enzyme method is simple, no need for complex metabolic regulation, no accumulation of by-products, and avoidance of the accumulation of by-products. The toxic effect of amyl two amine on microbial cells is avoided, but the enzyme activity of lysine decarboxylase is low and the stability of P H in alkaline environment is poor. It is a technical bottleneck in the process of industrial development. At present, most researchers are still not sufficient for the bioinformatics analysis of the multi species lysine decarboxylase, only the local link of the target gene expression is entered. In this study, the study selected Klebsiella acid decarboxylase, which can accumulate amyl amyl amines in nature, as the research object, systematically regulates the transcriptional process and translation process of the target gene expression by molecular biological means, and redirects the structure genes of the enzyme molecules: including CIS action. The effect of components on gene expression, especially the regulation of gene transcription initiation, the effect of translation process on expression, such as codon bias, SD sequence and other regulatory elements, to construct new enzyme proteins through molecular directed evolution. Through these methods, the expression of lysine decarboxylase, activity and stability in alkaline environment are improved. The process of synthesis of amyl two amine by new lysine decarboxylase was studied. The efficiency of lysine decarboxylase catalyzed synthesis of amyl amyl two amine was greatly improved, and the basis for the industrialization of amyl two amines was laid. The main contents and results were as follows: (1) a heterologous table was first made to the lysine decarboxylase of Klebsiella acid. The codon and promoter of the gene were optimized and the expression of lysine decarboxylase was significantly improved. The P UC18-KOldc plasmid was constructed by amplification of the lysine decarboxylase gene LDC of Klebsiella acid, and Escherichia coli K12 MG1655 was transferred to the recombinant bacteria E.coli LN18 to realize Klebsiella lysine production. The GC content of Klebsiella lysine decarboxylase gene was reduced from 54% to 49% by codon optimization, and the gene sequence was similar to that of the wild type lysine decarboxylase gene sequence. The content of amyl two amine in the fermentation broth of the codon E.coli LN20 reached 6.6 g/L fermentation broth. The strain E.coli LN18 increased by 50%, increased by 16% compared to the E.coli LN35 that overexpressed the lysine decarboxylase, which indicated that codon optimization significantly improved the expression efficiency of Klebsiella lysine decarboxylase in Escherichia coli, and also indicated that Klebsiella lysine decarboxylase was more active than lysine in Escherichia coli. The carboxyenzyme activity was high. The recombinant strains of different promoters were constructed, in which the concentration of amyl two amines synthesized by E.coli LN24 reached up to 7.6 g/L fermentation broth, indicating that Ptac promoter was more beneficial to the expression of Klebsiella lysine decarboxylase. (2) RBS (ribosome junction) based on high throughput screening of Klebsiella lysine decarboxylase gene. Sequence optimization, molecular directed evolution and related bioinformatics analysis. A high throughput screening method for mutant strains was established. The sequence of RBS was mutated with quickchange PCR, and 1000 strains of mutant strains were screened to get the highest level of amyl two amine, E.coli LN1103, which was 2.3 of the level of the synthesis of amyl two amine in E.coli LN18. RBS strength calculator analysis and analysis of soluble protein expression showed that the optimization of ribosome binding site sequence could enhance the translation efficiency of lysine decarboxylase m RNA. By random mutation of Klebsiella acid decarboxylase by error PCR, molecular directed evolution was carried out. 1000 strains of mutant strains were screened from 5 strains. The level of synthetic amyl two amine was significantly higher than that of the control recombinant. The optimal strain E.coli LN3014 synthesis of amyl amyl two amine reached 11.2 g/L fermented liquid, which was 2.5 times the level of E.coli LN18 synthesis amyl two amine. (3) the lysine decarboxylase after molecular directed evolution has a unique characteristic of enzymology and maintains high catalytic activity and stability under alkaline conditions. The lysine decarboxylase (E.coli LN3014) maintained 36h under P H value 8, the relative enzyme activity remained 85%, and was highly stable in the alkaline environment. The lysine decarboxylase Cad A of the Escherichia coli was not active in P H 8. When p H was increased from 6 to 8, the maximum reaction rate of the wild type lysine decarboxylase (E.coli) From 1.67 mmol/min to 0.65 mmol/min, the decrease was 60%; while the maximum reaction rate of lysine decarboxylase (E.coli LN3014) by molecular directed evolution at P H 8 was compared with P H6.0, only the decrease of 7.0%. from the enzyme's Michaelis constant, the point mutation of lysine decarboxylase did not affect the binding of protein and substrate L- lysine. It is indicated that E.coli LN18 lysine decarboxylase has high catalytic activity and stability under basic conditions. It can establish a new process of enzymatic synthesis of amyl amyl amyl amine, which has the feasibility of industrial implementation. (4) the enzyme production of recombinant strain E.coli LN3014 in the scale of shake flask and 10 L tank was studied. The process belongs to the growth correlation type. With the growth of microorganism, the enzyme activity is continuously improved. The conditions of the shake flask fermentation are optimized by orthogonal experiment. The lysine decarboxylase of E.coli LN3014 fermentation liquid is 128 U/g fermented liquid, and 16%. is further improved by the optimization of the batch fermentation of the recombinant strain E.coli LN3014 in the 10 L cans and the batch batch fermentation. The P H-Stat control strategy was adopted in the feeding batch fermentation process, with glucose and ammonia water added, the density OD600 of E.coli LN3014 could reach 82, the enzyme activity reached 240 U/g fermentation liquid, which was 1.9 times the result of shake flask fermentation, which realized the high density culture of E.coli LN3014, and was significantly higher than the reported enzyme activity. (5) the recombinant bacteria cells were catalyzed. The process parameters for the formation of amyl two amine were investigated and optimized. The reaction system of amyl two amine could be synthesized without adjusting the P H value. The increase of P H caused by the accumulation of amyl amyl amine made the enzyme inactivated, which was one of the technical bottlenecks in the preparation of amyl amines by enzyme method. The concentration of substrate, cell concentration, and the addition of coenzyme in the reaction system were taken as a bottleneck. The reaction factor was systematically investigated. After optimization, the formation rate of amyl amyl amine was 887mmol/ (G-cell. H). The catalyst and the catalytic time were greatly saved, the catalytic efficiency was greatly improved, and the P H was not regulated in the whole reaction process. The inorganic salt content of the reaction system was reduced, and the subsequent purification and product quality were well helped. The catalytic efficiency of the enzyme synthesis of amyl two amine is low and the increase of P H in the reaction process leads to the technical bottleneck of lysine decarboxylase. Immobilized E.coli LN3014 cells with polyvinyl alcohol and immobilized cells can reuse 30 times for catalytic reaction. The conversion rate of substrate to the product is not obviously decreased, and the immobilized cell Reys on the immobilized cells. The optimum P H was migrated to the alkaline region. At 4, the half-life of lysine decarboxylase of immobilized cells was 30 days, which was 2 times of that of free cells.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TQ925;Q55

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