【作者】 姜世民；

【导师】 姜卫红；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 微生物学， 2007， 博士

【摘要】 大肠杆菌重组表达系统的主要问题之一,是过量表达的目的蛋白常常以不可溶的形式存在,因而不具有生物学活性。这影响了许多蛋白的性质研究和工业应用,因此需要建立新型有效的改造方法。本论文以重要工业用酶D-氨甲酰水解酶（D-carbamoylase, DCase）为研究对象,通过共表达和基因改造技术提高其在大肠杆菌中的可溶性表达,并对突变体进行结构与功能研究。D-氨甲酰水解酶是β-内酰胺类抗生素中间体D-对羟基苯甘氨酸制备的关键酶。我们克隆了一个来源于皮氏伯克霍尔德氏菌（Burkholderia pickettii）的D-氨甲酰水解酶,但在大肠杆菌过量表达后,80%左右目的蛋白以包涵体的形式存在,所以基因工程菌的酶活力比较低,成为工业应用瓶颈。为提高D-氨甲酰水解酶在大肠杆菌中的可溶性表达,首先采用了分子伴侣共表达技术。将三种分子伴侣DnaK-DnaJ-GrpE（DnaKJE）、GroEL-GroES（GroELS）和trigger factor（TF）分别与D-氨甲酰水解酶共表达。结果显示,在分子伴侣GroESL的辅助下,D-氨甲酰水解酶在大肠杆菌表达系统中的可溶性明显增加,相比于野生型,酶活提高了3倍。然而在实际应用中会涉及分子伴侣诱导物可使用性问题,因此我们又尝试了基因定向进化的方法。定向进化技术是在实验室里模拟自然进化的过程,通过对编码蛋白质的基因进行随机突变,从而定向筛选出特定突变体的有效方法。我们首先使用易错PCR和DNA Shuffling操作,对DCase基因进行随机突变,再通过一种蛋白质可溶性结构互补监测系统将正突变检出,筛选获得了一系列可溶性表达增加的突变体。通过详细分析这些突变体的氨基酸序列,并结合定点突变鉴定这些突变位点,揭示了与DCase的可溶性表达密切相关的三个重要氨基酸残基A¹⁸、Y³⁰和K³⁴。同源四聚体和单体的结构模建结果显示,A¹⁸位于β-折叠片和α-螺旋之间的一个转角上,而Y³⁰和K³⁴位于同一个α-螺旋,且三个突变点都位于蛋白分子的表面,远离蛋白的催化和活性中心。进一步的定点替换突变分析表明,这三个氨基酸残基的电荷改变和（或）亲水性的变化是D-氨甲酰水解酶突变体在E. coli中可溶性表达增加的两个关键因素。突变酶A18T和Y30N可溶性的增加主要由于突变位点氨基酸残基亲水性的提高;而K34E突变酶可溶性的提高可能主要归功于蛋白负电荷的增加。在此基础上,又对这三个位点进行了组合突变。SDS-PAGE结果表明,其中一个命名为DCase-M3的三联突变体（A18T/Y30N/K34E）在E. coli过量表达时,80%以上的目的蛋白成为可溶状态,很好地体现了有效突变的叠加作用。此外,对纯化的野生型DCase和突变型DCase-M3进行了酶学性质测定和对比,二者在动力学常数、热力学稳定性、最适温度、最适pH以及热稳定性等方面相似,可见在突变提高可溶性表达的同时并没有改变D-氨甲酰水解酶原有的特性。在上述研究的基础上,为了进一步弄清所获得的可溶性显著提高的D-氨甲酰水解酶突变体在工业发酵中应用的可行性,我们进行了D-氨甲酰水解酶改造前后发酵活力的比较实验。首先对野生型D-氨甲酰水解酶和单点突变D-氨甲酰水解酶K34E进行多种组合的摇瓶发酵实验,包括使用三种不同的培养基（LB、TB和改良的工业培养基）和三种不同的诱导温度（22℃、30℃和37℃）。结果显示,在所使用的培养条件下,单点突变D-氨甲酰水解酶K34E的酶活都不同程度地优于野生型。同时我们综合考虑D-对羟基苯甘氨酸制备的两个关键酶:D-乙内酰脲酶（DHase）和D-氨甲酰水解酶（DCase）,通过引入突变DCas（eK34E）,串联构建了一菌两酶的大肠杆菌基因工程菌株E.coli BL21（DE3）/pCHWT（DHase/ DCase）和E.coli BL21（DE3）/pCHMU（DHase/ K34E）。发酵结果显示,突变工程菌E. coli BL21（DE3）/pCHMU表现出的DHase/DCase的综合催化效率明显高于E. coli BL21（DE3）/pCHWT,提高近一倍。此外,我们还对高可溶性表达的三突变酶DCase-M3进行了5升发酵罐发酵研究,结果说明,在放大发酵培养的条件下,突变酶DCase-M3与野生型DCase相比活力提高了近7倍,表现出很大的工业应用潜力。更多还原

【Abstract】 One of the greatest bottlenecks in producing recombinant proteins in Escherichia coli is that the target proteins, when expressed in a high amount, are often present in an insoluble form without detectable activities. This affects research on characterization of the target proteins and their application. D-carbamoylase （DCase）, catalyzing a rate-limiting step in a two-step reaction system for producing D-p-hydroxyphenylglycine （D-HPG）, is an important enzyme in semi-synthesis ofβ-lactam antibiotics in industry. We used a co-expression system with molecular chaperone and directed evolution techniques to improve soluble expression of DCase in Escherichia coli, followed by investigatng the relationship between structure and function of the enzyme.A DCase gene was cloned from Burkholderia pickettii, and over-expressed in E. coli as a recombinant protein. The resultant DCase activity was, however, very low because nearly 80% of recombinant DCase was partitioned into insoluble aggregates. To facilitate the expression of soluble and active DCase, three different of molecular chaperones, DnaK-DnaJ-GrpE （DnaKJE）, GroEL-GroES （GroELS） and trigger factor （TF）, were co-expressed with the DCase, respectively. The target protein aggregate from DCase overproduction were alleviated with the aid of GroELS, resulting in a three-fold increase in DCase enzyme activity compared to the wild-type strain. However, owing to problems related to application of expression inducer in above strategy, alternative method is used for directed evolution of the protein. Directed evolution incorporates Darwinian principles of mutation and selection into experimental strategies for improving biocatalyst or enzyme properties in the laboratory. In this study, error-prone PCR and DNA shuffling techniques are applied to randomly mutate its encoding sequence, followed by an efficient screening based on structural complementation. Several mutants of DCase with reduced aggregation are isolated. Solubility tests of these mutants and several other mutants generated by site-directed mutagenesis indicate that three amino acid residues of DCase （A18, Y30 and K34） are related to the DCase protein solubility in DCase. In the structure model of the DCase-M3 homotetramer and the DCase-M3 monomer, the amino acid of the 18th position is situated at a turn betweenβ-sheet andα-helix, and amino acids of both the 30th and 34th positions were are located inα-helix. The three residues are distributed on the surface of the target protein, and are located far from the catalytic sites (Glu⁴⁷, Lys¹²⁷ and Cys¹⁷²). In silico structural modeling analyses further suggested that hydrophilicity and/or negative charge at these three residues may be responsible for the increased solubility of DCase proteins produced in E. coli. Substitutions of A¹⁸ and Y³⁰ with selected hydrophilic amino acids revealed that solubility of A18T and Y30N muteins was clearly improved. Additional replacement at the K³⁴ position showed that increased negative charge of K34E mutein led to improvement of solubility of the mutated proteins. Based on the information, multiple engineering-designed mutants were constructed by site-directed mutagenesis; among them, a triple mutant A18T/Y30N/K34E （named as DCase-M3） was successfully over-expressed in E. coli with up to 80% of DCase-M3 proteins in soluble form. These results indicate that more soluble proteins can be obtained by combination of mutations. DCase-M3 was purified to homogeneity and a comparative analysis with WT DCase showed that DCase-M3 enzyme is similar to the native DCase in its biochemical properties, including kinetic and thermodynamic parameters, optimal temperature, optimal pH, and thermo-stability at 60℃and 65℃.Based on the data obtained from laboratory studies, the WT and the mutant DCase were fermented and the DCase activity was dertermined for industrial use. Firstly, we fermented the WT DCase and a single-point mutant DCase （K34E）, using three different culture media （LB, TB and improved industrial culture medium） and three inducing temperatures （22℃、30℃ and 37℃）, respectively. The results showed that K34E mutein had a higher activity than that of WT Dcase in each culture conditions. Considering that two enzymes （D-hydantoinase and D-carbamoylase） are involved in D-HPG production, it was interesting to see their combinational effect after mutation, DHase and mutant DCase （K34E） genes were cloned in Escherichia coli in rank to form E. coli BL21 （DE3）/pCHWT （DHase/ DCase） and E. coli BL21 （DE3） /pCHMU （DHase/ K34E）. The fermentation results showed that D-HPG production efficiency of E. coli BL21（DE3）/pCHMU was twice as much as that of E. coli BL21（DE3）/pCHWT. In addition, we enlarged the fermentation scale to 5L for WT DCase and DCase-M3 constructs. It showed that activity of DCase-M3 mutein was increased by seven-fold incomparison with that of WT DCase, indicating that DCase-M3 mutein has a great potential in industrial application.更多还原