重组工程介导的神经氨酸醛缩酶定向进化

N-acetyl-D-neuraminic acid is the crucial precursor of anti-flu medicine Zanamivir. Enzymatic catalysis of N-acetyl-D-neuraminic acid is achieved via enzymatic catalysis process, with first the epimerization of N-acetyl-D-glucosamine to N-acetyl-D-mannosamine by the epimerase; and the second step is the aldolase catalyzed condensation between N-acetyl-D-mannosamine and pyruvate. Because of the low activity, reversal reaction and substrates inhibition of the aldolase, the process suffers the shortcomings of long reaction time and relatively low yield. Recombineering is a DNA cloning and modification biotechnology based on the DNA homologous recombination. This study aims to establish a recombinerring-mediated directed evolution method. The experimental strategy is as follows. Firstly, the DNA mutant library generated through EP-PCR is used to electroporate the recombinase expressing strain harboring the target gene expression vector. After the recombinase catalyzed recombination, expression protein mutant library existing in the expression host is obtained, thus avoiding the cloning and transformation steps of the classic methods. The convenience, mutation coverage as well as the screening efficiency will be evaluated through the comparison with DNA shuffling, one of the most often used directed evolution methods. The obtained aldolase mutant with the highest catalytic activity will be used to establish a more efficient enzymatic catalysis route; structure-activity relationship of aldolase will be generated through the analysis of the correlation between amino acid sequence and the catalytic activity, which will set the stage for rational aldolase design.

N-乙酰-D-神经氨酸是抗流感药物扎那米韦的关键中间体。神经氨酸由酶法催化而得，即先由异构酶催化N-乙酰-D-葡萄糖胺得到N-乙酰-D-甘露糖胺，再以醛缩酶催化后者与丙酮酸进行缩合反应。由于醛缩酶的低活性、缩合反应的可逆性以及底物抑制等缺点，目前的反应路线有着反应时间长和收率较低等不足。重组工程是一种利用DNA之间同源重组而实现DNA克隆和修饰的生物技术。本研究拟建立重组工程介导的酶的定向进化方法，即将通过易错PCR所得到的DNA突变体电转化目的基因表达菌株，在重组酶催化下直接获得克隆在表达载体上的突变体库，这就避免了常规方法所需的克隆和转化步骤。本方法将与最为常用的定向进化方法之一的DNA shuffling相比较以评价其简便性、突变体库覆盖率和筛选效率。所获得的高活性醛缩酶突变体将用于建立更加有效的酶法催化新路线，突变体库的序列分析和催化活性之间的构效关系将为醛缩酶的理性优化奠定基础。

N-乙酰-D-神经氨酸是抗流感药物扎那米韦的关键中间体。酶法催化合成N-乙酰-D-神经氨酸涉及异构酶和醛缩酶。高催化特性的醛缩酶将对改善催化工艺用重要意义。本研究首先建立了一种基于DNA重组的重组工程介导的基因突变方法。点突变的效率高达83%，点饱和突变的效率达62%，达到了方法学的要求。其次表达异构酶和表达醛缩酶的两个全细胞催化合成神经氨酸的摩尔转化率高达75%。将异构酶基因和醛缩酶基因分别置于T7强启动子之下，并串联克隆在一个表达载体上，N-乙酰-D-神经氨酸的摩尔转化率为45.2%。我们将异构酶基因和醛缩酶基因以及氯霉素抗性基因一起通过重组工程法整合至异源表达宿主菌BL21(DE3)基因组后，获得基因工程菌株的摩尔转化率为35.8%。随后进行了一系列的基于重组工程的基因改造，即降解N-乙酰-D-葡萄糖胺相关基因的敲除和合成N-乙酰-D-葡萄糖胺相关基因的敲入和降解丙酮酸的相关的糖类基因的敲除。将目前所得菌株进行全细胞水平的催化合成，摩尔转化率为68.3%。所得工艺简化的菌株将用于进一步的优化和中试研究。

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