Directed evolution of an industrial biocatalyst: 2-deoxy-D-ribose 5-phosphate aldolase
Directed evolution of an industrial biocatalyst: 2-deoxy-D-ribose 5-phosphate aldolase
复制标题
DOI：
10.1002/biot.200600020
复制
发表时间：
2006-05-01
影响因子：
4.7
通讯作者：
Mink, Daniel
中科院分区：
文献类型：
--
作者：
Jennewein, Stefan;Schurmann, Martin;Mink, Daniel
醛缩酶正在成为工业合成手性分子的强大且经济高效的工具。它们催化对映选择性碳-碳键形成，在温和的反应条件下产生最多两个手性中心。尽管醛缩酶用途广泛，但底物范围窄，合成条件下的酶失活是醛缩酶大规模应用的主要障碍。本研究利用定向进化优化了大肠杆菌2脱氧- d -核糖5-磷酸醛缩酶（DERA）作为工业合成（3R,5S）6-氯-2,4,6-三脱氧六吡喃苷的生物催化剂。这种伐他汀类药物如立普妥（阿托伐他汀）的多用途手性前体是由DERA在氯乙醛和两种乙醛等效物的串联醛反应中合成的。然而，大肠杆菌DERA对氯乙醛的亲和力较低，并且在对生物催化有用的醛浓度下迅速失活。使用高通量筛选氯乙醛抗性和更高的生产力，已经确定了几个改进的变体。通过组合最有利的突变，我们获得了在工业相关条件下，与野生型DERA相比，(3R,5S)-6-氯-2,4,6-三脱氧六吡喃苷合成的10倍改进变体。
Aldolases are emerging as powerful and cost efficient tools for the industrial synthesis of chiral Received 10 March 2006 molecules. They catalyze enantioselective carbon-carbon bond formations, generating up to two Revised 4 April 2006 chiral centers under mild reaction conditions. Despite their versatility, narrow substrate ranges Accepted 5 April 2006 and enzyme inactivation under synthesis conditions represented major obstacles for large-scale applications of aldolases. In this study we applied directed evolution to optimize Escherichia coli 2deoxy-D-ribose 5-phosphate aldolase (DERA) as biocatalyst for the industrial synthesis of (3R,5S)6-chloro-2,4,6-trideoxyhexapyranoside. This versatile chiral precursor for vastatin drugs like Lipitor (atorvastatin) is synthesized by DERA in a tandem-aldol reaction from chloroacetalclehyde and two acetaldehyde equivalents. However, E coli DERA shows low affinity to chloroacetalclehyde and is rapidly inactivated at aldehyde concentrations useful for biocatalysis. Using high-throughput screenings for chloroacetaldehyde resistance and for higher productivity, several improved variants have been identified. By combination of the most beneficial mutations we obtained a tenfold improved variant compared to wild-type DERA with regard to (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside synthesis, under industrially relevant conditions.