A toolbox approach for the rapid evaluation of multi-step enzymatic syntheses comprising a 'mix and match' E. coli expression system with microscale experimentation
A toolbox approach for the rapid evaluation of multi-step enzymatic syntheses comprising a 'mix and match' E. coli expression system with microscale experimentation
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DOI：
10.3109/10242422.2011.609589
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发表时间：
2011-10-01
影响因子：
1.8
通讯作者：
Lye, G. J.
中科院分区：
文献类型：
--
作者：
Rios-Solis, L.;Halim, M.;Lye, G. J.
这项工作描述了一个实验“工具箱”的快速评估和优化的多步酶促合成，包括一个“混合和匹配”E。基于大肠杆菌的表达系统和自动化微孔规模实验。该方法说明了从头设计的途径，用于合成光学纯的氨基醇，使用酶转酮醇酶（TK）和转氨酶（TAm）催化不对称碳-碳键的形成和选择性手性胺基团加成分别。急诊大肠杆菌表达系统，基于两个兼容的质粒，使对酶从以前的工程和克隆的TK和TAm库进行评估的顺序转换不同的初始底物。这是由微孔实验，使不同的生物催化剂形式，使用不同的胺供体和底物进料策略的有效调查补充。使用该实验“工具箱”，设计并执行非对映异构体（2S，3S）-2-氨基戊烷-1，3-二醇（APD）和（2S，3R）-2-氨基-1，3，4-丁三醇（ABT）的一锅法合成，其在25小时内得到APD的最终产物产率为90%mol/mol，ABT的最终产物产率为87%mol/mol（相对于初始TK底物）。对于APD的合成，大肠杆菌TK突变体D469 E与来自紫色色杆菌2025的TAm配对，而对于ABT的合成，野生型大肠杆菌TK突变体D469 E与来自紫色色杆菌2025的TAm配对。大肠杆菌TK表现出最高的比活性，ee（对映体过量）> 95%。对于这两种反应，全细胞形式的TK-TAm生物催化剂比细胞裂解物表现更好，而异丙胺（IPA）是比甲基苄胺（MBA）更优选的胺供体，因为避免了与初始TK底物的副反应。TK和TAm酶的可用文库和微孔数据的可扩展性表明，该“工具箱”为化学和制药领域的早期生物转化过程设计提供了有效的方法。
This work describes an experimental 'toolbox' for the rapid evaluation and optimisation of multi-step enzymatic syntheses comprising a 'mix and match' E. coli-based expression system and automated microwell scale experimentation. The approach is illustrated with a de novo designed pathway for the synthesis of optically pure amino alcohols using the enzymes transketolase (TK) and transaminase (TAm) to catalyze asymmetric carbon-carbon bond formation and selective chiral amine group addition respectively. The E. coli expression system, based on two compatible plasmids, enables pairs of enzymes from previously engineered and cloned TK and TAm libraries to be evaluated for the sequential conversion of different initial substrates. This is complemented by the microwell experimentation which enables efficient investigation of different biocatalyst forms, use of different amine donors and substrate feeding strategies. Using this experimental 'toolbox', one-pot syntheses of the diastereoisomers (2S,3S)-2-aminopentane-1,3-diol (APD) and (2S,3R)-2-amino-1,3,4-butanetriol (ABT) were designed and performed, which gave final product yields of 90% mol/mol for APD and 87% mol/mol for ABT (relative to the initial TK substrates) within 25 hours. For the synthesis of APD, the E coli TK mutant D469E was paired with the TAm from Chromobacterium violaceum 2025 while for ABT synthesis the wild-type E. coli TK exhibited the highest specific activity and ee(enantiomeric excess) of >95%. For both reactions, whole-cell forms of the TK-TAm biocatalyst performed better than cell lysates while isopropylamine (IPA) was a preferable amine donor than methylbenzylamine (MBA) since side reactions with the initial TK substrates were avoided. The available libraries of TK and TAm enzymes and scalable nature of the microwell data suggest this 'toolbox' provides an efficient approach to early stage bioconversion process design in the chemical and pharmaceutical sectors.