Directed evolution of toluene dioxygenase from Pseudomonas putida for improved selectivity toward cis-indandiol during indene bioconversion.
Directed evolution of toluene dioxygenase from Pseudomonas putida for improved selectivity toward cis-indandiol during indene bioconversion.
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从恶臭假单胞菌中定向进化甲苯双加氧酶，以提高茚生物转化过程中对顺式茚满二醇的选择性。
DOI：
10.1006/mben.2000.0162
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发表时间：
2000
影响因子：
8.4
通讯作者：
C. Lee
中科院分区：
文献类型：
--
作者：
N. Zhang;B. G. Stewart;J. Moore;R. Greasham;D. Robinson;B. Buckland;C. Lee
恶臭假单胞菌F1的甲苯双加氧酶（TDO）将茚转化为顺式茚二醇（外消旋）、1-茚醇和1-茚酮的混合物。所需产物顺式-（1 S，2 R）-茚二醇是化学合成硫酸茚地那韦（Crixivan）的潜在关键中间体，硫酸茚地那韦是默克公司用于治疗艾滋病的HIV-1蛋白酶抑制剂。为了减少在茚生物转化过程中形成的不期望的副产物1-茚醇和1-茚酮，在大肠杆菌中表达的重组TDO使用易错聚合酶链反应（epPCR）方法通过定向进化进化而进化。开发了高通量荧光和分光光度测定法，用于在96孔格式中快速筛选突变体文库。鉴定了具有减少的1-茚醇副产物形成的突变体，并且通过HPLC确认了所选突变体的个体茚生物转化产物谱。通过分析基因的核苷酸序列来鉴定突变酶的氨基酸序列的变化。使用具有来自每个文库的最期望的产物谱的突变体进行每个后续轮的诱变，所述最期望的产物谱定义为最低的1-茚醇浓度和最高的顺式-（1 S，2 R）-茚二醇对映体过量。在三轮诱变和筛选后，突变体1C 4 -3G被鉴定为与野生型相比1-茚醇形成减少三倍（总产物的20%对60%），并且产物（顺式-茚二醇）产率增加40%。
Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S,2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S,2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.
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一种酶的定向进化和功能克隆的方法。
DOI：
10.1073/pnas.95.18.10523
发表时间：
1998
影响因子：
11.1
作者：
Pedersen,H;Hölder,S;Sutherlin,DP;Schwitter,U;King,DS;Schultz,PG
通讯作者：
Schultz,PG
影响因子：
5.7
作者：
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通讯作者：
Ramaswamy, S
影响因子：
17.3
作者：
Kuchner, O;Arnold, FH
通讯作者：
Arnold, FH
影响因子：
5.6
作者：
Moore, JC;Jin, HM;Arnold, FH
通讯作者：
Arnold, FH
铜绿假单胞菌 lasA 基因的核苷酸序列和在大肠杆菌中的表达。
影响因子：
3.2
作者：
Schad,PA;Iglewski,BH
通讯作者：
Iglewski,BH