New approach to multiply deuterated isoprenoids using triply engineered Escherichia coli and its potential as a tool for mechanistic enzymology
New approach to multiply deuterated isoprenoids using triply engineered Escherichia coli and its potential as a tool for mechanistic enzymology
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DOI：
10.1021/ja003390y
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发表时间：
2001-02-14
影响因子：
15
通讯作者：
Seto, H
中科院分区：
文献类型：
--
作者：
Kakinuma, K;Dekishima, Y;Seto, H
类异戊二烯在自然界具有多种化学性质，在生物体中普遍存在，在生物过程中起着至关重要的作用。这些类异戊二烯的生物合成通过甲羟戊酸和非甲羟戊酸途径进行，这取决于生物体和细胞器，二磷酸异戊烯酯（IPP）是这两种情况下的关键中间体。因此，代谢工程和这些途径的控制应该为研究涉及的有趣的化学和生物化学以及开发选择性化疗药物和类异戊二烯相关材料提供新的机会。本文介绍了一种制备玉米黄质等高氘化类异戊二烯的实用新方法，及其在大肠杆菌代谢工程基础上分析类异戊二烯生物合成机理的潜力。氘标记化合物在生物化学和生物有机以及物理化学研究中具有不可估量的价值。非甲羟戊酸途径，而不是甲羟戊酸途径，现在已知在大肠杆菌中起作用。最近，Seto等人3,4和Boronat等人5描述了双工程大肠杆菌的构建，他们破坏了参与非甲羟戊酸途径的某种酶的基因，并引入了甲羟戊酸途径中关键酶的基因。这类转化细胞只有在补充甲羟戊酸途径的代谢中间体的情况下才能存活。重要的是，这保证了细胞中形成的所有IPP必须来自补充的中间体。大肠杆菌DK2236 （ptmv20,7 pACCAR25ΔcrtX）以这种方式构建，但实际上是三重工程。即，1-脱氧-d -木糖5-磷酸还原异构酶（dxr）基因被破坏，并引入一个携带IPP形成的三种酶基因的质粒pTMV20。此外，还引入了另一种质粒pACCAR25ΔcrtX，该质粒携带合成玉米黄质所需的crtE、crtB、crtI、crtY和crtZ基因。该菌株在外源甲基戊酸存在下存活并产生黄色的玉米黄质色素。按照前面所述合成的完全氘化的甲羟戊内酯-d9 （MVL-d9）被补充到上述三重工程大肠杆菌的培养中。11生物合成的玉米黄质通过重复层析进行提取和纯化，达到均匀性。纯化产物的分子离子（M+）在M /z 605-622之间为一簇同位素离子。未检测到m/ z568（未标记的m +）离子的痕迹。所有形成的玉米黄质均来源于补充的MVL-d9。这是这种高度和多重氘化玉米黄质的第一个例子，并清楚地表明，目前的方法在制备各种同位素标记的类异戊二烯方面具有巨大的潜力。就生物合成机制而言，玉米黄质在上述工程大肠杆菌中生物合成的事实表明，包括IPP异构酶在内的整个甲羟戊酸途径是可行的。此外，聚集的M+离子清楚地表明分子间氘的含量是可变的。这似乎是由于在异构酶反应过程中，中间二甲基丙烯基二磷酸（DMAPP）的甲基交换了氢，IPP的C-4亚甲基也交换了氢。因此，一旦一个质子被纳入DMAPP的自由旋转的C-4甲基中，DMAPP到IPP的逆反应应该产生(E)和(Z)质子化的IPP，如图1所示。
Isoprenoids are chemically diverse in nature, ubiquitous in living organisms, and crucial in biological processes. The biosynthesis of such isoprenoids proceeds through mevalonate1 and non-mevalonate pathways2 depending upon organisms and cellular organella, isopentenyl diphosphate (IPP) being a key intermediate in both cases. Metabolic engineering and control of these pathways should thus provide new opportunities to study the intriguing chemistry and biochemistry involved and to develop selective chemotherapeutic agents and isoprenoid-related materials. This paper describes a new practical approach for the preparation of highly deuterated isoprenoids, such as zeaxanthin, and their potential for analyzing the biosynthetic mechanism of isoprenoids, based on the metabolic engineering of Escherichia coli. Deuteriumlabeled compounds are invaluable in biochemical and bioorganic as well as physicochemical research. The non-mevalonate pathway, but not the mevalonate pathway, is now known to function in E. coli. Recently, Seto et al. 3, 4 and Boronat et al. 5 described the construction of doubly engineered E. coli with the disruption of a gene of a certain enzyme involved in the non-mevalonate pathway and the introduction of the genes responsible for the key enzymes in the mevalonate pathway. These sort of transformant cells can survive only with supplementation of metabolic intermediates in the mevalonate pathway. Importantly, this warrants that all of the IPP formed in the cells must be derived from the supplemented intermediate. E. coli DK2236 (pTMV20, 7 pACCAR25ΔcrtX) was constructed in this manner, but was actually triply engineered. That is, the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr) gene was disrupted, and a plasmid pTMV20 carrying the genes of three enzymes responsible for the formation of IPP was introduced. In addition, another plasmid pACCAR25ΔcrtX was introduced, which carried the crtE, crtB, crtI, crtY, and crtZ genes required for the synthesis of zeaxanthin. 8 This strain survived and produced a yellow pigment of zeaxanthin in the presence of exogenous mevalonate. 9 Fully deuterated mevalonolactone-d9 (MVL-d9), which had been synthesized as described previously, 10 was supplemented to the culture of the above triply engineered E. coli. 11 The biosynthesized zeaxanthin was extracted and purified to homogeneity by repeated chromatography. The molecular ion (M+) of the purified product was observed as a cluster of isotopomer ions between m/z 605-622. No trace of an ion at m/z 568 (nonlabeled M+) was detected. All of the zeaxanthin formed was proved to be derived only from the supplemented MVL-d9. This was the first example of such highly and multiply deuterated zeaxanthin and clearly demonstrates the significant potential of the present approach for the preparation of various isotope-labeled isoprenoids.As to the biosynthetic mechanism, the fact that zeaxanthin was biosynthesized in the above engineered E. coli appears to suggest that the whole mevalonate pathway including IPP isomerase was viable. 12 In addition, the clustered M+ ion clearly implies variable deuterium content from molecule to molecule. This appears to be due to the exchange of hydrogen at the methyl group of intermediary dimethylallyl diphosphate (DMAPP) during the isomerase reaction and accordingly at the C-4 methylene group of IPP as well. Thus, once a proton is incorporated into the freely rotating C-4 methyl group of DMAPP, the reverse reaction from DMAPP to IPP should afford (E)-and (Z)-protonated IPP equally as shown in Figure 1.