Engineering Escherichia coli for anaerobic alkane activation: Biosynthesis of (1‐methylalkyl)succinates
Engineering Escherichia coli for anaerobic alkane activation: Biosynthesis of (1‐methylalkyl)succinates
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用于厌氧烷烃活化的工程大肠杆菌：(1-甲基烷基)琥珀酸酯的生物合成
DOI：
10.1002/bit.27956
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发表时间：
2021
影响因子：
3.8
通讯作者：
Cirino, Patrick C.
中科院分区：
文献类型：
--
作者：
Wang, Yixi;Nguyen, Nam;Lee, Seung H.;Wang, Qinxuan;May, Jeremy A.;Gonzalez, Ramon;Cirino, Patrick C.
在缺氧环境中，微生物激活烷烃进行后续代谢最常见的方式是将富马酸加到亚末端碳上，产生烷基琥珀酸酯。烷基琥珀酸脱氢酶是复杂的多亚基酶，其利用催化甘氨酰自由基并需要配偶体，活化酶以进行氢提取。虽然已经鉴定了许多编码推定的烷基琥珀酸脱氢酶的基因，主要来自硝酸盐还原菌和硫酸盐还原菌，但很少有基因被表征，并且没有报道在异源宿主中功能性表达。在这里，我们描述了（1-甲基烷基）琥珀酸合成酶（Mas）系统从固氮菌菌株HxN 1在重组大肠杆菌中的功能表达。质谱法证实了富马酸盐除己烷、丁烷和丙烷外的预期产物的厌氧生物合成。当masC、masD、masE、masB和masG都存在于表达质粒上时，观察到（1-甲基戊基）琥珀酸酯的最大产量;省略masC使产量减少66%，而省略任何其他基因则会消除产量。同时，将E.大肠杆菌铁硫簇操纵子）提高了产物滴度，在低温（18°C）下进行生物转化也是如此，这两者都表明烷基琥珀酸生物合成在很大程度上受到该酶系统的功能表达的限制。
In anoxic environments, microbial activation of alkanes for subsequent metabolism occurs most commonly through the addition of fumarate to a subterminal carbon, producing an alkylsuccinate. Alkylsuccinate synthases are complex, multi‐subunit enzymes that utilize a catalytic glycyl radical and require a partner, activating enzyme for hydrogen abstraction. While many genes encoding putative alkylsuccinate synthases have been identified, primarily from nitrate‐ and sulfate‐reducing bacteria, few have been characterized and none have been reported to be functionally expressed in a heterologous host. Here, we describe the functional expression of the (1‐methylalkyl)succinate synthase (Mas) system fromAzoarcus sp. strain HxN1 in recombinantEscherichia coli. Mass spectrometry confirms anaerobic biosynthesis of the expected products of fumarate addition to hexane, butane, and propane. Maximum production of (1‐methylpentyl)succinate is observed whenmasC,masD,masE,masB, andmasGare all present on the expression plasmid; omittingmasCreduces production by 66% while omitting any other gene eliminates production. Meanwhile, deletingiscR(encoding the repressor of theE. coliiron–sulfur cluster operon) improves product titer, as does performing the biotransformation at reduced temperature (18°C), both suggesting alkylsuccinate biosynthesis is largely limited by functional expression of this enzyme system.
影响因子：
3.9
作者：
Shisler, Krista A.;Broderick, Joan B.
通讯作者：
Broderick, Joan B.