Synthesis of Heterologous Mevalonic Acid Pathway Enzymes in Clostridium ljungdahlii for the Conversion of Fructose and of Syngas to Mevalonate and Isoprene.
Synthesis of Heterologous Mevalonic Acid Pathway Enzymes in Clostridium ljungdahlii for the Conversion of Fructose and of Syngas to Mevalonate and Isoprene.
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DOI：
10.1128/aem.01723-17
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发表时间：
2018-01-01
影响因子：
4.4
通讯作者：
Whited GM
中科院分区：
文献类型：
--
作者：
Diner BA;Fan J;Scotcher MC;Wells DH;Whited GM
人们对利用微生物发酵以环境友好的方式利用廉价的原料生产高需求、高纯度的化学品越来越感兴趣。这里探讨的一个例子是异戊二烯(C5H8)的生产，这是一种半萜，主要在轮胎的合成橡胶中聚合为聚异戊二烯，但也可以转化为C10和C15生物燃料。在本文描述的所有工作中使用的严格厌氧产乙酸梭状芽胞杆菌能够使用Embden-Meyerhof-Parns途径进行糖酵解，并使用Wood-Ljugdahl途径进行固碳。构建了梭状芽孢杆菌-大肠杆菌穿梭载体，每个穿梭载体携带2或3个不同的真核甲基戊酸(MVA)途径或真核细胞中的异戊烯焦磷酸异构酶(IDI)和异戊二烯合成酶(ISPs)基因，并将其电穿孔到永达氏梭菌中。这些质粒中有一到两个被导入宿主细胞，能够从果糖和合成气(H2、CO2和CO)合成甲戊酸和异戊二烯，并将甲羟戊酸转化为异戊二烯。Western blotting结果表明，MVA途径的所有异源酶以及IDI和ISPs在龙须菜中都得到了高水平的合成，体内产物的合成也证明了这些异源酶具有酶活性。这里生产的甲氧戊酸和异戊二烯的数量远远低于商业生产菌株所需的数量。然而，提出的建议可以使产品形成的质量产量大幅增加。重要性这项研究证明了在永达木中合成异源代谢途径的能力，这是一种能够同时代谢单糖或合成气或两者兼有的有机体(混合营养)。合成气是一种廉价的碳和还原当量来源，是一些工业废气的主要成分，可通过气化纤维素生物废物和城市固体废物产生。因此，将其转化为有用的产品会带来潜在的成本和环境效益。如果有足够的还原当量可用，永达氏假丝酵母混合营养生长的能力也使其能够重新捕获糖酵解释放的二氧化碳，潜在地增加了产物形成的质量产量。异戊二烯是萜类化合物中最简单的一种，因此它的产生是合成萜类化合物途径中价值更高的产品的第一步。
There is a growing interest in the use of microbial fermentation for the generation of high-demand, high-purity chemicals using cheap feedstocks in an environmentally friendly manner. One example explored here is the production of isoprene (C5H8), a hemiterpene, which is primarily polymerized to polyisoprene in synthetic rubber in tires but which can also be converted to C10 and C15 biofuels. The strictly anaerobic, acetogenic bacterium Clostridium ljungdahlii, used in all of the work described here, is capable of glycolysis using the Embden-Meyerhof-Parnas pathway and of carbon fixation using the Wood-Ljungdahl pathway. Clostridium-Escherichia coli shuttle plasmids, each bearing either 2 or 3 different heterologous genes of the eukaryotic mevalonic acid (MVA) pathway or eukaryotic isopentenyl pyrophosphate isomerase (Idi) and isoprene synthase (IspS), were constructed and electroporated into C. ljungdahlii. These plasmids, one or two of which were introduced into the host cells, enabled the synthesis of mevalonate and of isoprene from fructose and from syngas (H2, CO2, and CO) and the conversion of mevalonate to isoprene. All of the heterologous enzymes of the MVA pathway, as well as Idi and IspS, were shown to be synthesized at high levels in C. ljungdahlii, as demonstrated by Western blotting, and were enzymatically active, as demonstrated by in vivo product synthesis. The quantities of mevalonate and isoprene produced here are far below what would be required of a commercial production strain. However, proposals are made that could enable a substantial increase in the mass yield of product formation. IMPORTANCE This study demonstrates the ability to synthesize a heterologous metabolic pathway in C. ljungdahlii, an organism capable of metabolizing either simple sugars or syngas or both together (mixotrophy). Syngas, an inexpensive source of carbon and reducing equivalents, is produced as a major component of some industrial waste gas, and it can be generated by gasification of cellulosic biowaste and of municipal solid waste. Its conversion to useful products therefore offers potential cost and environmental benefits. The ability of C. ljungdahlii to grow mixotrophically also enables the recapture, should there be sufficient reducing equivalents available, of the CO2 released upon glycolysis, potentially increasing the mass yield of product formation. Isoprene is the simplest of the terpenoids, and so the demonstration of its production is a first step toward the synthesis of higher-value products of the terpenoid pathway.