ABSTRACTThere 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 C10and C15biofuels. The strictly anaerobic, acetogenic bacteriumClostridium 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 colishuttle 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 intoC. 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 inC. ljungdahlii, as demonstrated by Western blotting, and were enzymatically active, as demonstrated byin vivoproduct 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.IMPORTANCEThis study demonstrates the ability to synthesize a heterologous metabolic pathway inC. 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 ofC. ljungdahliito grow mixotrophically also enables the recapture, should there be sufficient reducing equivalents available, of the CO2released 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.
Electron availability in CO
2
, CO and H
2
mixtures constrains flux distribution, energy management and product formation in
Clostridium ljungdahlii
