Derek H Wells, Bruce A Diner, Gregory M Whited, Miles C Scotcher, Janine Fan
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 Emben-Meyerhof-Parnas pathway and of carbon fixation using the Wood-Ljungdahl pathway. Clostridium-E. 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 different of which were introduced into the host cells, enabled the synthesis of mevalonate and of isoprene from fructose and from syngas (H2, CO2, CO) and the conversion of mevalonate to isoprene. All of the heterologous enzymes of the mevalonic acid (MVA) pathway as well as isopentenyl pyrophosphate isomerase (Idi) and isoprene synthase (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. Proposals are made, however, 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 is an inexpensive source of carbon and reducing equivalents and is produced as a major component of some industrial waste gas, and 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 CO2 released upon glycolysis, potentially increasing the mass yield of product formation. Isoprene is the simplest of the terpenoids and so demonstration of its production is a first step toward synthesis of higher-value products of the terpenoid pathway.