Synthesis of Heterologous Mevalonic Acid Pathway Enzymes in Clostridium ljungdahlii for the Conversion of Fructose and of Syngas to Mevalonate and Isoprene

Abstract

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.

Keywords: Clostridium ljungdahlii; Wood-Ljungdahl pathway; acetogenic bacteria; bioenergetics; fructose; isoprene; metabolic engineering; mevalonate; mevalonic acid pathway; syngas.