Formation of Isobutene from 3-Hydroxy-3-Methylbutyrate by Diphosphomevalonate Decarboxylase

Abstract

ABSTRACT Isobutene is an important commercial chemical used for the synthesis of butyl rubber, terephthalic acid, specialty chemicals, and a gasoline performance additive known as alkylate. Currently, isobutene is produced from petroleum and hence is nonrenewable. Here, we report that the Saccharomyces cerevisiae mevalonate diphosphate decarboxylase ( Sc MDD) can convert 3-hydroxy-3-methylbutyrate (3-HMB) to isobutene. Whole cells of Escherichia coli producing Sc MDD with an N-terminal 6×His tag (His 6 - Sc MDD) formed isobutene from 3-HMB at a rate of 154 pmol h −1 g cells −1 . In contrast, no isobutene was detected from control cells lacking Sc MDD. His 6 - Sc MDD was purified by nickel affinity chromatography and shown to produce isobutene from 3-HMB at a rate of 1.33 pmol min −1 mg −1 protein. Controls showed that both His 6 - Sc MDD and 3-HMB were required for detectable isobutene formation. Isobutene was identified by gas chromatography (GC) with flame ionization detection as well as by GC-mass spectrometry (MS). Sc MDD was subjected to error-prone PCR, and two improved variants were characterized, Sc MDD1 (I145F) and Sc MDD2 (R74H). Whole cells of E. coli producing Sc MDD1 and Sc MDD2 produced isobutene from 3-HMB at rates of 3,000 and 5,888 pmol h −1 g cells −1 , which are 19- and 38-fold increases compared to rates for cells producing His 6 - Sc MDD. This showed that genetic modifications can be used to increase the rate at which Sc MDD converts 3-HMB to isobutene. Because 3-HMB can be produced from l -leucine, Sc MDD has a potential application for the production of renewable isobutene. Moreover, isobutene is a gas, which might simplify its purification from a fermentation medium, substantially reducing production costs.