Formation of isobutene from 3-hydroxy-3-methylbutyrate by diphosphomevalonate decarboxylase.
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 (ScMDD) can convert 3-hydroxy-3-methylbutyrate (3-HMB) to isobutene. Whole cells of Escherichia coli producing ScMDD with an N-terminal 6×His tag (His(6)-ScMDD) 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 ScMDD. His(6)-ScMDD 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)-ScMDD 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). ScMDD was subjected to error-prone PCR, and two improved variants were characterized, ScMDD1 (I145F) and ScMDD2 (R74H). Whole cells of E. coli producing ScMDD1 and ScMDD2 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)-ScMDD. This showed that genetic modifications can be used to increase the rate at which ScMDD converts 3-HMB to isobutene. Because 3-HMB can be produced from l-leucine, ScMDD 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.
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
David S
DS
Gogerty
Thomas A
TA
Bobik
D000475:Alkenes / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D002849:Chromatography, Gas; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D045782:Hemiterpenes; D007700:Kinetics; D008958:Models, Molecular; D010421:Pentanoic Acids / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000475:Alkenes; D045782:Hemiterpenes; D010421:Pentanoic Acids; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C008216:isovaleric acid; D002262:Carboxy-Lyases; C022503:pyrophosphomevalonate decarboxylase; C008176:isobutylene