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.

Escherichia coli was engineered for the production of even-and odd-chain fatty acids (FAs) by fermentation. Co-production of thiolase, hydroxybutyryl-CoA dehydrogenase, crotonase and trans-enoyl-CoA reductase from a synthetic operon allowed the production of butyrate, hexanoate and octanoate. Elimination of native fermentation pathways by genetic deletion (DldhA, DadhE, DackA, Dpta, DfrdC) helped eliminate undesired by-products and increase product yields. Initial butyrate production rates were high (0.7 g l "1 h "1 ) but quickly levelled off and further study suggested this was due to product toxicity and/or acidification of the growth medium. Results also showed that endogenous thioesterases significantly influenced product formation. In particular, deletion of the yciA thioesterase gene substantially increased hexanoate production while decreasing the production of butyrate. E. coli was also engineered to co-produce enzymes for even-chain FA production (described above) together with a coenzyme B 12 -dependent pathway for the production of propionyl-CoA, which allowed the production of odd-chain FAs (pentanoate and heptanoate). The B 12 -dependent pathway used here has the potential to allow the production of odd-chain FAs from a single growth substrate (glucose) in a more energy-efficient manner than the prior methods.

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Glutamate‐267 is highly conserved in alcohol dehydrogenases and is suggested to be involved in a protein promoting vibration that facilitates catalysis. The residue is buried in the coenzyme binding domain. The Glu267Asn and Glu267His substitutions increase modestly the turnover numbers with ethanol and acetaldehyde as substrates, greatly increase the dissociation constants for the coenzymes NAD+ and NADH, and significantly decrease catalytic efficiencies. Large substrate isotope effects for oxidation of ethanol or benzyl alcohol suggest that hydride transfer is a major rate‐limiting step in catalysis. The enzymes are activated relative to wild‐type enzyme because coenzyme is released faster. X‐Ray crystallography shows that the structures of the mutated enzymes are similar to the open conformation of the wild‐type apo‐enzyme and that Asn‐267 or His‐267 are accommodated with local changes in the structure. Interestingly, NAD or some degradation product of NAD is found in the coenzyme binding site, but electron density is not definitive for positioning the nicotinamide ribose moiety. Furthermore, when the Glu267His enzyme is co‐crystallized with added NAD+ and 2,2,2‐trifluoroethanol, the structure is also in the open conformation. The substitutions of Glu‐267 appears to prevent the conformational change to a closed form that is observed with wild‐type enzyme when it binds coenzyme. Affinity for coenzyme binding may be decreased because of small alterations in the binding pocket and impaired coupling of the binding with the conformational change. The studies support an important role for Glu‐267, but the modest changes in the rate constant for hydride transfer show that this residue is not critical for catalytic rate enhancement. (Supported by NIH grant AA00279)