Abstract
Cyanobacteria can be utilized as a platform for direct phototrophic conversion of CO2 to produce several types of carbon-neutral biofuels. One promising compound to be produced photobiologically in cyanobacteria is isobutene. As a volatile compound, isobutene will quickly escape the cells without building up to toxic levels in growth medium or get caught in the membranes. Unlike liquid biofuels, gaseous isobutene may be collected from the headspace and thus avoid the costly extraction of a chemical from culture medium or from cells. Here we investigate a putative synthetic pathway for isobutene production suitable for a photoautotrophic host. First, we expressed α-ketoisocaproate dioxygenase from Rattus norvegicus (RnKICD) in Escherichia coli. We discovered isobutene formation with the purified RnKICD with the rate of 104.6 ± 9 ng (mg protein)-1 min-1 using α-ketoisocaproate as a substrate. We further demonstrate isobutene production in the cyanobacterium Synechocystis sp. PCC 6803 by introducing the RnKICD enzyme. Synechocystis strain heterologously expressing the RnKICD produced 91 ng l−1 OD750−1 h−1. Thus, we demonstrate a novel sustainable platform for cyanobacterial production of an important building block chemical, isobutene. These results indicate that RnKICD can be used to further optimize the synthetic isobutene pathway by protein and metabolic engineering efforts.
Highlights
Isobutene (2-Methylpropene) is a small volatile and highly reactive alkene
We hypothesized that by expressing an α-ketoisocaproate dioxygenase from Rattus norvegicus (RnKICD) and a known isobutene forming enzyme, mevalonate-3-kinase from Picrophilus torridus (PtM3K), we would be able to introduce a pathway from the native metabolite KIC to isobutene via HMB
Since RnKICD is oxygen dependent, the bio-catalysis is preferably done in an oxygenic host such as Synechocystis
Summary
Isobutene (2-Methylpropene) is a small volatile and highly reactive alkene. As a platform chemical, isobutene is an important starting material for the production of alkylate and polymer gasoline, butyl rubber and speciality chemicals (Geilen et al, 2014). Attempts to produce isobutene from renewable sources have focused on two approaches: (i) production of bio-based isobutanol via bacterial fermentation followed by dehydration of isobutanol to isobutene using metal catalysts (Taylor et al, 2010); (ii) introducing a complete artificial metabolic pathway to convert glucose to isobutene by bacterial fermentation (van Leeuwen et al, 2012). Both of the described bio-isobutene routes start with biomass-derived sugars and are limited by the availability of sustainably produced biomass. It was estimated that more than 70% of the costs for isobutene production at commercial scale are due to the utilization of fermentable sugars as the feedstock for the heterotrophic micro-organisms. (van Leeuwen et al, 2012)
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