Abstract
The aim of this study was to establish isobutanol production on chemically defined medium in Escherichia coli. By individually expressing each gene of the pathway, we constructed a plasmid library for isobutanol production. Strain screening on chemically defined medium showed successful production in the robust E. coli W strain, and expression vector IB 4 was selected as the most promising construct due to its high isobutanol yields and efficient substrate uptake. The investigation of different aeration strategies in combination with strain improvement and the implementation of a pulsed fed-batch were key for the development of an efficient production process. E. coli W ΔldhA ΔadhE Δpta ΔfrdA enabled aerobic isobutanol production at 38% of the theoretical maximum. Use of cheese whey as raw material resulted in longer process stability, which allowed production of 20 g l−1 isobutanol. Demonstrating isobutanol production on both chemically defined medium and a residual waste stream, this study provides valuable information for further development of industrially relevant isobutanol production processes.
Highlights
Second-generation biofuels, which are produced from lignocellulosic biomass or waste streams, are considered as strategically important sustainable fuels due to their renewability, Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Isobutanol is a metabolite not naturally synthesized by any organism
The production of isobutanol has been demonstrated in several organisms, including Escherichia coli [5, 6], Saccharomyces cerevisiae [7, 8], Corynebacterium glutamicum [9], Clostridium thermocellum [10] and even autotrophic organisms like Ralstonia eutropha [11] and Synecococcus elongatus [12]
Overexpression of α-ketoisovalerate decarboxylase from Lactococcus lactis was shown to result in high isobutanol yields [13]
Summary
Isobutanol is produced by an alcohol dehydrogenase (AdhA). Two of these enzymes, ilvC and ilvD, are native to E. coli [5]. Acetolactate synthetase is the first enzyme in the 2,3-butanediol production pathway of natural producers. E. coli cannot naturally produce isobutanol because it lacks an α-ketoisovalerate decarboxylase [16]. E. coli has a native alcohol dehydrogenase yqhD, but overexpression of adhA from L. lactis is advantageous due to utilization of NADH rather than NADPH as a cofactor [17]. In a mutated form of ilvC, the cofactor was exchanged from NADPH to NADH These mutations enabled anaerobic isobutanol production at 100% of the theoretical yield [18]
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