Abstract
BackgroundIsobutanol, a C4 branched-chain higher alcohol, is regarded as an attractive next-generation transport fuel. Metabolic engineering for efficient isobutanol production has been achieved in many studies. BmoR, an alcohol-regulated transcription factor, mediates a σ54-dependent promoter Pbmo of alkane monooxygenase in n-alkane metabolism of Thauera butanivorans and displays high sensitivity to C4–C6 linear alcohols and C3–C5 branched-chain alcohols. In this study, to achieve the high-level production of isobutanol, we established a screening system which relied on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway and then employed it to screen isobutanol overproduction strains from an ARTP mutagenesis library.ResultsFirstly, we constructed and verified a GFP-based BmoR-Pbmo device responding to the isobutanol produced by the host. Then, this screening system was employed to select three mutants which exhibited higher GFP/OD600 values than that of wild type. Significantly, GFP/OD600 of mutant 10 was 190.7 ± 4.8, a 1.4-fold higher value than that of wild type. Correspondingly, the isobutanol titer of that strain was 1597.6 ± 129.6 mg/L, 2.0-fold higher than the wild type. With the overexpression of upstream pathway genes, the isobutanol production from mutant 10 reached 14.0 ± 1.0 g/L after medium optimization in shake flask. The isobutanol titer reached 56.5 ± 1.8 g/L in a fed-batch production experiment.ConclusionsThis work screened out isobutanol overproduction strains from a mutagenesis library by using a screening system which depended on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway. Optimizing fermentation condition and reinforcing upstream pathway could realize the increase of isobutanol production from the overproducer. Lastly, fed-batch fermentation of the mutant enhanced the isobutanol production to 56.5 ± 1.8 g/L.
Highlights
Isobutanol, a C4 branched-chain higher alcohol, is regarded as an attractive next-generation transport fuel
Assembling the endogenous 2-KIV biosynthetic pathway and exogenous ketoisovalerate decarboxylase (Kivd) and alcohol dehydrogenase (AdhA) could realize the biosynthesis of isobutanol from glucose
Characterization of BmoR‐Pbmo biosensor via feeding isobutanol To verify the response of the BmoR-based biosensor to alcohols, gfp was chosen as a reporter gene under the control of promoter Pbmo
Summary
Isobutanol, a C4 branched-chain higher alcohol, is regarded as an attractive next-generation transport fuel. To achieve the high-level production of isobutanol, we established a screening system which relied on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway and employed it to screen isobutanol overproduction strains from an ARTP mutagenesis library. Many microorganisms such as Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Saccharomyces cerevisiae (S. cerevisiae) and Corynebacterium glutamicum (C. glutamicum) have been engineered to produce isobutanol [8, 13,14,15]. In these studies, the ketoisovalerate decarboxylase (Kivd) and alcohol dehydrogenase (AdhA) would catalyze 2-KIV to isobutyraldehyde and convert isobutyraldehyde to isobutanol, respectively. To broaden the carbon source for higher alcohols production, Huo et al employed single-cell protein wastes as carbon source and reached 56% of the theoretical yield [16]
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