Establishment of BmoR-based biosensor to screen isobutanol overproducer.

ABSTRACT: BACKGROUND:Isobutanol, 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 ?54-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. RESULTS:Firstly, 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. CONCLUSIONS:This 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.

SUBMITTER: Yu H 

PROVIDER: S-EPMC6366067 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature