数字农科院2.0

Establishment Of Bmor-Based Biosensor To S.creen Isobutanol Overproducer

文献类型： 外文期刊

作者： Wang, Ning;Yu, Huan;Chen, Zhenya;Yang, Yu;Huo, Wenbo;Huo, Yi-Xin;Zhang, Yuhong;Zhang, Wei

作者机构：

关键词： BmoR; Biosensor; Screening; Isobutanol; Mutagenesis

期刊名称： MICROBIAL CELL FACTORIES

ISSN： 1475-2859

年卷期： 2019 年 18 卷

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摘要： 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 sigma(54)-dependent promoter P-bmo 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-P-bmo 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 190.74.8, a 1.4-fold higher value than that of wild type. Correspondingly, the isobutanol titer of that strain was 1597.6 +/- 129.6mg/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 +/- 1.0g/L after medium optimization in shake flask. The isobutanol titer reached 56.5 +/- 1.8g/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 +/- 1.8g/L.

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