全基因組序列定序技術與反向工程方法應用於研究異丁醇高耐受度大腸桿菌

Whole genome sequencing and reverse engineering isobutanol tolerant strain in Escherichia coli

吳東耘(Tung-Yun Wu)

指導教授 ： 鄭西顯

國立清華大學/工學院/化學工程學系/博士(2010年)

https://doi.org/10.6843/NTHU.2010.00184

摘要

Recently, interest in the production of isobutanol as a potential substitute for gasoline has risen. Previously, Escherichia coli has been metabolically engineered to produce isobutanol, which reached more than 20g/L in the optimized pathways. Here we aim to identify genotype-phenotype relationships in a strain of E. coli tolerant to high concentrations of isobutanol. In this study, we combined cell evolution with whole genome sequencing technology to identify genotypes potentially conferring isobutanol tolerance phenotype from isolated mutant and the experimental verification confirms gene acrA, gatY, tnaA, marC-marRAB, yhbJ are responsible for isobutanol tolerance phenotype. Further investigation showed inactivation of the AcrAB/TolC multidrug efflux transport and MarC-MarRAB multiple antibiotic resistance systems increase isobutanol tolerance. Additionally, the inactivation of YhbJ, which regulates the synthesis of glucosamine-6-phosphate, leads to increased isobutanol tolerance. Two additional gene knockouts, gatY and tnaA, were also identified as key elements for isobutanol tolerance. We successfully reconstructed E. coli strains with increased tolerance to isobutanol by combining these five deletions. In addition, the comparison of isobutanol response and other solvent stress is discussed. Lastly, our isobutanol production data showed no elevated productivity in isobutanol tolerant mutant, which suggests the productivity may not correlate to the tolerance at the growth phase but in stationary phase. The approach described here could apply to comprehensive chemical tolerances of microorganisms and provide a general framework to design and construct tolerant mutants.

關鍵字

生質能源 ； 異丁醇 ； 耐受度 ； 全基因組定序

並列摘要

無資料

並列關鍵字

biofuels ； isobutanol ； tolerance ； whole genome sequencing

參考文獻

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