Comprehensive sequence-flux mapping of a levoglucosan utilization pathway in <i>E. coli</i> [electronic resource]

• Ngôn ngữ: eng
• Ký hiệu phân loại: 547.6 Aromatic compounds
• Thông tin xuất bản: Washington, D.C. : Oak Ridge, Tenn. : United States. Dept. of Energy. Office of Science ; Distributed by the Office of Scientific and Technical Information, U.S. Dept. of Energy, 2015
• Mô tả vật lý: Size: p. 1235-1243 : , digital, PDF file.
• Bộ sưu tập: Metadata
• ID: 263599
• Chủ đề: 1. 09 biomass fuels; 2. 59 basic biological sciences; 3. Biological science; 4. Energy sciences

Tóm tắt

 Synthetic metabolic pathways often suffer from low specific productivity, and new methods that quickly assess pathway functionality for many thousands of variants are urgently needed. Here we present an approach that enables the rapid and parallel determination of sequence effects on flux for complete gene-encoding sequences. We show that this method can be used to determine the effects of over 8000 single point mutants of a pyrolysis oil catabolic pathway implanted in <
 i>
 Escherichia coli<
 /i>
 . Experimental sequence-function data sets predicted whether fitness-enhancing mutations to the enzyme levoglucosan kinase resulted from enhanced catalytic efficiency or enzyme stability. A structure of one design incorporating 38 mutations elucidated the structural basis of high fitness mutations. One design incorporating 15 beneficial mutations supported a 15-fold improvement in growth rate and greater than 24-fold improvement in enzyme activity relative to the starting pathway. Lastly, this technique can be extended to improve a wide variety of designed pathways.