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Engineering nonphosphorylative metabolism to generate lignocellulose-derived products

Abstract

Conversion of lignocellulosic biomass into value-added products provides important environmental and economic benefits. Here we report the engineering of an unconventional metabolism for the production of tricarboxylic acid (TCA)-cycle derivatives from D-xylose, L-arabinose and D-galacturonate. We designed a growth-based selection platform to identify several gene clusters functional in Escherichia coli that can perform this nonphosphorylative assimilation of sugars into the TCA cycle in less than six steps. To demonstrate the application of this new metabolic platform, we built artificial biosynthetic pathways to 1,4-butanediol (BDO) with a theoretical molar yield of 100%. By screening and engineering downstream pathway enzymes, 2-ketoacid decarboxylases and alcohol dehydrogenases, we constructed E. coli strains capable of producing BDO from D-xylose, L-arabinose and D-galacturonate. The titers, rates and yields were higher than those previously reported using conventional pathways. This work demonstrates the potential of nonphosphorylative metabolism for biomanufacturing with improved biosynthetic efficiencies.

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Figure 1: Assimilation pathways of lignocellulosic sugars through the nonphosphorylative metabolism.
Figure 2: The growth platform to test functional nonphosphorylative gene clusters in E. coli.
Figure 3: BDO production using different combinations of 2-ketoacid decarboxylases (KDCs) and alcohol dehydrogenases (ADHs), and Kivd variants.
Figure 4: Production of BDO from D-xylose, L-arabinose and D-galacturonate in 1.3-l bioreactors.
Figure 5: Growth platform to mine putative nonphosphorylative clusters in E. coli and BDO production using these operons.

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Acknowledgements

This work was supported by research grants from the Office for Technology Commercialization of the University of Minnesota, the McKnight Land Grant Professorship Program and the National Science Foundation through the Center for Sustainable Polymers (CHE-1413862). We thank M. McClintock and K. Fox for assisting with revisions that greatly improved the article.

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Y.-S.T., M.X., P.J. and K.Z. designed experiments. Y.-S.T., M.X., P.J., Jilong W., Jingyu W. and C.S. performed experiments. Y.-S.T., M.X., P.J. and K.Z. analyzed data. Y.-S.T., M.X., P.J., Jilong W., Jingyu W., C.S. and K.Z. wrote and edited the paper.

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Correspondence to Kechun Zhang.

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Competing interests

Y.-S.T., M.X., P.J. and K.Z. are co-inventors on the patent applications “Biosynthetic pathways and methods” (patent application WO2014100173) and “Recombinant cells and methods for nonphosphorylative metabolism” (US Provisional Application 62/255,856).

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Tai, YS., Xiong, M., Jambunathan, P. et al. Engineering nonphosphorylative metabolism to generate lignocellulose-derived products. Nat Chem Biol 12, 247–253 (2016). https://doi.org/10.1038/nchembio.2020

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