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Engineering nonphosphorylative metabolism to generate lignocellulose-derived products


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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  1. Graham-Rowe, D. Agriculture: Beyond food versus fuel. Nature 474, S6–S8 (2011).

    Article  CAS  Google Scholar 

  2. Perlack, R.D. et al. Biomass as feedstock for a bioenergy and bioproducts industry: the technical feasibility of a billion-ton annual supply (Oak Ridge National Laboratory, 2005).

  3. Li, C. et al. Influence of physico-chemical changes on enzymatic digestibility of ionic liquid and AFEX pretreated corn stover. Bioresour. Technol. 102, 6928–6936 (2011).

    Article  CAS  Google Scholar 

  4. Himmel, M.E., Baker, J.O. & Overend, R.P. Enzymatic Conversion of Biomass for Fuels Production (American Chemical Society, Washington, DC, 1994).

  5. Wei, N., Quarterman, J., Kim, S.R., Cate, J.H. & Jin, Y.S. Enhanced biofuel production through coupled acetic acid and xylose consumption by engineered yeast. Nat. Commun. 4, 2580 (2013).

    Article  Google Scholar 

  6. Shaw, A.J. et al. Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield. Proc. Natl. Acad. Sci. USA 105, 13769–13774 (2008).

    Article  CAS  Google Scholar 

  7. Gao, D. et al. Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis. Proc. Natl. Acad. Sci. USA 110, 10922–10927 (2013).

    Article  CAS  Google Scholar 

  8. Edwards, M.C. et al. Addition of genes for cellobiase and pectinolytic activity in Escherichia coli for fuel ethanol production from pectin-rich lignocellulosic biomass. Appl. Environ. Microbiol. 77, 5184–5191 (2011).

    Article  CAS  Google Scholar 

  9. Cirino, P.C., Chin, J.W. & Ingram, L.O. Engineering Escherichia coli for xylitol production from glucose-xylose mixtures. Biotechnol. Bioeng. 95, 1167–1176 (2006).

    Article  CAS  Google Scholar 

  10. Steen, E.J. et al. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463, 559–562 (2010).

    Article  CAS  Google Scholar 

  11. Niu, W., Molefe, M.N. & Frost, J.W. Microbial synthesis of the energetic material precursor 1,2,4-butanetriol. J. Am. Chem. Soc. 125, 12998–12999 (2003).

    Article  CAS  Google Scholar 

  12. Yim, H. et al. Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. Nat. Chem. Biol. 7, 445–452 (2011).

    Article  CAS  Google Scholar 

  13. Pharkya, P., Burgard, A.P. & Maranas, C.D. Exploring the overproduction of amino acids using the bilevel optimization framework OptKnock. Biotechnol. Bioeng. 84, 887–899 (2003).

    Article  CAS  Google Scholar 

  14. Weimberg, R. Pentose oxidation by Pseudomonas fragi. J. Biol. Chem. 236, 629–635 (1961).

    CAS  PubMed  Google Scholar 

  15. Stephens, C. et al. Genetic analysis of a novel pathway for D-xylose metabolism in Caulobacter crescentus. J. Bacteriol. 189, 2181–2185 (2007).

    Article  CAS  Google Scholar 

  16. Brouns, S.J.J. et al. Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment. J. Biol. Chem. 281, 27378–27388 (2006).

    Article  CAS  Google Scholar 

  17. Novick, N.J. & Tyler, M.E. L-arabinose metabolism in Azospirillum brasiliense. J. Bacteriol. 149, 364–367 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Boer, H., Maaheimo, H., Koivula, A., Penttilä, M. & Richard, P. Identification in Agrobacterium tumefaciens of the D-galacturonic acid dehydrogenase gene. Appl. Microbiol. Biotechnol. 86, 901–909 (2010).

    Article  CAS  Google Scholar 

  19. Andberg, M. et al. Characterization of a novel Agrobacterium tumefaciens galactarolactone cycloisomerase enzyme for direct conversion of D-galactarolactone to 3-deoxy-2-keto-L-threo-hexarate. J. Biol. Chem. 287, 17662–17671 (2012).

    Article  CAS  Google Scholar 

  20. Hosoya, S., Yamane, K., Takeuchi, M. & Sato, T. Identification and characterization of the Bacillus subtilis D-glucarate/galactarate utilization operon ycbCDEFGHJ. FEMS Microbiol. Lett. 210, 193–199 (2002).

    CAS  PubMed  Google Scholar 

  21. Liu, H. & Lu, T. Autonomous production of 1,4-butanediol via a de novo biosynthesis pathway in engineered Escherichia coli. Metab. Eng. 29, 135–141 (2015).

    Article  CAS  Google Scholar 

  22. Liu, H. et al. Biosynthesis of ethylene glycol in Escherichia coli. Appl. Microbiol. Biotechnol. 97, 3409–3417 (2013).

    Article  CAS  Google Scholar 

  23. Meijnen, J.P., de Winde, J.H. & Ruijssenaars, H.J. Establishment of oxidative D-xylose metabolism in Pseudomonas putida S12. Appl. Environ. Microbiol. 75, 2784–2791 (2009).

    Article  CAS  Google Scholar 

  24. Radek, A. et al. Engineering of Corynebacterium glutamicum for minimized carbon loss during utilization of D-xylose containing substrates. J. Biotechnol. 192, 156–160 (2014).

    Article  CAS  Google Scholar 

  25. Lin, Y., Shen, X., Yuan, Q. & Yan, Y. Microbial biosynthesis of the anticoagulant precursor 4-hydroxycoumarin. Nat. Commun. 4, 2603 (2013).

    Article  Google Scholar 

  26. Djurdjevic, I., Zelder, O. & Buckel, W. Production of glutaconic acid in a recombinant Escherichia coli strain. Appl. Environ. Microbiol. 77, 320–322 (2011).

    Article  CAS  Google Scholar 

  27. Zhang, K. & Xiong, M. Biosynthetic pathways and methods for TCA derivatives. Patent PCT/US2013/076118 (WO 2014100173 A1) (2014).

  28. Moore, R.A. et al. Contribution of gene loss to the pathogenic evolution of Burkholderia pseudomallei and Burkholderia mallei. Infect. Immun. 72, 4172–4187 (2004).

    Article  CAS  Google Scholar 

  29. Stoolmiller, A.C. & Abeles, R.H. Formation of α-ketoglutaric semialdehyde from L-2-keto-3-deoxyarabonic acid and isolation of L-2-keto-3-deoxyarabonate dehydratase from Pseudomonas saccharophila. J. Biol. Chem. 241, 5764–5771 (1966).

    CAS  PubMed  Google Scholar 

  30. Duncan, M.J. L-Arabinose metabolism in Rhizobia. J. Gen. Microbiol. 113, 177–179 (1979).

    Article  CAS  Google Scholar 

  31. Yoon, S.H., Moon, T.S., Iranpour, P., Lanza, A.M. & Prather, K.J. Cloning and characterization of uronate dehydrogenases from two pseudomonads and Agrobacterium tumefaciens strain C58. J. Bacteriol. 191, 1565–1573 (2009).

    Article  CAS  Google Scholar 

  32. de la Plaza, M., Fernández de Palencia, P., Peláez, C. & Requena, T. Biochemical and molecular characterization of alpha-ketoisovalerate decarboxylase, an enzyme involved in the formation of aldehydes from amino acids by Lactococcus lactis. FEMS Microbiol. Lett. 238, 367–374 (2004).

    CAS  Google Scholar 

  33. Xiong, M. et al. A bio-catalytic approach to aliphatic ketones. Sci. Rep. 2, 311 (2012).

    Article  Google Scholar 

  34. Iding, H. et al. Benzoylformate decarboxylase from Pseudomonas putida as stable catalyst for the synthesis of chiral 2-hydroxy ketones. Chemistry 6, 1483–1495 (2000).

    Article  CAS  Google Scholar 

  35. Bastian, S. et al. Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli. Metab. Eng. 13, 345–352 (2011).

    Article  CAS  Google Scholar 

  36. Larroy, C., Fernández, M.R., González, E., Parés, X. & Biosca, J.A. Characterization of the Saccharomyces cerevisiae YMR318C (ADH6) gene product as a broad specificity NADPH-dependent alcohol dehydrogenase: relevance in aldehyde reduction. Biochem. J. 361, 163–172 (2002).

    Article  CAS  Google Scholar 

  37. Oshima, T. & Biville, F. Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon. Microbiology 152, 2129–2135 (2006).

    Article  CAS  Google Scholar 

  38. Berthold, C.L. et al. Structure of the branched-chain keto acid decarboxylase (KdcA) from Lactococcus lactis provides insights into the structural basis for the chemoselective and enantioselective carboligation reaction. Acta Crystallogr. D Biol. Crystallogr. 63, 1217–1224 (2007).

    Article  CAS  Google Scholar 

  39. Xiong, M., Schneiderman, D.K., Bates, F.S., Hillmyer, M.A. & Zhang, K. Scalable production of mechanically tunable block polymers from sugar. Proc. Natl. Acad. Sci. USA 111, 8357–8362 (2014).

    Article  CAS  Google Scholar 

  40. Zhang, K., Sawaya, M.R., Eisenberg, D.S. & Liao, J.C. Expanding metabolism for biosynthesis of nonnatural alcohols. Proc. Natl. Acad. Sci. USA 105, 20653–20658 (2008).

    Article  CAS  Google Scholar 

  41. Görke, B. & Stülke, J. Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat. Rev. Microbiol. 6, 613–624 (2008).

    Article  Google Scholar 

  42. Datsenko, K.A. & Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640–6645 (2000).

    Article  CAS  Google Scholar 

  43. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 0008 (2006).

    Article  Google Scholar 

  44. Zhang, K., Woodruff, A.P., Xiong, M., Zhou, J. & Dhande, Y.K. A synthetic metabolic pathway for production of the platform chemical isobutyric acid. ChemSusChem 4, 1068–1070 (2011).

    Article  CAS  Google Scholar 

  45. Engler, C., Kandzia, R. & Marillonnet, S. A one pot, one step, precision cloning method with high throughput capability. PLoS One 3, e3647 (2008).

    Article  Google Scholar 

  46. Watanabe, S., Shimada, N., Tajima, K., Kodaki, T. & Makino, K. Identification and characterization of L-arabonate dehydratase, L-2-keto-3-deoxyarabonate dehydratase, and L-arabinolactonase involved in an alternative pathway of L-arabinose metabolism. Novel evolutionary insight into sugar metabolism. J. Biol. Chem. 281, 33521–33536 (2006).

    Article  CAS  Google Scholar 

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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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Authors and Affiliations



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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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).

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