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Engineering a thermoregulated intein-modified xylanase into maize for consolidated lignocellulosic biomass processing

Abstract

Plant cellulosic biomass is an abundant, low-cost feedstock for producing biofuels and chemicals. Expressing cell wall–degrading (CWD) enzymes (e.g. xylanases) in plant feedstocks could reduce the amount of enzymes required for feedstock pretreatment and hydrolysis during bioprocessing to release soluble sugars. However, in planta expression of xylanases can reduce biomass yield and plant fertility. To overcome this problem, we engineered a thermostable xylanase (XynB) with a thermostable self-splicing bacterial intein to control the xylanase activity. Intein-modified XynB (iXynB) variants were selected that have <10% wild-type enzymatic activity but recover >60% enzymatic activity upon intein self-splicing at temperatures >59 °C. Greenhouse-grown xynB maize expressing XynB has shriveled seeds and low fertility, but ixynB maize had normal seeds and fertility. Processing dried ixynB maize stover by temperature-regulated xylanase activation and hydrolysis in a cocktail of commercial CWD enzymes produced >90% theoretical glucose and >63% theoretical xylose yields.

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Figure 1: Development of the thermoregulated intein-modified XynB.
Figure 2: Dynamics of iXynB activation and splicing.
Figure 3: Amino acid residues that modulate thermoregulated intein splicing.
Figure 4: xynB and ixynB seed development.
Figure 5: Glucose and xylose yields from processing xynB and ixynB stover.

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Acknowledgements

The authors gratefully acknowledge F. Ausubel, M. Ladisch and reviewers for valuable comments on the manuscript. We thank J. Donald for assistance in preparing figures.

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Authors

Contributions

B.S. conceived, developed and performed protein engineering experiments, and led enzyme development and data analysis. X.S. and X.Z. constructed genes, and screened and characterized variants. T.S., M.R. and M.P. validated variants. J.A. conducted structural analysis and modeling. O.B. and V.S. oversaw and conducted plant transformation. E.H., H.L., B.G. and N.A.E. conducted plant analysis. D.Z. and J.C.S.J. oversaw and conducted processing experiments. G.L. and R.M.R. managed the overall project, helped design experiments, organized efforts and contributed intellectually. B.S. and R.M.R. wrote the paper.

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Correspondence to R Michael Raab.

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This research was funded by Agrivida, Inc.

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Shen, B., Sun, X., Zuo, X. et al. Engineering a thermoregulated intein-modified xylanase into maize for consolidated lignocellulosic biomass processing. Nat Biotechnol 30, 1131–1136 (2012). https://doi.org/10.1038/nbt.2402

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