Employing a biochemical protecting group for a sustainable indigo dyeing strategy

Abstract

Indigo is an ancient dye uniquely capable of producing the signature tones in blue denim; however, the dyeing process requires chemical steps that are environmentally damaging. We describe a sustainable dyeing strategy that not only circumvents the use of toxic reagents for indigo chemical synthesis but also removes the need for a reducing agent for dye solubilization. This strategy utilizes a glucose moiety as a biochemical protecting group to stabilize the reactive indigo precursor indoxyl to form indican, preventing spontaneous oxidation to crystalline indigo during microbial fermentation. Application of a β-glucosidase removes the protecting group from indican, resulting in indigo crystal formation in the cotton fibers. We identified the gene coding for the glucosyltransferase PtUGT1 from the indigo plant Polygonum tinctorium and solved the structure of PtUGT1. Heterologous expression of PtUGT1 in Escherichia coli supported high indican conversion, and biosynthesized indican was used to dye cotton swatches and a garment.

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Figure 1: A glucosyl protecting group enables control over the timing and location of indigo dyeing.
Figure 2: The crystal structure of PtUGT1 with bound indoxyl sulfate.
Figure 3: Heterologous expression of PtUGT1 stabilizes indoxyl before it dimerizes, producing indican.
Figure 4: Production and growth curves for indican and indigo production.
Figure 5: Bio-indican can be used as an effective, reductant-free cotton textile dye.

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Acknowledgements

The authors thank the rest of the Berkeley iGEM 2013 team, C. Somerville, N. Sorek, S. Bauer, N. Harris, D. Savage, B. Sights, S. Wagner, and the Dueber Lab, especially S. Bhakta, D. Stanley, L. Latimer, P. Grewal, and S. Halperin, for valuable discussions and experimental assistance. The UC Berkeley Vincent J. Coates Genomics Sequencing Laboratory and Proteomics/Mass Spectrometry Laboratory provided transcriptome and protein sequencing. The M. Chang laboratory (University of California, Berkeley) provided the base E. coli strain. This work was supported by Bakar Fellows Program (fellowship to J.E.D.), NSF CBET 1605465 (J.E.D.), a generous gift from Levi Strauss & Co. (J.E.D.), the US Department of Defense (fellowship to T.M.H. and Z.N.R.) and Agilent (iGEM undergraduate fellowships). Crystallographic experiments were performed as part of the DOE Joint BioEnergy Institute (http://www.jbei.org) which is supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy. We thank the Berkeley Center for Structural Biology beamline staff for technical assistance during data collection. The BCSB is supported in part by the National Institutes of Health, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under Contract No. DE-AC02-05CH11231.

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T.M.H., Z.N.R., and J.E.D. designed the research. T.M.H., D.H.W., Z.N.R., B.C., and R.L.P. collected data. D.H.W. solved the crystallographic structure. T.M.H., D.H.W., Z.N.R., P.D.A., and J.E.D. analyzed the data. T.M.H., D.H.W., and J.E.D. wrote the paper.

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Correspondence to John E Dueber.

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The authors declare competing financial interests in the form of a pending patent application, US application no. 62/127,778.

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Hsu, T., Welner, D., Russ, Z. et al. Employing a biochemical protecting group for a sustainable indigo dyeing strategy. Nat Chem Biol 14, 256–261 (2018). https://doi.org/10.1038/nchembio.2552

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