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A marine bacterial enzymatic cascade degrades the algal polysaccharide ulvan

Abstract

Marine seaweeds increasingly grow into extensive algal blooms, which are detrimental to coastal ecosystems, tourism and aquaculture. However, algal biomass is also emerging as a sustainable raw material for the bioeconomy. The potential exploitation of algae is hindered by our limited knowledge of the microbial pathways—and hence the distinct biochemical functions of the enzymes involved—that convert algal polysaccharides into oligo- and monosaccharides. Understanding these processes would be essential, however, for applications such as the fermentation of algal biomass into bioethanol or other value-added compounds. Here, we describe the metabolic pathway that enables the marine flavobacterium Formosa agariphila to degrade ulvan, the main cell wall polysaccharide of bloom-forming Ulva species. The pathway involves 12 biochemically characterized carbohydrate-active enzymes, including two polysaccharide lyases, three sulfatases and seven glycoside hydrolases that sequentially break down ulvan into fermentable monosaccharides. This way, the enzymes turn a previously unexploited renewable into a valuable and ecologically sustainable bioresource.

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Data availability

All data that support the findings of this study are available from the corresponding authors upon reasonable request. The protein structures are deposited in the PDB under 6HHM, 6HHN, 6HPD and 6HR5. Mass spectrometry data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository64 with the dataset identifier PXD009299.

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Acknowledgements

We thank the German Research Foundation (DFG) for funding through the Research Unit FOR2406 ‘Proteogenomics of Marine Polysaccharide Utilization’ (POMPU) (by grant nos. BO 1862/17-1 to U.T.B., HE 7217/2-1 to J.-H.H. and SCHW 595/10-1 to T.S.). J.-H.H. acknowledges funding by the Emmy-Noether Program of the DFG (grant no. HE 7217/1-1). G.M. is grateful to the French National Research Agency (ANR) for its support with regards to the investment expenditure program IDEALG (grant no. ANR-10-BTBR-04) and the Blue Enzymes project (reference ANR-14-CE19-0020-01). M.-K.Z. and F.U. were supported by scholarships from the Institute of Marine Biotechnology e.V. We thank C. Leroux for mass spectrometry analyses and M. Czjzek and A. Boraston for helpful discussions. We are indebted to the local contacts for their support during X-ray data collection at the PROXIMA-1 and PROXIMA-2 beamlines (SOLEIL Synchrotron, Saint Aubin, France) and the P11 beamline (DESY, Hamburg, Germany). We thank A. Otto, S. Junker and T. Sura for help with the metabolic labeling approach and T. Hinzke for support with analyses of the proteome data. We thank F. Lesourd (Agrival, Plouenan, France) for the gift of the ‘Agrival’ ulvan sample.

Author information

J.-H.H., T.S., G.M. and U.T.B. initiated the study and directed the project. L.R., A.P., R.L. and M.B. cloned the genes and expressed and purified the enzymes for the degradation reactions. M.B., J.-H.H. and L.R. isolated ulvan and purified oligomers. Metabolites were analyzed by C.S. via NMR and HPLC–ELS–MS for which M.D.M. provided resources. L.R. and M.B. performed biocatalyses for the analyses in gel-based assays. A.P. together with M.B. performed HPAEC–PAD analyses. M.-K.Z. with support from S.M., F.U. and A.T.-S. performed the proteome analyses for which D.B. provided the resources. N.G., C.S.R. and T.R. performed crystallographic experiments and solved the protein structures. G.M. analyzed the crystal structure of the l-rhamnose mutarotase and of the sulfatases. S.T. performed the computational analyses of PUL predictions. J.-H.H. and L.R. wrote the paper with input from U.T.B., G.M., S.M., M.-K.Z. and T.S. All authors read and approved the final manuscript.

Correspondence to Thomas Schweder or Uwe T. Bornscheuer or Jan-Hendrik Hehemann.

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Supplementary information

Supplementary Information

Supplementary Tables 1–11, Supplementary Figures 1–48 and Supplementary Note

Reporting Summary

Supplementary Dataset 1

Summary of all proteins covered by the metabolic labeling approach.

Supplementary Dataset 2

Summary of all proteins covered by the subproteome fractionation.

Supplementary Dataset 3

Sequence-based prediction of subcellular protein localization for ulvan PUL-encoded proteins.

Supplementary Dataset 4

Multimodular proteins of ulvan PUL.

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Fig. 1: Genomic overview of putative ulvan PUL in marine Bacteroidetes and the proteomic response of the F. agariphila PUL to ulvan and rhamnose.
Fig. 2: List of PUL H-encoded and relevant non-PUL H-encoded proteins, corresponding locus tags and functional annotation as well as their relative abundance (mean log2 ratio) with the respective carbon source.
Fig. 3: Structural analyses of ulvan-specific sulfatases.
Fig. 4: Zooming into the degradation of ulvan fragments.
Fig. 5: Structure of the l-rhamnose mutarotase P21_mutarotase.
Fig. 6: Model of the ulvan degradation pathway in F. agariphila as suggested by the proteogenomic, biochemical and structural biological analyses in this study.