Identification and Quantification of Glycans in Whole Cells: Architecture of Microalgal Polysaccharides Described by Solid-State Nuclear Magnetic Resonance

Abstract

Microalgae are photosynthetic organisms widely distributed in nature and serve as a sustainable source of bioproducts. Their carbohydrate components are also promising candidates for bioenergy production and bioremediation, but the structural characterization of these heterogeneous polymers in cells remains a formidable problem. Here we present a widely applicable protocol for identifying and quantifying the glycan content using magic-angle-spinning (MAS) solid-state NMR (ssNMR) spectroscopy, with validation from glycosyl linkage and composition analysis deduced from mass-spectrometry (MS). Two-dimensional 13C–13C correlation ssNMR spectra of a uniformly 13C-labeled green microalga Parachlorella beijerinckii reveal that starch is the most abundant polysaccharide in a naturally cellulose-deficient strain, and this polymer adopts a well-organized and highly rigid structure in the cell. Some xyloses are present in both the mobile and rigid domains of the cell wall, with their chemical shifts partially aligned with the flat-ribbon 2-fold xylan identified in plants. Surprisingly, most other carbohydrates are largely mobile, regardless of their distribution in glycolipids or cell walls. These structural insights correlate with the high digestibility of this cellulose-deficient strain, and the in-cell ssNMR methods will facilitate the investigations of other economically important algae species.

Solid-State NMR Investigations of Extracellular Matrixes and Cell Walls of Algae, Bacteria, Fungi, and Plants

“…In addition, starch was found to adopt a well-organized structure in the cell, as shown by the extensive intermolecular contacts between its constituent molecules (Figure B) . Some xyloses were found to exist in both the mobile and rigid regions of the cell wall, with their chemical shifts partially aligned with the flat-ribbon 2-fold xylan that was initially recognized in plants.…”

“…Recently, we have introduced a broadly applicable protocol for quantifying the content and linkages of glycans in microalgae using ssNMR spectroscopy . The method was demonstrated on a naturally cellulose-deficient strain (CK-5) of the green microalga Parachlorella beijerinckii , which is a promising candidate for producing high-value fermentable carbohydrates.…”

“…The deconvoluted (blue) peaks are plotted underneath the simulated (magenta) and measured (black) spectra. Adapted with permission from ref . Copyright 2021 American Chemical Society.…”

Solid-State NMR Dipolar and Chemical Shift Anisotropy Recoupling Techniques for Structural and Dynamical Studies in Biological Systems

“…The structural studies are thus more restricted in the assembly modes among and within different types of polysaccharides such as cellulose, pectin, and hemicellulose and their contacts with other components including lignin, protein, water, and more . Consequently, long-range correlations obtained via SD- and TSAR-based recoupling methods have been important tools for the structural analysis of the carbohydrates-rich biosystems . Moreover, the effective 13 C recoupling bandwidth should cover the 60–110 ppm region for the C–OH and C–O–C species, which are of central importance in carbohydrates.…”

Solid-state NMR analysis of unlabeled fungal cell walls from Aspergillus and Candida species

“…The fungal cell walls, especially those of C. albicans , are substantially more mobile than the plant materials and are considered unfavorable for MAS-DNP. The success of natural-abundance DNP thus opens the frontier to interrogate other cellular systems with similar dynamical characteristics, such as microalgae, bacteria, and human and animal cells ( Takahashi, 2013 , Overall and Barnes, 2021 , Albert et al, 2018 , Romaniuk and Cegelski, 2015 , Poulhazan, 2021 , Poulhazan et al, 2018 , Arnold et al, 2018 , Ghosh et al, 2021 ), without isotopic enrichment.

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