Building custom polysaccharides in vitro with an efficient, broad-specificity xyloglucan glycosynthase and a fucosyltransferase

Abstract

The current drive for applications of biomass-derived compounds, for energy and advanced materials, has led to a resurgence of interest in the manipulation of plant polymers. The xyloglucans, a family of structurally complex plant polysaccharides, have attracted significant interest due to their intrinsic high affinity for cellulose, both in muro and in technical applications. Moreover, current cell wall models are limited by the lack of detailed structure-property relationships of xyloglucans, due to a lack of molecules with well-defined branching patterns. Here, we have developed a new, broad-specificity "xyloglucan glycosynthase", selected from active-site mutants of a bacterial endoxyloglucanase, which catalyzed the synthesis of high molar mass polysaccharides, with complex side-chain structures, from suitable glycosyl fluoride donor substrates. The product range was further extended by combination with an Arabidopsis thaliana α(1→2)-fucosyltransferase to achieve the in vitro synthesis of fucosylated xyloglucans typical of dicot primary cell walls. These enzymes thus comprise a toolkit for the controlled enzymatic synthesis of xyloglucans that are otherwise impossible to obtain from native sources. Moreover, this study demonstrates the validity of a chemo-enzymatic approach to polysaccharide synthesis, in which the simplicity and economy of glycosynthase technology is harnessed together with the exquisite specificity of glycosyltransferases to control molecular complexity.

Figure 1

General structure of XXXG-type xyloglucans and structures of α-glycosyl fluoride substrates for xyloglucan glycosynthases. (a) Variable substitution of the Xyl₃Glc₄ core repeat in xyloglucans. The standard xyloglucan structural abbreviations(55) are given below the corresponding motifs, assuming full branch substitution. For example, the galactoxyloglucan from tamarind seeds is predominantly comprised of XXXG, XXLG, and XLLG units, with minor amounts of XLXG units.(56) In contrast, the fucogalactoxyloglucan from Arabidopsis thaliana primary cell walls contains primarily XXXG, XXFG, and XLFG units, with minor amounts of other structures.(48) (b) α-Glycosyl fluoride substrates for xyloglucan glycosynthases.

Figure 2

General scheme for the production of xyloglucans with defined, homogoenous side-chain structure using a combined glycosynthase/glycosyltransferase approach. Oligosaccharide nomenclature is identical to that shown in Figure 1.

Figure 3

PpXG5 E323G activity on α-xyloglucosyl fluorides. (A) Initial rate kinetics of PpXG5 E323G with XXXGαF; (B) initial rate kinetics of PpXG5 E323G with XLLGαF. Error bars represent standard deviations of duplicate measurements.

Figure 4

HPSEC-ELS analysis of the PpXG5 E323G glycosynthase reactions with XXXGαF or XLLGαF as substrates. Dashed line, XXXGαF products formed after overnight incubation at 30 °C; solid line, XLLGαF products formed after overnight incubation at 30 °C. The eluent was DMSO, and molar mass calibration was performed with pullulan standards.

Figure 5

Fucosylation of (XLLG)_n-based xyloglucans by AtFUT1. MALDI-TOF MS of pure XLLG standard (A) and endo-xyloglucanase limit digests of (XLLG)nαF (M_w 12 000, M_w/M_n 1.45) incubated with AtFUT1 and GDP-l-Fuc for 0 min (B), 6 h (C), and 17 h (D).