Deletion of a single glycosyltransferase in Caldicellulosiruptor bescii eliminates protein glycosylation and growth on crystalline cellulose

Abstract

Protein glycosylation pathways have been identified in a variety of bacteria and are best understood in pathogens and commensals in which the glycosylation targets are cell surface proteins, such as S layers, pili, and flagella. In contrast, very little is known about the glycosylation of bacterial enzymes, especially those secreted by cellulolytic bacteria. Caldicellulosiruptor bescii secretes several unique synergistic multifunctional biomass-degrading enzymes, notably cellulase A which is largely responsible for this organism’s ability to grow on lignocellulosic biomass without the conventional pretreatment. It was recently discovered that extracellular CelA is heavily glycosylated. In this work, we identified an O-glycosyltransferase in the C. bescii chromosome and targeted it for deletion. The resulting mutant was unable to grow on crystalline cellulose and showed no detectable protein glycosylation. Multifunctional biomass-degrading enzymes in this strain were rapidly degraded. With the genetic tools available in C. bescii, this system represents a unique opportunity to study the role of bacterial enzyme glycosylation as well an investigation of the pathway for protein glycosylation in a non-pathogen.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1266-x) contains supplementary material, which is available to authorized users.

Insights into Thermophilic Plant Biomass Hydrolysis from Caldicellulosiruptor Systems Biology

“…Glycosylation of CelA with galactose disaccharides was confirmed by overexpression in the C. bescii ∆celA strain [89,90], only extracellular CelA was observed as being glycosylated [89] and the glycotransferase (GT) located in the GDL is required for CelA glycosylation [79,91]. Initially, deletion of the GT responsible for glycosylation of CelA resulted in proteolytic clipping of extracellular enzymes and lower cell densities observed by plate counts from planktonic cells growing on microcrystalline cellulose [91]. In contrast, deletion of the same GT from a different parental C. bescii strain did not result in a dramatic effect, and the GT deletion strain was still capable of solubilizing microcrystalline cellulose [79].…”

“…In contrast, Conway et al monitored cellulose solubilization in vivo, and other factors aside from glycosylation may impact enzyme efficiency [79]. Lastly, while fewer C. bescii GT knock out colonies were observed by Russell et al, [91], no comparison in growth on a solid medium was made between planktonic cells grown on cellobiose versus cellulose. It is possible that in addition to reduced stability of extracellular hydrolytic enzymes, the glycosylation deficient C. bescii strains are also deficient in their ability to form colonies on solid medium, resulting in a false negative result for growth on microcrystalline cellulose.…”

“…Linker regions present in modular enzymes are rich in proline-threonine repeats that indicate that CelA is likely glycosylated, and one report on the native purification of CelA indicated that the purified protein stained positively for glycosylation [19]. Glycosylation of CelA with galactose disaccharides was confirmed by overexpression in the C. bescii ∆celA strain [89,90], only extracellular CelA was observed as being glycosylated [89] and the glycotransferase (GT) located in the GDL is required for CelA glycosylation [79,91]. Initially, deletion of the GT responsible for glycosylation of CelA resulted in proteolytic clipping of extracellular enzymes and lower cell densities observed by plate counts from planktonic cells growing on microcrystalline cellulose [91].…”

“…An important first step in increasing cellulolytic activity in C. bescii was demonstrated by chromosomal expression of a β-glucanase and/or endoglucanse from A. cellulolyticus, which resulted in synergistic increases in cellulose hydrolysis, and over a 17-fold improvement of C. bescii growth on cellulose [137]. Heterologous expression of a cellobiose phosphorylase from Thermotoga maritima to relieve cellobiose inhibition improved the performance of the C. bescii endoglucanase knock-in strain [91], and the best gains in cellulolytic capacity were observed when all three enzymes (β-glucanase, endoglucanse, and cellobiose phosphorylase) were co-expressed in C. bescii [138]. Clearly, even though the GDL of C. bescii had evolved to maximize synergy between the endogenous C. bescii enzymes, there are opportunities to engineer superior cellulolytic strains.…”

Investigation of a thermostable multi-domain xylanase-glucuronoyl esterase enzyme from Caldicellulosiruptor kristjanssonii incorporating multiple carbohydrate-binding modules

“…As we were unable to assay the activity of CkGE15A at elevated temperatures due to the temperature sensitivity of the substrates, this comparison is likely not a good reflection of the enzyme's true catalytic efficiency at high temperatures. Additionally, Caldicellulosiruptor bacteria have been shown to glycosylate CAZymes [59,60], and possibly also CkXyn10C-GE15A is glycosylated in vivo and requires these post-translational modifications for full activity, as it has previously been shown that glycosylation is of great importance to the activity of other esterases, increasing their reaction rates by several orders of magnitude [61]. All of these factors considered, it is likely that the true kinetic parameters for CkGE15A is significantly higher than reported here.…”