Compensatory guaiacyl lignin biosynthesis at the expense of syringyl lignin in 4CL1-knockout poplar

The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated 4-coumarate:CoA ligases (4CLs) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. Here we show that CRISPR-knockout of 4CL1 in poplar (Populus tremula x alba) preferentially reduced syringyl (S) lignin, with negligible effects on biomass recalcitrance. Concordant with reduced S-lignin was downregulation of ferulate 5-hydroxylases (F5Hs). Lignification was largely sustained by 4CL5, a low-affinity paralog of 4CL1 typically with only minor xylem expression or activity. Levels of caffeate, the preferred substrate of 4CL5, increased in line with significant upregulation of caffeoyl shikimate esterase1. Upregulation of caffeoyl-CoA O-methyltransferase1 and downregulation of F5Hs are consistent with preferential funneling of 4CL5 products toward G-lignin biosynthesis at the expense of S-lignin. Thus, transcriptional and metabolic adaptations to 4CL1-knockout appear to have enabled 4CL5 catalysis at a level sufficient to sustain lignification. Finally, genes involved in sulfur assimilation, the glutathione-ascorbate cycle, and various antioxidant systems were upregulated in the mutants, suggesting cascading responses to perturbed thioesterification in lignin biosynthesis

Novel Rhamnogalacturonan I and Arabinoxylan Polysaccharides of Flax Seed Mucilage1[C][OA]

The viscous seed mucilage of flax (Linum usitatissimum) is a mixture of rhamnogalacturonan I and arabinoxylan with novel side group substitutions. The rhamnogalacturonan I has numerous single nonreducing terminal residues of the rare sugar l-galactose attached at the O-3 position of the rhamnosyl residues instead of the typical O-4 position. The arabinoxylan is highly branched, primarily with double branches of nonreducing terminal l-arabinosyl units at the O-2 and O-3 positions along the xylan backbone. While a portion of each polysaccharide can be purified by anion-exchange chromatography, the side group structures of both polysaccharides are modified further in about one-third of the mucilage to form composites with enhanced viscosity. Our finding of the unusual side group structures for two well-known cell wall polysaccharides supports a hypothesis that plants make a selected few ubiquitous backbone polymers onto which a broad spectrum of side group substitutions are added to engender many possible functions. To this end, modification of one polymer may be accompanied by complementary modifications of others to impart functions to heterocomposites not present in either polymer alone

<i>Kingella kingae</i> Expresses Four Structurally Distinct Polysaccharide Capsules That Differ in Their Correlation with Invasive Disease

<div><p><i>Kingella kingae</i> is an encapsulated gram-negative organism that is a common cause of osteoarticular infections in young children. In earlier work, we identified a glycosyltransferase gene called <i>csaA</i> that is necessary for synthesis of the [3)-β-Gal<i>p</i>NAc-(1→5)-β-Kdo<i>p</i>-(2→] polysaccharide capsule (type a) in <i>K</i>. <i>kingae</i> strain 269–492. In the current study, we analyzed a large collection of invasive and carrier isolates from Israel and found that <i>csaA</i> was present in only 47% of the isolates. Further examination of this collection using primers based on the sequence that flanks <i>csaA</i> revealed three additional gene clusters (designated the <i>csb</i>, <i>csc</i>, and <i>csd</i> loci), all encoding predicted glycosyltransferases. The <i>csb</i> locus contains the <i>csbA</i>, <i>csbB</i>, and <i>csbC</i> genes and is associated with a capsule that is a polymer of [6)-α-Glc<i>p</i>NAc-(1→5)-β-(8-OAc)Kdo<i>p</i>-(2→] (type b). The <i>csc</i> locus contains the <i>cscA</i>, <i>cscB</i>, and <i>cscC</i> genes and is associated with a capsule that is a polymer of [3)-β-Rib<i>f</i>-(1→2)-β-Rib<i>f</i>-(1→2)-β-Rib<i>f</i>-(1→4)-β-Kdo<i>p</i>-(2→] (type c). The <i>csd</i> locus contains the <i>csdA</i>, <i>csdB</i>, and <i>csdC</i> genes and is associated with a capsule that is a polymer of [P-(O→3)[β-Gal<i>p</i>-(1→4)]-β-Glc<i>p</i>NAc-(1→3)-α-Glc<i>p</i>NAc-1-] (type d). Introduction of the <i>csa</i>, <i>csb</i>, <i>csc</i>, and <i>csd</i> loci into strain KK01Δ<i>csa</i>, a strain 269–492 derivative that lacks the native <i>csaA</i> gene, was sufficient to produce the type a capsule, type b capsule, type c capsule, and type d capsule, respectively, indicating that these loci are solely responsible for determining capsule type in <i>K</i>. <i>kingae</i>. Further analysis demonstrated that 96% of the invasive isolates express either the type a or type b capsule and that a disproportionate percentage of carrier isolates express the type c or type d capsule. These results establish that there are at least four structurally distinct <i>K</i>. <i>kingae</i> capsule types and suggest that capsule type plays an important role in promoting <i>K</i>. <i>kingae</i> invasive disease.</p></div

PCR screening of capsule synthesis genes reveals four loci.

<p>(A) PCR amplification across the variable synthesis region using flanking primers produces four amplicon sizes. (B) NruI digest of <i>arg/hemB</i> amplicon. (C) Illustration of the four capsule synthesis loci and the engineered empty locus. Each locus shows the capsule synthesis genes unique to each capsule type (white) and the highly homologous flanking regions shared among all strains (gray). Solid arrows (size not to scale) denote the approximate location of the screening primers that anneal to the homologous flanking regions used to amplify across the flanking genes irrespective of internal sequence (as shown in Panel A). The dashed arrows above each locus denote the approximate location of the locus-specific screening primers that generate amplicons for (D) the <i>csa</i> locus (~2400 bp), (E) the <i>csb</i> locus (~2200 bp), (F) the <i>csc</i> locus (~2750 bp), and (G) the <i>csd</i> locus (~4100 bp). Lane 1, ladder; lane 2, KK01; lane 3, PYKK98; lane 4, PYKK93; lane 5, PYKK89; lane 6, PYKK121; lane 7, PYKK58; lane 8, PYKK59; lane 9, PYKK60; lane 10, D7674; lane 11, E3339; lane 12, D7453; lane 13, BB270.</p

Comparative molar ratio of main glycosyl residues in polysaccharide purified from the surface of the capsule swap strains as detected by 1-D Proton NMR.

<p>Comparative molar ratio of main glycosyl residues in polysaccharide purified from the surface of the capsule swap strains as detected by 1-D Proton NMR.</p

A library of chemically defined human N-glycans synthesized from microbial oligosaccharide precursors

Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and reference standards. Here, we describe a combined biological/enzymatic synthesis that is capable of efficiently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, our approach involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplified purification scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases including key N-acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated and subsequently used to develop a glycan microarray for high-throughput, fluorescence-based screening of glycan-binding proteins. Taken together, these results confirm our combined synthesis strategy as a new, user-friendly route for supplying chemically defined human glycans simply by combining biosynthetically-derived precursors with enzymatic remodeling

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Association between capsule locus screening and capsule composition.

<p>Association between capsule locus screening and capsule composition.</p

Comparison of capsule migration pattern between capsule types.

<p>Alcian blue stained gel depicting the migration pattern of capsule material purified from the surface of the source strains (lanes 2–5), capsule locus deletion mutants (lane 6–9), and the capsule complements (lanes 10–13). Lane 1, ladder; lane 2, KK01; lane 3, PYKK58; lane 4, PYKK60; lane 5, BB270; lane 6, KK01Δ<i>csa</i>; lane 7, PYKK58Δ<i>csb</i>; lane 8, PYKK60Δ<i>csc</i>; lane 9 BB270Δ<i>csd</i>; lane 10, KK01Δ<i>csa(csa)</i>; lane 11 PYKK58Δ<i>csb(csb)</i>; lane 12, PYKK60Δ<i>csc(csc)</i>; lane 13 BB270Δ<i>csd(csd)</i>.</p

Chemical shift assignments of the type d capsular polysaccharide purified from BB270.

<p>Chemical shift assignments of the type d capsular polysaccharide purified from BB270.</p