In Vitro Analysis of d‑Lactyl-CoA-Polymerizing Polyhydroxyalkanoate Synthase in Polylactate and Poly(lactate-<i>co</i>-3-hydroxybutyrate) Syntheses

Engineered d-lactyl-coenzyme A (LA-CoA)-polymerizing polyhydroxyalkanoate synthase (PhaC1_PsSTQK) efficiently produces poly­(lactate-<i>co</i>-3-hydroxybutyrate) [P­(LA-<i>co</i>-3HB]) copolymer in recombinant Escherichia coli, while synthesizing tiny amounts of poly­(lactate) (PLA)-like polymers in recombinant Corynebacterium glutamicum. To elucidate the mechanisms underlying the interesting phenomena, <i>in vitro</i> analysis of PhaC1_PsSTQK was performed using homo- and copolymerization conditions of LA-CoA and 3-hydroxybutyryl-CoA. PhaC1_PsSTQK polymerized LA-CoA as a sole substrate. However, the extension of PLA chains completely stalled at a molecular weight of ∼3000, presumably due to the low mobility of the generated polymer. The copolymerization of these substrates only proceeded with a low concentration of LA-CoA. In fact, the intracellular LA-CoA concentration in P­(LA-<i>co</i>-3HB)-producing E. coli was below the detection limit, while that in C. glutamicum was as high as acetyl-CoA levels. Therefore, it was concluded that the mobility of polymerized products and LA-CoA concentration are dominant factors characterizing PLA and P­(LA-<i>co</i>-3HB) biosynthetic systems

A protease/peptidase from culture medium of <i>Flammulina velutipes</i> that acts on arabinogalactan-protein

<p>Arabinogalactan-proteins (AGPs) are highly diverse plant proteoglycans found on the plant cell surface. AGPs have large arabinogalactan (AG) moieties attached to a core-protein rich in hydroxyproline (Hyp). The AG undergoes hydrolysis by various glycoside hydrolases, most of which have been identified, whereas the core-proteins is presumably degraded by unknown proteases/peptidases secreted from fungi and bacteria in nature. Although several enzymes hydrolyzing other Hyp-rich proteins are known, the enzymes acting on the core-proteins of AGPs remain to be identified. The present study describes the detection of protease/peptidase activity toward AGP core-proteins in the culture medium of winter mushroom (<i>Flammulina velutipes</i>) and partial purification of the enzyme by several conventional chromatography steps. The enzyme showed higher activity toward Hyp residues than toward proline and alanine residues and acted on core-proteins prepared from gum arabic. Since the activity was inhibited in the presence of Pefabloc SC, the enzyme is probably a serine protease.</p> <p>The degradation of the core-protein of AGPs by a protease/peptidase from winter mushroom.</p

Dynamic Changes of Intracellular Monomer Levels Regulate Block Sequence of Polyhydroxyalkanoates in Engineered <i>Escherichia coli</i>

Biological polymer synthetic systems, which utilize no template molecules, normally synthesize random copolymers. We report an exception, a synthesis of block polyhydroxyalkanoates (PHAs) in an engineered <i>Escherichia coli</i>. Using an engineered PHA synthase, block copolymers poly­[(<i>R</i>)-2-hydroxybutyrate­(2HB)-<i>b</i>-(<i>R</i>)-3-hydroxybutyrate­(3HB)] were produced in <i>E. coli</i>. The covalent linkage between P­(2HB) and P­(3HB) segments was verified with solvent fractionation and microphase separation. Notably, the block sequence was generated under the simultaneous consumption of two monomer precursors, indicating the existence of a rapid monomer switching mechanism during polymerization. Based on <i>in vivo</i> metabolic intermediate analysis and the relevant <i>in vitro</i> enzymatic activities, we propose a model in which the rapid intracellular 3HB-CoA fluctuation during polymer synthesis is a major factor in generating block sequences. The dynamic change of intracellular monomer levels is a novel regulatory principle of monomer sequences of biopolymers

Wood decay characteristics.

<p>Comparative weight loss of parental strain 11061 and single basidiospore derivatives on colonized loblolly pine wood (<i>Pinus taeda</i>) wood wafers were determined after 4, 8 and 12 weeks incubation (bottom left panel) as described in Methods. Single basidiospore strain 5–6 also aggressively decayed birch and spruce (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004759#pgen.1004759.s057" target="_blank">Text S1</a>) and was selected for sequencing. Upper panels show scanning electron microscopy <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004759#pgen.1004759-Blanchette2" target="_blank">[68]</a> of radial (left) and transverse (right) sections of pine wood tracheids that were substantially eroded or completely degraded by <i>P. gigantea</i> strain 5–6 by week twelve. Transverse section of sound wood (bottom photo) provides comparison. (Bar  = 40 µm).</p

Genes encoding LC-MS/MS detected proteins and exhibiting>2-fold regulation in comparisons of NELP and ELP cultures.¹

<p>Genes encoding LC-MS/MS detected proteins and exhibiting>2-fold regulation in comparisons of NELP and ELP cultures.^{<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004759#nt108" target="_blank">1</a>}</p

Schematic representations of lignocellulose components in cell walls of pine wood.

<p>Panel A: The extractives (long chain fatty acids, triglycerides, resin acids and terpenes) are found primarily in the resin ducts, but damage to pine wood causes the release of these compounds across wounded areas. Panel B: In tracheid cell walls, the amorphous, phenylpropanoid polymer lignin (brown) form a matrix around the more structured carbohydrate polymers, hemicellulose (yellow and green) and cellulose (blue).</p

Transcripts accumulating>4-fold in non-extracted loblolly pine wood (NELP) relative to extracted loblolly pine wood (ELP).¹

<p>Transcripts accumulating>4-fold in non-extracted loblolly pine wood (NELP) relative to extracted loblolly pine wood (ELP).^{<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004759#nt105" target="_blank">1</a>}</p

<i>P. gigantea</i> transcriptome.

<p>Scatterplot (A) shows the distribution of RNA-seq RPKM values (log₂) for 11,376 <i>P. gigantea</i> genes when grown on basal salts containing acetone-extracted loblolly pine wood (ELP) or non-extracted loblolly pine wood (NELP). Lines define 2-fold borders and best fit regression. Darkened points represent 44 transcripts accumulating>4-fold at p<0.01. Venn diagram (B) illustrates genes with RPKM signals>10 and upregulated>4-fold in NELP relative to ELP. Twenty-two genes showed significant transcript accumulation in NELP relative to ELP suggesting potential response to resin and pitch content. Under these stringent thresholds (p<0.01;>4-fold), only one gene, a MCO model Phlgi1_129839, showed significant transcript accumulation in ELP relative to NELP. Additional detail appears in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004759#pgen-1004759-t001" target="_blank">Tables 1</a>-<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004759#pgen-1004759-t003" target="_blank">3</a>. Detailed methods and complete data are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004759#pgen.1004759.s057" target="_blank">Text S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004759#pgen.1004759.s059" target="_blank">Dataset S2</a>.</p

Differentially regulated genes in media containing non-extracted loblolly pine wood (NELP), solvent extracted loblolly pine wood (ELP), or glucose (Glc) as sole carbon source.

<p>Differentially regulated genes in media containing non-extracted loblolly pine wood (NELP), solvent extracted loblolly pine wood (ELP), or glucose (Glc) as sole carbon source.</p

Glyoxalate shunt and proposed relationship to lipid oxidation when <i>P. gigantea</i> is cultivated on wood-containing media (ELP or NELP) relative to Glc medium.

<p>Enzymes encoded by upregulated genes are black highlighted and associated with thickened arrows. Abbreviations: ABC-G1, ABC transporter associated with monoterpene tolerance; ADH/AO, Acyl-CoA dehydrogenase/oxidase; AH, Aconitate hydratase; CoA ligase, long fatty acid-CoA ligase; DLAT, Dihydrolipoyllysine-residue acetyltransferase; DLST, Dihydrolipoyllysine-residue succinyltransferase; EH, Enoyl-CoA hydratase; FDH, Formate dehydrogenase; FH, Fumarate hydratase; KT, Ketothiolase (acetyl-CoA C-acyltransferase); HAD, 3-Hydroxyacyl-CoA dehydrogenase; ICL, Isocitrate lyase; IDH, Isocitrate dehydrogenase; MDH, Malate dehydrogenase; MS, Malate synthase; ODH, Oxoglutarate dehydrogenase; OXA, Oxaloacetase; OXDC, Oxalate decarboxylase; OXO, Oxalate oxidase; PC, Pyruvate carboxylase; PDH, Pyruvate dehydrogenase; PEP, Phosphoenolpyruvate; PEPCK, Phosphoenolpyruvate carboxykinase; PEPK, Phosphoenolpyruvate kinase; SDH, succinate dehydrogenase. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004759#pgen.1004759.s059" target="_blank">Dataset S2</a> for detailed gene expression data.</p