Ethyl Carbamate Formation Regulated by Lactic Acid Bacteria and Nonconventional Yeasts in Solid-State Fermentation of Chinese <i>Moutai</i>-Flavor Liquor

This study aimed to identify specific microorganisms related to the formation of precursors of EC (ethyl carbamate) in the solid-state fermentation of Chinese <i>Moutai</i>-flavor liquor. The EC content was significantly correlated with the urea content during the fermentation process (<i>R</i>² = 0.772, <i>P</i> < 0.01). Differences in urea production and degradation were found at both species and functional gene levels by metatranscriptomic sequencing and culture-dependent analysis. <i>Lactobacillus</i> spp. could competitively degrade arginine through the arginine deiminase pathway with yeasts, and most <i>Lactobacillus</i> species were capable of degrading urea. Some dominant nonconventional yeasts, such as <i>Pichia</i>, <i>Schizosaccharomyces</i>, and <i>Zygosaccharomyces</i> species, were shown to produce low amounts of urea relative to <i>Saccharomyces cerevisiae</i>. Moreover, unusual urea degradation pathways (urea carboxylase, allophanate hydrolase, and ATP-independent urease) were identified. Our results indicate that EC precursor levels in the solid-state fermentation can be controlled using lactic acid bacteria and nonconventional yeasts

Enumeration of Virtual Libraries of Combinatorial Modular Macrocyclic (Bracelet, Necklace) Architectures and Their Linear Counterparts

A wide variety of cyclic molecular architectures are built of modular subunits and can be formed combinatorially. The mathematics for enumeration of such objects is well-developed yet lacks key features of importance in chemistry, such as specifying (i) the structures of individual members among a set of isomers, (ii) the distribution (i.e., relative amounts) of products, and (iii) the effect of nonequal ratios of reacting monomers on the product distribution. Here, a software program (<i>Cyclaplex</i>) has been developed to determine the number, identity (including isomers), and relative amounts of linear and cyclic architectures from a given number and ratio of reacting monomers. The program includes both mathematical formulas and generative algorithms for enumeration; the latter go beyond the former to provide desired molecular-relevant information and data-mining features. The program is equipped to enumerate four types of architectures: (i) linear architectures with directionality (macroscopic equivalent = electrical extension cords), (ii) linear architectures without directionality (batons), (iii) cyclic architectures with directionality (necklaces), and (iv) cyclic architectures without directionality (bracelets). The program can be applied to cyclic peptides, cycloveratrylenes, cyclens, calixarenes, cyclodextrins, crown ethers, cucurbiturils, annulenes, expanded meso-substituted porphyrin­(ogen)­s, and diverse supramolecular (e.g., protein) assemblies. The size of accessible architectures encompasses up to 12 modular subunits derived from 12 reacting monomers or larger architectures (e.g. 13–17 subunits) from fewer types of monomers (e.g. 2–4). A particular application concerns understanding the possible heterogeneity of (natural or biohybrid) photosynthetic light-harvesting oligomers (cyclic, linear) formed from distinct peptide subunits

Sequence logos of the multiple alignments of the 214 CYP93 proteins in plants.

<p>The sequence logos of plant CYP93 proteins based on amino acid alignment using MAFFT are shown. The logos were generated using Weblogo. The bit score indicates the information content for each position in the sequence. The height of the letter designating the amino acid residue at each position represents the degree of conservation. The key conserved motifs are underlined; the red lines indicate the less conserved regions; the black ones, the P450 motifs; and the blue ones, the substrate recognition sites (SRSs). The white triangles indicate the conserved intron insertion location of plant CYP93 genes; the numbers within the triangles indicate the splicing phase of the intron (0 refers to phase 0). The red and black dots indicate the conserved amino acid insertion or deletion sites, respectively, in a given subfamily and/or clade; the number below each dot indicates the corresponding subfamily, i.e., B indicates the CYP93B subfamily.</p

Genome-Wide Analysis, Classification, Evolution, and Expression Analysis of the Cytochrome P450 93 Family in Land Plants

<div><p>Cytochrome P450 93 family (CYP93) belonging to the cytochrome P450 superfamily plays important roles in diverse plant processes. However, no previous studies have investigated the evolution and expression of the members of this family. In this study, we performed comprehensive genome-wide analysis to identify CYP93 genes in 60 green plants. In all, 214 CYP93 proteins were identified; they were specifically found in flowering plants and could be classified into ten subfamilies—CYP93A–K, with the last two being identified first. CYP93A is the ancestor that was derived in flowering plants, and the remaining showed lineage-specific distribution—CYP93B and CYP93C are present in dicots; CYP93F is distributed only in Poaceae; CYP93G and CYP93J are monocot-specific; CYP93E is unique to legumes; CYP93H and CYP93K are only found in <i>Aquilegia coerulea</i>, and CYP93D is Brassicaceae-specific. Each subfamily generally has conserved gene numbers, structures, and characteristics, indicating functional conservation during evolution. Synonymous nucleotide substitution (<i>d</i>_N/<i>d</i>_S) analysis showed that CYP93 genes are under strong negative selection. Comparative expression analyses of CYP93 genes in dicots and monocots revealed that they are preferentially expressed in the roots and tend to be induced by biotic and/or abiotic stresses, in accordance with their well-known functions in plant secondary biosynthesis.</p></div

A putative motif conserved in group G9 proteins.

<p>Black and gray shading indicate the presence of identical and conserved amino acid residues, respectively, in >75% of the aligned sequences. Consensus amino acid residues are shown below the alignment. The ‘‘x’’ indicates no conservation at this position.</p

Phylogenetic relationships, intron pattern, expression pattern, architecture of conserved protein motifs, and subgroup designations in typical R2R3-MYB proteins from maize (Zm).

<p>A, The neighbor-joining (NJ) tree on the left includes 157 typical R2R3-MYB proteins from maize. The tree shows the 18 phylogenetic subgroups (S1–S18) marked with colored backgrounds, to facilitate subfamily identification with high predictive value. The numbers beside the branches represent bootstrap values (50%) based on 1000 replications. Eight proteins did not fit well into clusters. The colorful marker in the tree indicates the corresponding intron distribution patterns, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037463#pone-0037463-g003" target="_blank">Figure 3</a>, below. B, The gene structure is presented by green exon (s), red MYB domain (s), blue UTR (s), and spaces between the colourful boxes corresponding to introns. The sizes of exons and introns can be estimated using the horizontal lines; the number indicated the phases of corresponding introns. C, The expression patterns of MYB genes in maize. The letter R above the column of expression data refers to root, ST refers to stem, L refers to leaf, FC refers to female catkins, MC refers to male catkins, and S refers to seed. D, Architecture of conserved protein motifs in 18 subfamilies. The motifs on the right were detected using MEME and are graphically represented as white boxes drawn to scale for a representative plant MYB protein of each subfamily.</p

Schematic of the intron distribution patterns within the maize R2R3-MYB DNA-binding domains.

<p>Alignment of DNA-binding domains is representative of 12 intron patterns, named from a to l. Locations of introns are indicated by white triangles. The number within each triangle indicates the splicing phases of the MYB domain sequences: 0 refers to phase 0; 1 refers to phase 1; and 2 refers to phase 2. The number of ZmMYB proteins with each pattern is given on the right. The correlation of intron distribution patterns and phylogenetic subfamilies is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037463#pone-0037463-g002" target="_blank">Figure 2</a>.</p

Phylogenetic tree of the R2R3-MYB proteins from maize (Zm), <i>Arabidopsis</i> (At), and other plant species.

<p>The neighbor-joining tree includes 158 R2R3-MYB proteins from maize, 126 R2R3-MYB proteins from <i>Arabidopsis</i>, and a further 52 R2R3-MYB proteins from other plant species. The proteins are clustered into 37 subgroups (triangles), designated with a subgroup number (e.g., G1). Subfamilies are represented as collapsed triangles, with depth and width proportional to sequence divergence and size, respectively. The black triangles indicate that the subgroup includes ZmMYBs and AtMYBs; the hatched and white triangles indicate that the subgroup includes or excludes ZmMYBs, respectively. Bootstrap values <50% are not shown in the phylogenetic tree. Four proteins did not fit well into clusters. The first 25 subgroups were designated as previously reports of AtMYBs by Stracke et al. (2001) and Dubos et al. (2010). The subgroups were listed in round bracket with annotated functions, for reference. 12 new subgroups were added because of the increased data set.</p

Cloning and Phylogenetic Analysis of <i>Brassica napus</i> L. <i>Caffeic Acid O-Methyltransferase 1</i> Gene Family and Its Expression Pattern under Drought Stress

<div><p>For many plants, regulating lignin content and composition to improve lodging resistance is a crucial issue. Caffeic acid O-methyltransferase (COMT) is a lignin monomer-specific enzyme that controls S subunit synthesis in plant vascular cell walls. Here, we identified 12 <i>BnCOMT1</i> gene homologues, namely <i>BnCOMT1-1</i> to <i>BnCOMT1-12</i>. Ten of 12 genes were composed of four highly conserved exons and three weakly conserved introns. The length of intron I, in particular, showed enormous diversification. Intron I of homologous <i>BnCOMT1</i> genes showed high identity with counterpart genes in <i>Brassica rapa</i> and <i>Brassica oleracea</i>, and intron I from positional close genes in the same chromosome were relatively highly conserved. A phylogenetic analysis suggested that <i>COMT</i> genes experience considerable diversification and conservation in <i>Brassicaceae</i> species, and some <i>COMT1</i> genes are unique in the <i>Brassica</i> genus. Our expression studies indicated that <i>BnCOMT1</i> genes were differentially expressed in different tissues, with <i>BnCOMT1-4</i>, <i>BnCOMT1-5</i>, <i>BnCOMT1-8</i>, and <i>BnCOMT1-10</i> exhibiting stem specificity. These four <i>BnCOMT1</i> genes were expressed at all developmental periods (the bud, early flowering, late flowering and mature stages) and their expression level peaked in the early flowering stage in the stem. Drought stress augmented and accelerated lignin accumulation in high-lignin plants but delayed it in low-lignin plants. The expression levels of <i>BnCOMT1s</i> were generally reduced in water deficit condition. The desynchrony of the accumulation processes of total lignin and <i>BnCOMT1</i>s transcripts in most growth stages indicated that <i>BnCOMT1s</i> could be responsible for the synthesis of a specific subunit of lignin or that they participate in other pathways such as the melatonin biosynthesis pathway.</p></div

Phylogenetic relationships of the 60 species investigated in the present study.

<p>Phylogenetic relationships (branch lengths are arbitrary) among these species have been described previously (<a href="http://www.phytozome.net/" target="_blank">http://www.phytozome.net/</a>). The total number of cytochrome P450 93 (CYP93) proteins identified in each genome is indicated on the right.</p