Supplementary material for 'Genome mining of cryptic bisabolenes that were biosynthesized by intramembrane terpene synthases from Antrodia cinnamomea'

Terpenoids represent the largest structural family of natural products (NPs) and have various applications in the pharmaceutical, food and fragrance industries. Their diverse scaffolds are generated via a multi-step cyclization cascade of linear isoprene substrates catalysed by terpene synthases (TPSs). Bisabolene NPs, which are sesquiterpenes (C15), have wide applications in medicines and biofuels and serve as bioactive substances in ecology. Despite the discovery of some canonical class I TPSs that synthesize bisabolenes from plants, bacteria and insects, it remained unknown whether any bisabolene synthases from fungi could produce bisabolenes as a main product. Antrodia cinnamomea, a Basidiomycota fungus, is a medicinal mushroom indigenous to Taiwan and a known prolific producer of bioactive terpenoids, but little is known regarding the enzymes involved in the biosynthetic pathways. Here, we applied a genome mining approach against A. cinnamomea and discovered two non-canonical UbiA-type TPSs that both synthesize (+)-(S,Z)-α-bisabolene (1). It was determined that two tailoring enzymes, a P450 monooxygenase and a methyltransferase, install a C14-methyl ester on the bisabolene scaffold. In addition, four new bisabolene derivatives, 2 and 4‒6, were characterized from heterologous reconstitution in Saccharomyces cerevisiae. Our study uncovered enzymatic tools to generate structurally diverse bisabolene NPs.This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’

Evaluation of <i>Drosophila</i> Metabolic Labeling Strategies for <i>in Vivo</i> Quantitative Proteomic Analyses with Applications to Early Pupa Formation and Amino Acid Starvation

Although stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was first developed as a cell culture-based technique, stable isotope-labeled amino acids have since been successfully introduced <i>in vivo</i> into select multicellular model organisms by manipulating the feeding diets. An earlier study by others has demonstrated that heavy lysine labeled <i>Drosophila melanogaster</i> can be derived by feeding with an exclusive heavy lysine labeled yeast diet. In this work, we have further evaluated the use of heavy lysine and/or arginine for metabolic labeling of fruit flies, with an aim to determine its respective quantification accuracy and versatility. <i>In vivo</i> conversion of heavy lysine and/or heavy arginine to several nonessential amino acids was observed in labeled flies, leading to distorted isotope pattern and underestimated heavy to light ratio. These quantification defects can nonetheless be rectified at protein level using the normalization function. The only caveat is that such a normalization strategy may not be suitable for every biological application, particularly when modified peptides need to be individually quantified at peptide level. In such cases, we showed that peptide ratios calculated from the summed intensities of all isotope peaks are less affected by the heavy amino acid conversion and therefore less sequence-dependent and more reliable. Applying either the single Lys8 or double Lys6/Arg10 metabolic labeling strategy to flies, we quantitatively mapped the proteomic changes during the onset of metamorphosis and upon amino acid deprivation. The expression of a number of steroid hormone 20-hydroxyecdysone regulated proteins was found to be changed significantly during larval–pupa transition, while several subunits of the V-ATPase complex and components regulating actomyosin were up-regulated under starvation-induced autophagy conditions

Evaluation of <i>Drosophila</i> Metabolic Labeling Strategies for <i>in Vivo</i> Quantitative Proteomic Analyses with Applications to Early Pupa Formation and Amino Acid Starvation

Although stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was first developed as a cell culture-based technique, stable isotope-labeled amino acids have since been successfully introduced <i>in vivo</i> into select multicellular model organisms by manipulating the feeding diets. An earlier study by others has demonstrated that heavy lysine labeled <i>Drosophila melanogaster</i> can be derived by feeding with an exclusive heavy lysine labeled yeast diet. In this work, we have further evaluated the use of heavy lysine and/or arginine for metabolic labeling of fruit flies, with an aim to determine its respective quantification accuracy and versatility. <i>In vivo</i> conversion of heavy lysine and/or heavy arginine to several nonessential amino acids was observed in labeled flies, leading to distorted isotope pattern and underestimated heavy to light ratio. These quantification defects can nonetheless be rectified at protein level using the normalization function. The only caveat is that such a normalization strategy may not be suitable for every biological application, particularly when modified peptides need to be individually quantified at peptide level. In such cases, we showed that peptide ratios calculated from the summed intensities of all isotope peaks are less affected by the heavy amino acid conversion and therefore less sequence-dependent and more reliable. Applying either the single Lys8 or double Lys6/Arg10 metabolic labeling strategy to flies, we quantitatively mapped the proteomic changes during the onset of metamorphosis and upon amino acid deprivation. The expression of a number of steroid hormone 20-hydroxyecdysone regulated proteins was found to be changed significantly during larval–pupa transition, while several subunits of the V-ATPase complex and components regulating actomyosin were up-regulated under starvation-induced autophagy conditions

Evaluation of <i>Drosophila</i> Metabolic Labeling Strategies for <i>in Vivo</i> Quantitative Proteomic Analyses with Applications to Early Pupa Formation and Amino Acid Starvation

Although stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was first developed as a cell culture-based technique, stable isotope-labeled amino acids have since been successfully introduced <i>in vivo</i> into select multicellular model organisms by manipulating the feeding diets. An earlier study by others has demonstrated that heavy lysine labeled <i>Drosophila melanogaster</i> can be derived by feeding with an exclusive heavy lysine labeled yeast diet. In this work, we have further evaluated the use of heavy lysine and/or arginine for metabolic labeling of fruit flies, with an aim to determine its respective quantification accuracy and versatility. <i>In vivo</i> conversion of heavy lysine and/or heavy arginine to several nonessential amino acids was observed in labeled flies, leading to distorted isotope pattern and underestimated heavy to light ratio. These quantification defects can nonetheless be rectified at protein level using the normalization function. The only caveat is that such a normalization strategy may not be suitable for every biological application, particularly when modified peptides need to be individually quantified at peptide level. In such cases, we showed that peptide ratios calculated from the summed intensities of all isotope peaks are less affected by the heavy amino acid conversion and therefore less sequence-dependent and more reliable. Applying either the single Lys8 or double Lys6/Arg10 metabolic labeling strategy to flies, we quantitatively mapped the proteomic changes during the onset of metamorphosis and upon amino acid deprivation. The expression of a number of steroid hormone 20-hydroxyecdysone regulated proteins was found to be changed significantly during larval–pupa transition, while several subunits of the V-ATPase complex and components regulating actomyosin were up-regulated under starvation-induced autophagy conditions

Evaluation of <i>Drosophila</i> Metabolic Labeling Strategies for <i>in Vivo</i> Quantitative Proteomic Analyses with Applications to Early Pupa Formation and Amino Acid Starvation

Although stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was first developed as a cell culture-based technique, stable isotope-labeled amino acids have since been successfully introduced <i>in vivo</i> into select multicellular model organisms by manipulating the feeding diets. An earlier study by others has demonstrated that heavy lysine labeled <i>Drosophila melanogaster</i> can be derived by feeding with an exclusive heavy lysine labeled yeast diet. In this work, we have further evaluated the use of heavy lysine and/or arginine for metabolic labeling of fruit flies, with an aim to determine its respective quantification accuracy and versatility. <i>In vivo</i> conversion of heavy lysine and/or heavy arginine to several nonessential amino acids was observed in labeled flies, leading to distorted isotope pattern and underestimated heavy to light ratio. These quantification defects can nonetheless be rectified at protein level using the normalization function. The only caveat is that such a normalization strategy may not be suitable for every biological application, particularly when modified peptides need to be individually quantified at peptide level. In such cases, we showed that peptide ratios calculated from the summed intensities of all isotope peaks are less affected by the heavy amino acid conversion and therefore less sequence-dependent and more reliable. Applying either the single Lys8 or double Lys6/Arg10 metabolic labeling strategy to flies, we quantitatively mapped the proteomic changes during the onset of metamorphosis and upon amino acid deprivation. The expression of a number of steroid hormone 20-hydroxyecdysone regulated proteins was found to be changed significantly during larval–pupa transition, while several subunits of the V-ATPase complex and components regulating actomyosin were up-regulated under starvation-induced autophagy conditions

Evaluation of <i>Drosophila</i> Metabolic Labeling Strategies for <i>in Vivo</i> Quantitative Proteomic Analyses with Applications to Early Pupa Formation and Amino Acid Starvation

Although stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was first developed as a cell culture-based technique, stable isotope-labeled amino acids have since been successfully introduced <i>in vivo</i> into select multicellular model organisms by manipulating the feeding diets. An earlier study by others has demonstrated that heavy lysine labeled <i>Drosophila melanogaster</i> can be derived by feeding with an exclusive heavy lysine labeled yeast diet. In this work, we have further evaluated the use of heavy lysine and/or arginine for metabolic labeling of fruit flies, with an aim to determine its respective quantification accuracy and versatility. <i>In vivo</i> conversion of heavy lysine and/or heavy arginine to several nonessential amino acids was observed in labeled flies, leading to distorted isotope pattern and underestimated heavy to light ratio. These quantification defects can nonetheless be rectified at protein level using the normalization function. The only caveat is that such a normalization strategy may not be suitable for every biological application, particularly when modified peptides need to be individually quantified at peptide level. In such cases, we showed that peptide ratios calculated from the summed intensities of all isotope peaks are less affected by the heavy amino acid conversion and therefore less sequence-dependent and more reliable. Applying either the single Lys8 or double Lys6/Arg10 metabolic labeling strategy to flies, we quantitatively mapped the proteomic changes during the onset of metamorphosis and upon amino acid deprivation. The expression of a number of steroid hormone 20-hydroxyecdysone regulated proteins was found to be changed significantly during larval–pupa transition, while several subunits of the V-ATPase complex and components regulating actomyosin were up-regulated under starvation-induced autophagy conditions

Additional file 7: Table S2. of Coevolution of Siglec-11 and Siglec-16 via gene conversion in primates

Primer sequences for genomic PCR. (XLSX 9 kb

Additional file 2: Figure S2. of Coevolution of Siglec-11 and Siglec-16 via gene conversion in primates

Phylogenetic relationships of the A/A’ regions of SIGLEC11 and SIGLEC16. The partial sequence of bonobo SIGLEC11 was obtained previously (GenBank accession no. AB211392; [11]) and used in the tree construction. The tree topology is identical to that shown in Fig. 2A, with the exception of genes of the genus Pan. As for the lineage of the genus Pan, bonobo SIGLEC11 is most closely related to chimpanzee SIGLEC11 but the genes of genus Pan form a cluster in the tree. This suggests that gene conversion between SIGLEC11 and SIGLEC16 occurred before the divergence of chimpanzee and bonobo in the lineage of the genus Pan. Numbers on the phylogenetic tree represent bootstrap values based on 1000 replications. Hsa, Homo sapiens; Ptr, Pan troglodytes; Ppa, Pan paniscus; Ggo, Gorilla gorilla; Hla, Hylobates lar; Pan, Papio anubis; Cja, Callithrix jacchus. (PDF 45 kb

Additional file 4: Figure S4. of Coevolution of Siglec-11 and Siglec-16 via gene conversion in primates

Relaxed evolution of exons that underwent gene conversions. The phylogenetic tree of introns was used as a reference to examine functional constraints. The topology of the intron tree is similar to that of the Ac/Ac’ region, representing that gene conversion occurred in each primate lineage (Figure 2A). Lineage-specific ratios of nonsynonymous substitutions per site to silent substitutions per site (at both synonymous and intron sites) are shown on each branch. A significant difference between nonsynonymous substitutions per site and neutral substitutions per site is found in only three branches, one leading to gorilla SIGLEC11, one leading to baboon SIGLEC11, and the other leading to two baboon genes (P<0.02, Z-test). These branches are represented by bold lines. Hsa, Homo sapiens; Ptr, Pan troglodytes; Ggo, Gorilla gorilla; Hla, Hylobates lar; Pan, Papio anubis; Cja, Callithrix jacchus. (PDF 40 kb

Additional file 3: Figure S3. of Coevolution of Siglec-11 and Siglec-16 via gene conversion in primates

Alignment of amino acid sequences of Siglec-11 and Siglec-16 proteins in primates. Amino acid sequences of primate Siglec-11 and Siglec-16 (signal peptide + first and second immunoglobulin-like domains) were aligned by ClustalO. The amino acid position fully conserved among all aligned Siglec-11 and Siglec-16 is marked with asterisk (*), and the position conserved within groups of strongly or weakly similar amino acids (based on the properties of side chain) is marked with colon (:) or period (.), respectively. Functional human Siglec-16 sequence was used for the alignment. Hsa: Homo sapiens; Ptr: Pan troglodytes; Ggo: Gorilla gorilla; Hla: Hylobates lar; Pan: Papio anubis; Cja: Callithrix jacchus. Residues important for sialic acid recognition are marked with colored squares. Because the atomic level structure of Siglec-11/-16 is not available at present, amino acid residues known to be important for glycan recognition by Siglecs in common (based on the atomic level structures of Siglec-1, -2, -4, -5, and -7 in complex with respective ligand) are indicated. Red square: essential arginine residue interacting with the carboxyl group of sialic acid; Orange squares: aromatic amino acid residues involved in the coordination of sialic acid. (PDF 36 kb