Clade classification of monolignol biosynthesis gene family members reveals target genes to decrease lignin in Lolium perenne

In monocots, lignin content has a strong impact on the digestibility of the cell wall fraction. Engineering lignin biosynthesis requires a profound knowledge of the role of paralogues in the multigene families that constitute the monolignol biosynthesis pathway. We applied a bioinformatics approach for genome‐wide identification of candidate genes in Lolium perenne that are likely to be involved in the biosynthesis of monolignols. More specifically, we performed functional subtyping of phylogenetic clades in four multigene families: 4CL, COMT, CAD and CCR. Essential residues were considered for functional clade delineation within these families. This classification was complemented with previously published experimental evidence on gene expression, gene function and enzymatic activity in closely related crops and model species. This allowed us to assign functions to novel identified L. perenne genes, and to assess functional redundancy among paralogues. We found that two 4CL paralogues, two COMT paralogues, three CCR paralogues and one CAD gene are prime targets for genetic studies to engineer developmentally regulated lignin in this species. Based on the delineation of sequence conservation between paralogues and a first analysis of allelic diversity, we discuss possibilities to further study the roles of these paralogues in lignin biosynthesis, including expression analysis, reverse genetics and forward genetics, such as association mapping. We propose criteria to prioritise paralogues within multigene families and certain SNPs within these genes for developing genotyping assays or increasing power in association mapping studies. Although L. perenne was the target of the analyses presented here, this functional subtyping of phylogenetic clades represents a valuable tool for studies investigating monolignol biosynthesis genes in other monocot species.     

Geographically variable selection in Ambystoma tigrinum virus (Iridoviridae) throughout the western USA

We investigated spatially variable selection in Ambystoma tigrinum virus (ATV) which causes frequent and geographically widespread epizootics of the tiger salamander, Ambystoma tigrinum. To test for evidence of selection, we sequenced several coding and noncoding regions from virus strains isolated from epizootics throughout western North America. Three of the sequenced regions contained homologues for genes putatively involved in host immune evasion and virulence: eIF‐2α, caspase activation and recruitment domain (CARD) and β‐OH‐steroid oxidoreductase. Selection analysis showed evidence of very strong purifying selection on eIF‐2α, purifying selection within certain viral clades on CARD and positive selection on β‐OH‐steroid oxidoreductase within certain clades. Analysis using multidivtime and Tajima’s relative rate tests indicate accelerated rates of evolution within clades associated with anthropogenic movement. These clades also demonstrate greater spatial variability in selection, suggesting a lack of local adaptation (i.e. locally adapted populations should exhibit little to no selection because of absent or reduced variation in fitness once a fitness optimum is reached). Increased transfer of non‐native viral strains to naïve salamander populations, in conjunction with local maladaptation as a result of local selection pressures, may explain the spread and emergence of ATV epizootics in A. tigrinum in western North America.     

A Novel Synthetic Procedure for “Reverse” C-Nucleosides

The Colletotrichum lagenarium PKS1 gene was expressed in the heterologous fungal host, Aspergillus oryzae, under the starch-inducible α-amylase promoter to identify the direct product of polyketide synthase (PKS) encoded by the PKS1 gene. The main compound produced by an A. oryzae transformant was isolated and characterized to be 1,3,6,8-tetrahydroxynaphthalene (T4HN) as its tetraacetate. Since the PKS1 gene was cloned from C. lagenarium to complement the nonmelanizing albino mutant, T4HN was assumed to be an initial biosynthetic intermediate, and thus the product of the PKS reaction, but had not been isolated from the fungus. The production of T4HN by the PKS1 transformant unambiguously identified the gene to encode a PKS of pentaketide T4HN. In addition, tetraketide orsellinic acid and pentaketide isocoumarin were isolated, the latter being derived from a pentaketide monocyclic carboxylic acid, as by-products of the PKS1 PKS reaction. Production of the pentaketide carboxylic acid provided insights into the mechanism for the PKS1 polyketide synthase reaction to form T4HN.     

Phylogenetic analysis of type I polyketide synthase and nonribosomal peptide synthetase genes in Antarctic sediment

The modular polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) have been found to be involved in natural product synthesis in many microorganisms. Study on their diversities in natural environment may provide important ecological insights, in addition to opportunities for antibacterial drugs development. In this study, the PKS and NRPS gene diversities in two coast sediments near China Zhongshan Station were studied. The phylogenetic analysis of amino acid (AA) sequences indicated that the identified ketosynthase (KS) domains were clustered with those from diverse bacterial groups, including Proteobacteria, Firmicutes, Planctomycetes, Cyanobacteria, Actinobacteria, and some uncultured symbiotic bacteria. One new branch belonging to hybrid PKS/NRPS enzyme complexes and five independent clades were found on the phylogenetic tree. The obtained adenylation (A) domains were mainly clustered within the Cyanobacteria and Proteobacteria group. Most of the identified KS and A domains showed below 80 and 60% identities at the AA level to their closest matches in GenBank, respectively. The diversities of both KS and A domains in natural environmental sample were different from those in sewage-contaminated sample. These results revealed the great diversity and novelty of both PKS and NRPS genes in Antarctic sediment.     

Approaches to stabilization of inter-domain recombination in polyketide synthase gene expression plasmids

Regions of extremely high sequence identity are recurrent in modular polyketide synthase (PKS) genes. Such sequences are potentially detrimental to the stability of PKS expression plasmids used in the combinatorial biosynthesis of polyketide metabolites. We present two different solutions for circumventing intra-plasmid recombination within the megalomicin PKS genes in Streptomyces coelicolor. In one example, a synthetic gene was used in which the codon usage was reengineered without affecting the primary amino acid sequence. The other approach utilized a heterologous subunit complementation strategy to replace one of the problematic regions. Both methods resulted in PKS complexes capable of 6-deoxyerythronolide B analogue biosynthesis in S. coelicolor CH999, permitting reproducible scale-up to at least 5-l stirred-tank fermentation and a comparison of diketide precursor incorporation efficiencies between the erythromycin and megalomicin PKSs. Electronic Publication     

Functional Evolution of a Multigene Family: Orthologous and Paralogous Pheromone Receptor Genes in the Turnip Moth,Agrotis segetum

Lepidopteran pheromone receptors (PRs), for which orthologies are evident among closely related species, provide an intriguing example of gene family evolution in terms of how new functions may arise. However, only a limited number of PRs have been functionally characterized so far and thus evolutionary scenarios suffer from elements of speculation. In this study we investigated the turnip moth Agrotis segetum, in which female moths produce a mixture of chemically related pheromone components that elicit specific responses from receptor cells on male antennae. We cloned nine A. segetum PR genes and the Orco gene by degenerate primer based RT-PCR. The nine PR genes, named as AsegOR1 and AsegOR3-10, fall into four distinct orthologous clusters of known lepidopteran PRs, of which one contains six paralogues. The paralogues are under relaxed selective pressure, contrasting with the purifying selection on other clusters. We identified the receptors AsegOR9, AsegOR4 and AsegOR5, specific for the respective homologous pheromone components (Z)-5-decenyl, (Z)-7-dodecenyl and (Z)-9-tetradecenyl acetates, by two-electrode voltage clamp recording from Xenopus laevis oocytes co-expressing Orco and each PR candidate. These receptors occur in three different orthologous clusters. We also found that the six paralogues with high sequence similarity vary dramatically in ligand selectivity and sensitivity. Different from AsegOR9, AsegOR6 showed a relatively large response to the behavioural antagonist (Z)-5-decenol, and a small response to (Z)-5-decenyl acetate. AsegOR1 was broadly tuned, but most responsive to (Z)-5-decenyl acetate, (Z)-7-dodecenyl acetate and the behavioural antagonist (Z)-8-dodecenyl acetate. AsegOR8 and AsegOR7, which differ from AsegOR6 and AsegOR1 by 7 and 10 aa respectively, showed much lower sensitivities. AsegOR10 showed only small responses to all the tested compounds. These results suggest that new receptors arise through gene duplication, and relaxed evolutionary constraints or positive selection among paralogues allow functional divergence to occur in spite of purifying selection being the norm.     

Phylogenetic analysis of polyketide synthase genes fromAspergillus ochraceus

A number of polyketide synthase gene sequences fromAspergillus ochraceus were isolated by both SSH-PCR and degenerate PCR. The deduced amino acid sequences of the corresponding clonedpks DNA fragments were then aligned with the amino acid sequences of other polyketide synthase enzymes. One of thesepks genes is essential for ochratoxin A biosynthesis (OTA-PKS). The OTA-PKS was most similar to methylsalicylic acid synthase (MSAS) type PKS proteins based on the alignment of the ketosynthase domains while if the acyl transferase domains were aligned it appeared to be more similar to PKS enzymes fromCochliobolus heterostrophus. The three PKS proteins identified by degenerate PCR were all from different PKS types, one was a MSAS type enzyme, the second was similar to the PKS proteins involved in lovastatin biosynthesis while the third was not similar to any of the other phylogenetic groupings. Data is presented which suggests that the use of phylogenetic analysis to predict the function of PKS proteins/genes is likely to be significantly enhanced by analyzing more than one domain of the protein. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: Irish Government under the National Development Plan 2000–2006     

Identifying concerted evolution and gene conversion in mammalian gene pairs lasting over 100 million years

Background  

Concerted evolution occurs in multigene families and is characterized by stretches of homogeneity and higher sequence similarity between paralogues than between orthologues. Here we identify human gene pairs that have undergone concerted evolution, caused by ongoing gene conversion, since at least the human-mouse divergence. Our strategy involved the identification of duplicated genes with greater similarity within a species than between species. These genes were required to be present in multiple mammalian genomes, suggesting duplication early in mammalian divergence. To eliminate genes that have been conserved due to strong purifying selection, our analysis also required at least one intron to have retained high sequence similarity between paralogues.     

Targeted gene replacements in a Streptomyces polyketide synthase gene cluster: role for the acyl carrier protein

A methodology was developed to construct any desired chromosomal mutation in the gene cluster that encodes the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2). A positive selection marker (resistance gene) is first introduced by double crossing-over into the chromosomal site of interest by use of an unstable delivery plasmid. This marker is subsequently replaced by the desired mutant allele via a second high-frequency double recombination event. The technology has been used to: (i) explore the significance of translational coupling between two adjacent PKS genes; (ii) prove that the acyl carrier protein (ACP) encoded by a gene in the cluster is necessary for the function of the actinorhodin PKS; (iii) provide genetic evidence supporting the hypothesis that serine 42 is the site of phosphopantetheinylation in the ACP of the actinorhodin PKS; and (iv) demonstrate that this ACP can be replaced by a Saccharopolyspora fatty acid synthase ACP to generate an active hybrid PKS.     

Evolutionary dynamics of Rh2 opsins in birds demonstrate an episode of accelerated evolution in the New World warblers (Setophaga)

Low rates of sequence evolution associated with purifying selection can be interrupted by episodic changes in selective regimes. Visual pigments are a unique system in which we can investigate the functional consequences of genetic changes, therefore connecting genotype to phenotype in the context of natural and sexual selection pressures. We study the RH2 and RH1 visual pigments (opsins) across 22 bird species belonging to two ecologically convergent clades, the New World warblers (Parulidae) and Old World warblers (Phylloscopidae) and evaluate rates of evolution in these clades along with data from 21 additional species. We demonstrate generally slow evolution of these opsins: both Rh1 and Rh2 are highly conserved across Old World and New World warblers. However, Rh2 underwent a burst of evolution within the New World genus Setophaga, where it accumulated substitutions at 6 amino acid sites across the species we studied. Evolutionary analyses revealed a significant increase in d_N/d_S in Setophaga, implying relatively strong selective pressures to overcome long‐standing purifying selection. We studied the effects of each substitution on spectral tuning and found they do not cause large spectral shifts. Thus, substitutions may reflect other aspects of opsin function, such as those affecting photosensitivity and/or dark–light adaptation. Although it is unclear what these alterations mean for colour perception, we suggest that rapid evolution is linked to sexual selection, given the exceptional plumage colour diversification in Setophaga.     

Isolation and characterization of a reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima

The reducing polyketide synthases found in filamentous fungi are involved in the biosynthesis of many drugs and toxins. Lichens produce bioactive polyketides, but the roles of reducing polyketide synthases in lichens remain to be clearly elucidated. In this study, a reducing polyketide synthase gene (U1PKS3) was isolated and characterized from a cultured mycobiont of Usnea longissima. Complete sequence information regarding U1PKS3 (6,519 bp) was obtained by screening a fosmid genomic library. A U1PKS3 sequence analysis suggested that it contains features of a reducing fungal type I polyketide synthase with β-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoacyl reducatse (KR), and acyl carrier protein (ACP) domains. This domain structure was similar to the structure of ccRadsl, which is known to be involved in resorcylic acid lactone biosynthesis in Chaetomium chiversii. The results of phylogenetic analysis located U1PKS3 in the clade of reducing polyketide synthases. RT-PCR analysis results demonstrated that UIPKS3 had six intervening introns and that UIPKS3 expression was upregulated by glucose, sorbitol, inositol, and mannitol.     

Strong Purifying Selection on the Odysseus Gene in Two Clades of Sibling Species of the Drosophila montium Species Subgroup

The Odysseus (OdsH) gene was duplicated from its ancestral neuron-expressed gene, unc-4, and then evolved very rapidly under strong positive Darwinian selection as a speciation gene causing hybrid-male sterility between closely related species of the Drosophila simulans clade. Has OdsH also experienced similar positive selection between Drosophila sibling species other than those of the simulans clade? We cloned and sequenced OdsH and unc-4 from two clades of the Drosophila montium species subgroup, the Drosophila lini and the Drosophila kikkawai clades. The ratios of Ka/Ks for OdsH were remarkably low between sibling species of these two clades, suggesting that OdsH has been subjected to strong purifying selection in these two clades.     

Biogeography of actinomycete communities and type II polyketide synthase genes in soils collected in New Jersey and Central Asia

Soil microbial communities are believed to be comprised of thousands of different bacterial species. One prevailing idea is that "everything is everywhere, and the environment selects," implying that all types of bacteria are present in all environments where their growth requirements are met. We tested this hypothesis using actinomycete communities and type II polyketide synthase (PKS) genes found in soils collected from New Jersey and Uzbekistan (n = 91). Terminal restriction fragment length polymorphism analysis using actinomycete 16S rRNA and type II PKS genes was employed to determine community profiles. The terminal fragment frequencies in soil samples had a lognormal distribution, indicating that the majority of actinomycete phylotypes and PKS pathways are present infrequently in the environment. Less than 1% of peaks were detected in more than 50% of samples, and as many as 18% of the fragments were unique and detected in only one sample. Actinomycete 16S rRNA fingerprints clustered by country of origin, indicating that unique populations are present in North America and Central Asia. Sequence analysis of type II PKS gene fragments cloned from Uzbek soil revealed 35 novel sequence clades whose levels of identity to genes in the GenBank database ranged from 68 to 92%. The data indicate that actinomycetes are patchily distributed but that distinct populations are present in North American and Central Asia. These results have implications for microbial bioprospecting and indicate that the cosmopolitan actinomycete species and PKS pathways may account for only a small proportion of the total diversity in soil.     

Biogeography of Actinomycete Communities and Type II Polyketide Synthase Genes in Soils Collected in New Jersey and Central Asia

Soil microbial communities are believed to be comprised of thousands of different bacterial species. One prevailing idea is that “everything is everywhere, and the environment selects,” implying that all types of bacteria are present in all environments where their growth requirements are met. We tested this hypothesis using actinomycete communities and type II polyketide synthase (PKS) genes found in soils collected from New Jersey and Uzbekistan (n = 91). Terminal restriction fragment length polymorphism analysis using actinomycete 16S rRNA and type II PKS genes was employed to determine community profiles. The terminal fragment frequencies in soil samples had a lognormal distribution, indicating that the majority of actinomycete phylotypes and PKS pathways are present infrequently in the environment. Less than 1% of peaks were detected in more than 50% of samples, and as many as 18% of the fragments were unique and detected in only one sample. Actinomycete 16S rRNA fingerprints clustered by country of origin, indicating that unique populations are present in North America and Central Asia. Sequence analysis of type II PKS gene fragments cloned from Uzbek soil revealed 35 novel sequence clades whose levels of identity to genes in the GenBank database ranged from 68 to 92%. The data indicate that actinomycetes are patchily distributed but that distinct populations are present in North American and Central Asia. These results have implications for microbial bioprospecting and indicate that the cosmopolitan actinomycete species and PKS pathways may account for only a small proportion of the total diversity in soil.     

A cryptic type I polyketide synthase (cpk) gene cluster in Streptomyces coelicolor A3(2)

The chromosome of Streptomyces coelicolor A3(2), a model organism for the genus Streptomyces, contains a cryptic type I polyketide synthase (PKS) gene cluster which was revealed when the genome was sequenced. The ca. 54-kb cluster contains three large genes, cpkA, cpkB and cpkC, encoding the PKS subunits. In silico analysis showed that the synthase consists of a loading module, five extension modules and a unique reductase as a terminal domain instead of a typical thioesterase. All acyltransferase domains are specific for a malonyl extender, and have a B-type ketoreductase. Tailoring and regulatory genes were also identified within the gene cluster. Surprisingly, some genes show high similarity to primary metabolite genes not commonly identified in any antibiotic biosynthesis cluster. Using western blot analysis with a PKS subunit (CpkC) antibody, CpkC was shown to be expressed in S. coelicolor at transition phase. Disruption of cpkC gave no obvious phenotype.     

Evolution of follistatin in teleosts revealed through phylogenetic, genomic and expression analyses

Follistatin (Fst) inhibits transforming growth factor-β (TGF-B) proteins and is a known regulator of amniote myogenesis. Here, we used phylogenetic, genomic and experimental approaches to study its evolution in teleosts. Phylogenetic analyses suggested that one fst gene (fst1) is common to euteleosts, but a second gene (fst2) is conserved specifically within the Ostariophysi. Zebrafish fst1/2 respectively appear on chromosomes 5 and 10 in two genomic regions, each with conserved synteny to a single region in tetrapods. Interestingly, other teleosts have two corresponding chromosomal regions with a similar repertoire of paralogues. Phylogenetic reconstruction clustered these gene duplicates into two sister clades branching from tetrapod sequences. We suggest that an ancestral fst-containing chromosome was duplicated during the teleost whole genome duplication, but that fst2 was lost in lineages external to the Ostariophysi. We show that Fst1 of teleosts/mammals has evolved under strong purifying selection, but the N-terminal of Fst2 may have evolved under positive selection. Furthermore, the tissue-specific expression of zebrafish fst2 was restricted to fewer tissues compared to its paralogue and the single fst1 orthologue of Atlantic salmon (Salmo salar). Zebrafish fst1/2 may have subfunctionalized relative to non-duplicated vertebrate lineages, as several regions in the fst promoter of tetrapods were conserved with one paralogue, but not both. Finally, we examined the embryonic expression of fst1 in a teleost outside the Ostariophysi (Atlantic salmon). During segmentation, fst1 was expressed in the anterior somite compartment but was excluded from muscle progenitors that strongly expressed myogenic regulatory factors (MRFs). Later, fst1 was expressed in myogenic progenitors of the pectoral fin buds and also within the pax7 ⁺ cell layer external to the myotome. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A gene cluster involved in nogalamycin biosynthesis fromStreptomyces nogalater: sequence analysis and complementation of early-block mutations in the anthracycline pathway

We have analyzed an anthracycline biosynthesis gene cluster fromStreptomyces nogalater. Based on sequence analysis, a contiguous region of 11 kb is deduced to include genes for the early steps in anthracycline biosynthesis, a regulatory gene (snoA) promoting the expression of the biosynthetic genes, and at least one gene whose product might have a role in modification of the glycoside moiety. The three ORFs encoding a minimal polyketide synthase (PKS) are separated from the regulatory gene (snoA) by a comparatively AT-rich region (GC content 60%). Subfragments of the DNA region were transferred toStreptomyces galilaeus mutants blocked in aclacinomycin biosynthesis, and to a regulatory mutant ofS. nogalater. TheS. galilaeus mutants carrying theS. nogalater minimal PKS genes produced auramycinone glycosides, demonstrating replacement of the starter unit for polyketide biosynthesis. The product ofsnoA seems to be needed for expression of at least the genes for the minimal PKS.     

Diversity of non-reducing polyketide synthase genes in the Pertusariales (lichenized Ascomycota): a phylogenetic perspective

Lichenized fungi synthesize a great variety of secondary metabolites. These are typically crystalline compounds, which are deposited extracellularly on the fungal hyphae. While we know a lot about the chemical properties and structures of these substances, we have very little information on the molecular background of their biosynthesis. In the current study we analyze the diversity of non-reducing polyketide synthase (PKS) genes in members of the lichenized Pertusariales. This order primarily contains fully oxidized secondary metabolites from different substance classes, and is chemically and phylogenetically well studied. Using a degenerate primer approach with subsequent cloning we detected up to five non-reducing PKS sequences in a single PCR product. Eighty-five new KS sequence fragments were obtained for this study. Analysis of the 157 currently available fungal KS sequence fragments in a Bayesian phylogenetic framework revealed 18 highly supported clades that included only lichenized taxa, only non-lichenized taxa, or both. Some Pertusarialean groupings of PKS sequences corresponded partly to phylogenetic groupings based on ribosomal DNA. This is reasonable, because a correlation between well-supported phylogenetic lineages and the occurrence of secondary metabolites in the Pertusariales has been observed before. However, no clear linkage was found between the PKS genes analyzed and the ability to produce a particular secondary substance. Several PKS clades did not reveal obvious patterns of secondary compound distribution or phylogenetic association. Compared with earlier phylogenetic analyses of KS sequences the increased sampling in the current study allowed us to detect many new groupings within the fungal non-reducing PKSs.     

Chimeric 6-methylsalicylic acid synthase with domains of acyl carrier protein and methyltransferase from Pseudallescheria boydii shows novel biosynthetic activity

Polyketides are important secondary metabolites, many of which exhibit potent pharmacological applications. Biosynthesis of polyketides is carried out by a single polyketide synthase (PKS) or multiple PKSs in successive elongations of enzyme-bound intermediates related to fatty acid biosynthesis. The polyketide gene PKS306 from Pseudallescheria boydii NTOU2362 containing domains of ketosynthase (KS), acyltransferase (AT), dehydratase (DH), acyl carrier protein (ACP) and methyltransferase (MT) was cloned in an attempt to produce novel chemical compounds, and this PKS harbouring green fluorescent protein (GFP) was expressed in Saccharomyces cerevisiae. Although fluorescence of GFP and fusion protein analysed by anti-GFP antibody were observed, no novel compound was detected. 6-methylsalicylic acid synthase (6MSAS) was then used as a template and engineered with PKS306 by combinatorial fusion. The chimeric PKS containing domains of KS, AT, DH and ketoreductase (KR) from 6MSAS with ACP and MT from PKS306 demonstrated biosynthesis of a novel compound. The compound was identified with a deduced chemical formula of C₇H₁₀O₃, and the chemical structure was named as 2-hydroxy-2-(propan-2-yl) cyclobutane-1,3-dione. The novel compound synthesized by the chimeric PKS in this study demonstrates the feasibility of combinatorial fusion of PKS genes to produce novel polyketides.