Systematic mining of fungal chimeric terpene synthases using an efficient precursor-providing yeast chassis

Chimeric terpene synthases, which consist of C-terminal prenyltransferase (PT) and N-terminal class I terpene synthase (TS) domains (termed PTTSs here), is unique to fungi and produces structurally diverse di- and sesterterpenes. Prior to this study, 20 PTTSs had been functionally characterized. Our understanding of the origin and functional evolution of PTTS genes is limited. Our systematic search of sequenced fungal genomes among diverse taxa revealed that PTTS genes were restricted to Dikarya. Phylogenetic findings indicated different potential models of the origin and evolution of PTTS genes. One was that PTTS genes originated in the common Dikarya ancestor and then underwent frequent gene loss among various subsequent lineages. To understand their functional evolution, we selected 74 PTTS genes for biochemical characterization in an efficient precursor-providing yeast system employing chassis-based, robot-assisted, high-throughput automatic assembly. We found 34 PTTS genes that encoded active enzymes and collectively produced 24 di- and sesterterpenes. About half of these di- and sesterterpenes were also the products of the 20 known PTTSs, indicating functional conservation, whereas the PTTS products included the previously unknown sesterterpenes, sesterevisene (1), and sesterorbiculene (2), suggesting that a diversity of PTTS products awaits discovery. Separating functional PTTSs into two monophyletic groups implied that an early gene duplication event occurred during the evolution of the PTTS family followed by functional divergence with the characteristics of distinct cyclization mechanisms.

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Biochemical Characterization and Function of Eight Microbial Type Terpene Synthases from Lycophyte Selaginella moellendorffii

International Journal of Molecular Sciences ◽

10.3390/ijms22020605 ◽

2021 ◽

Vol 22 (2) ◽

pp. 605

Author(s):

Yapei Zhao ◽

Tian Hu ◽

Ruiqi Liu ◽

Zhiqiang Hao ◽

Guoyan Liang ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Biochemical Characterization ◽

Vascular Plant ◽

Terpene Synthase ◽

Tomato Dc3000 ◽

Terpene Synthases ◽

Geranyl Diphosphate ◽

And Function ◽

Selaginella Moellendorffii ◽

And Staphylococcus Aureus

Selaginella moellendorffii is a lycophyte, a member of an ancient vascular plant lineage. Two distinct types of terpene synthase (TPS) genes were identified from this species, including S. moellendorffii TPS genes (SmTPSs) and S. moellendorffii microbial TPS-like genes (SmMTPSLs). The goal of this study was to investigate the biochemical functions of SmMTPSLs. Here, eight full-length SmMTPSL genes (SmMTPSL5, -15, -19, -23, -33, -37, -46, and -47) were functionally characterized from S. moellendorffii. Escherichia coli-expressed recombinant SmMTPSLs were tested for monoterpenes synthase and sesquiterpenes synthase activities. These enzymatic products were typical monoterpenes and sesquiterpenes that have been previous shown to be generated by typical plant TPSs when provided with geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) as the substrates. Meanwhile, SmMTPSL23, -33, and -37 were up-regulated when induced by alamethicin (ALA) and methyl jasmonate (MeJA), suggesting a role for these genes in plants response to abiotic stresses. Furthermore, this study pointed out that the terpenoids products of SmMTPSL23, -33, and -37 have an antibacterial effect on Pseudomonas syringae pv. tomato DC3000 and Staphylococcus aureus. Taken together, these results provide more information about the catalytic and biochemical function of SmMTPSLs in S. moellendorffii plants.

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Computational-guided discovery and characterization of a sesquiterpene synthase from Streptomyces clavuligerus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1505127112 ◽

2015 ◽

Vol 112 (18) ◽

pp. 5661-5666 ◽

Cited By ~ 32

Author(s):

Jeng-Yeong Chow ◽

Bo-Xue Tian ◽

Gurusankar Ramamoorthy ◽

Brandan S. Hillerich ◽

Ronald D. Seidel ◽

...

Keyword(s):

Biochemical Characterization ◽

Homology Model ◽

Streptomyces Clavuligerus ◽

Major Product ◽

Terpene Synthase ◽

Terpene Synthases ◽

Guided Discovery ◽

Functional Assignment ◽

Reaction Channels

Terpenoids are a large structurally diverse group of natural products with an array of functions in their hosts. The large amount of genomic information from recent sequencing efforts provides opportunities and challenges for the functional assignment of terpene synthases that construct the carbon skeletons of these compounds. Inferring function from the sequence and/or structure of these enzymes is not trivial because of the large number of possible reaction channels and products. We tackle this problem by developing an algorithm to enumerate possible carbocations derived from the farnesyl cation, the first reactive intermediate of the substrate, and evaluating their steric and electrostatic compatibility with the active site. The homology model of a putative pentalenene synthase (Uniprot: B5GLM7) from Streptomyces clavuligerus was used in an automated computational workflow for product prediction. Surprisingly, the workflow predicted a linear triquinane scaffold as the top product skeleton for B5GLM7. Biochemical characterization of B5GLM7 reveals the major product as (5S,7S,10R,11S)-cucumene, a sesquiterpene with a linear triquinane scaffold. To our knowledge, this is the first documentation of a terpene synthase involved in the synthesis of a linear triquinane. The success of our prediction for B5GLM7 suggests that this approach can be used to facilitate the functional assignment of novel terpene synthases.

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Joint profiling of gene expression and chromatin accessibility of amphioxus development at single cell resolution

10.21203/rs.3.rs-504113/v1 ◽

2021 ◽

Author(s):

Pengcheng Ma ◽

Xingyan Liu ◽

Huimin Liu ◽

Zaoxu Xu ◽

Xiangning Ding ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cell Fate ◽

Regulatory Networks ◽

Functional Divergence ◽

Expression Patterns ◽

Genetic Regulatory Networks ◽

Public Access ◽

Origin And Evolution ◽

Key Species

Abstract Vertebrate evolution was accompanied with two rounds of whole genome duplication followed by functional divergence in terms of regulatory circuits and gene expression patterns. As a basal and slow-evolving chordate species, amphioxus is an ideal paradigm for exploring the origin and evolution of vertebrates. Single cell sequencing has been widely employed to construct the developmental cell atlas of several key species of vertebrates (human, mouse, zebrafish and frog) and tunicate (sea squirts). Here, we performed single-nucleus RNA sequencing (snRNA-seq) and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) for different stages of amphioxus (covering embryogenesis and adult tissues). With the datasets generated we constructed the developmental tree for amphioxus cell fate commitment and lineage specification, and revealed the underlying key regulators and genetic regulatory networks. The generated data were integrated into an online platform, AmphioxusAtlas, for public access at http://120.79.46.200:81/AmphioxusAtlas.

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Transcriptome and proteome of the corm, leaf and flower of Hypoxis hemerocallidea (African potato)

PLoS ONE ◽

10.1371/journal.pone.0253741 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0253741

Author(s):

Mihai-Silviu Tomescu ◽

Selisha Ann Sooklal ◽

Thuto Ntsowe ◽

Previn Naicker ◽

Barbara Darnhofer ◽

...

Keyword(s):

De Novo ◽

Differential Expression Analysis ◽

Terpene Synthase ◽

Future Research ◽

Terpene Synthases ◽

Illumina Hiseq ◽

Prostate Hyperplasia ◽

Inflammatory Conditions ◽

Urinary Infections ◽

Hypoxis Hemerocallidea

The corm of Hypoxis hemerocallidea, commonly known as the African potato, is used in traditional medicine to treat several medical conditions such as urinary infections, benign prostate hyperplasia, inflammatory conditions and testicular tumours. The metabolites contributing to the medicinal properties of H. hemerocallidea have been identified in several studies and, more recently, the active terpenoids of the plant were profiled. However, the biosynthetic pathways and the enzymes involved in the production of the terpene metabolites in H. hemerocallidea have not been characterised at a transcriptomic or proteomic level. In this study, total RNA extracted from the corm, leaf and flower tissues of H. hemerocallidea was sequenced on the Illumina HiSeq 2500 platform. A total of 143,549 transcripts were assembled de novo using Trinity and 107,131 transcripts were functionally annotated using the nr, GO, COG, KEGG and SWISS-PROT databases. Additionally, the proteome of the three tissues were sequenced using LC-MS/MS, revealing aspects of secondary metabolism and serving as data validation for the transcriptome. Functional annotation led to the identification of numerous terpene synthases such as nerolidol synthase, germacrene D synthase, and cycloartenol synthase amongst others. Annotations also revealed a transcript encoding the terpene synthase phytoalexin momilactone A synthase. Differential expression analysis using edgeR identified 946 transcripts differentially expressed between the three tissues and revealed that the leaf upregulates linalool synthase compared to the corm and the flower tissues. The transcriptome as well as the proteome of Hypoxis hemerocallidea presented here provide a foundation for future research.

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Identification of Sesquiterpene Synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. Strain PCC 7120

Journal of Bacteriology ◽

10.1128/jb.00759-08 ◽

2008 ◽

Vol 190 (18) ◽

pp. 6084-6096 ◽

Cited By ~ 96

Author(s):

Sean A. Agger ◽

Fernando Lopez-Gallego ◽

Thomas R. Hoye ◽

Claudia Schmidt-Dannert

Keyword(s):

Functional Expression ◽

Terpene Synthase ◽

Putative Hybrid ◽

Nostoc Punctiforme ◽

Terpene Synthases ◽

E Coli ◽

Reductase Gene ◽

Pcc 7120 ◽

Defense Compound

ABSTRACT Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified in Nostoc sp. strain PCC 7120 (terpene synthase NS1) and Nostoc punctiforme PCC 73102 (terpene synthases NP1 and NP2). The second terpene synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-α-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-α-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene synthases. Coexpression of P450 genes with their adjacent located terpene synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.

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Biochemical characterization of microbial type terpene synthases in two closely related species of hornworts, Anthoceros punctatus and Anthoceros agrestis

Phytochemistry ◽

10.1016/j.phytochem.2018.02.011 ◽

2018 ◽

Vol 149 ◽

pp. 116-122 ◽

Cited By ~ 9

Author(s):

Wangdan Xiong ◽

Jianyu Fu ◽

Tobias G. Köllner ◽

Xinlu Chen ◽

Qidong Jia ◽

...

Keyword(s):

Related Species ◽

Biochemical Characterization ◽

Terpene Synthases ◽

Closely Related Species ◽

Anthoceros Punctatus

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A Comprehensive Survey on the Terpene Synthase Gene Family Provides New Insight into Its Evolutionary Patterns

Genome Biology and Evolution ◽

10.1093/gbe/evz142 ◽

2019 ◽

Vol 11 (8) ◽

pp. 2078-2098 ◽

Cited By ~ 8

Author(s):

Shu-Ye Jiang ◽

Jingjing Jin ◽

Rajani Sarojam ◽

Srinivasan Ramachandran

Keyword(s):

Gene Family ◽

Large Scale ◽

Family Members ◽

Terpene Synthase ◽

Limited Information ◽

Terpene Synthases ◽

Genome Wide ◽

A Genome ◽

Family Expansion ◽

Insight Into

Abstract Terpenes are organic compounds and play important roles in plant growth and development as well as in mediating interactions of plants with the environment. Terpene synthases (TPSs) are the key enzymes responsible for the biosynthesis of terpenes. Although some species were employed for the genome-wide identification and characterization of the TPS family, limited information is available regarding the evolution, expansion, and retention mechanisms occurring in this gene family. We performed a genome-wide identification of the TPS family members in 50 sequenced genomes. Additionally, we also characterized the TPS family from aromatic spearmint and basil plants using RNA-Seq data. No TPSs were identified in algae genomes but the remaining plant species encoded various numbers of the family members ranging from 2 to 79 full-length TPSs. Some species showed lineage-specific expansion of certain subfamilies, which might have contributed toward species or ecotype divergence or environmental adaptation. A large-scale family expansion was observed mainly in dicot and monocot plants, which was accompanied by frequent domain loss. Both tandem and segmental duplication significantly contributed toward family expansion and expression divergence and played important roles in the survival of these expanded genes. Our data provide new insight into the TPS family expansion and evolution and suggest that TPSs might have originated from isoprenyl diphosphate synthase genes.

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Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

Analytical Chemistry Insights ◽

10.4137/aci.s40292 ◽

2016 ◽

Vol 11s1 ◽

pp. ACI.S40292

Author(s):

Tom Manczak ◽

Henrik Toft Simonsen

Keyword(s):

Kinetic Parameters ◽

Biochemical Characterization ◽

Radioactive Isotopes ◽

Published Data ◽

Enzymatic Characterization ◽

Turnover Number ◽

Terpene Synthases ◽

Thapsia Garganica ◽

Insight Into

A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with reduced reagent usage, it allows further reduction in the use of radioactive isotopes and flammable organic solvents. The sesquiterpene synthases previously characterized were expressed in yeast, and the plant-derived Thapsia garganica kunzeaol synthase TgTPS2 was tested in this method. KM for TgTPS2 was found to be 0.55 μM; the turnover number, kcat, was found to be 0.29 s−1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/ KM was found to be 0.53 s−1 μM−1 for TgTPS2. The kinetic parameters were in agreement with previously published data.

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Origin and evolution of transporter substrate specificity within the NPF family

eLife ◽

10.7554/elife.19466 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 26

Author(s):

Morten Egevang Jørgensen ◽

Deyang Xu ◽

Christoph Crocoll ◽

Heidi Asschenfeldt Ernst ◽

David Ramírez ◽

...

Keyword(s):

Substrate Specificity ◽

Biochemical Characterization ◽

Biosynthetic Pathways ◽

Cyanogenic Glucosides ◽

Evolutionary Path ◽

Substrate Specificities ◽

Origin And Evolution ◽

Glucosinolate Biosynthesis ◽

Phylogenetic Lineage

Despite vast diversity in metabolites and the matching substrate specificity of their transporters, little is known about how evolution of transporter substrate specificities is linked to emergence of substrates via evolution of biosynthetic pathways. Transporter specificity towards the recently evolved glucosinolates characteristic of Brassicales is shown to evolve prior to emergence of glucosinolate biosynthesis. Furthermore, we show that glucosinolate transporters belonging to the ubiquitous NRT1/PTR FAMILY (NPF) likely evolved from transporters of the ancestral cyanogenic glucosides found across more than 2500 species outside of the Brassicales. Biochemical characterization of orthologs along the phylogenetic lineage from cassava to A. thaliana, suggests that alterations in the electrogenicity of the transporters accompanied changes in substrate specificity. Linking the evolutionary path of transporter substrate specificities to that of the biosynthetic pathways, exemplify how transporter substrate specificities originate and evolve as new biosynthesis pathways emerge.

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