Isopentenyl Pyrophosphate Isomerase Research Articles

Investigation of sex expression profiles and the cantharidin biosynthesis genes in two blister beetles.

Cantharidin (CTD) is a well-established defensive toxin synthesized by blister beetles, displaying both therapeutic potential and toxicity. Among these beetles, Hycleus cichorii and Hycleus phaleratus are the two most commercially significant species due to their capacity to produce CTD in males. In this investigation, we conducted a gene expression profiling analysis of male and female individuals of these two species, utilizing the Illumina Hiseq4000 platform. We identified 7,983 expressed genes, including 2,823 differentially expressed genes (DEGs) shared by both male and female blister beetles. Nineteen genes related to CTD biosynthesis in the terpenoid backbone biosynthesis pathway were identified, including hydroxymethylglutaryl-CoA reductase (HMGR; EC:1.1.1.34), which demonstrated a significant correlation with CTD content. Furthermore, hydroxymethylglutaryl-CoA synthase (HMGS; EC:2.3.3.10) and isopentenyl-diphosphate Delta-isomerase (IDI; EC:5.3.3.2) were also found to be significantly up-regulated in males. Comparative analysis revealed that NADP+-dependent farnesol dehydrogenase (FOHSDR; EC:1.1.1.216) and farnesyl diphosphate synthase (FDPS; EC:2.5.1.1) had the highest copy number in these beetles, significantly higher than the copy number of the other four non-Meloidae insects. The analysis of the protein-protein interaction network of genes related to CTD biosynthesis revealed that the acetyl-CoA C-acetyltransferase (ACAT; EC:2.3.1.9) gene was the central gene, exhibiting greater expression in male blister beetles than in females. This study offers novel insights into the mechanisms of CTD biosynthesis in blister beetles and enhances our comprehensions of the association between particular genes and CTD content.

Proteins from toad’s parotoid macroglands: do they play a role in gland functioning and chemical defence?

BackgroundParotoid gland secretion of bufonid toads is a rich source of toxic molecules that are used against predators, parasites and pathogens. Bufadienolides and biogenic amines are the principal compounds responsible for toxicity of parotoid secretion. Many toxicological and pharmacological analyses of parotoid secretions have been performed, but little is known about the processes related to poison production and secretion. Therefore, our aim was to investigate protein content in parotoids of the common toad, Bufo bufo, to understand the processes that regulate synthesis and excretion of toxins as well as functioning of parotoid macroglands.ResultsApplying a proteomic approach we identified 162 proteins in the extract from toad’s parotoids that were classified into 11 categories of biological functions. One-third (34.6%) of the identified molecules, including acyl-CoA-binding protein, actin, catalase, calmodulin, and enolases, were involved in cell metabolism. We found many proteins related to cell division and cell cycle regulation (12.0%; e.g. histone and tubulin), cell structure maintenance (8.4%; e.g. thymosin beta-4, tubulin), intra- and extracellular transport (8.4%), cell aging and apoptosis (7.3%; e.g. catalase and pyruvate kinase) as well as immune (7.0%; e.g. interleukin-24 and UV excision repair protein) and stress (6.3%; including heat shock proteins, peroxiredoxin-6 and superoxide dismutase) response. We also identified two proteins, phosphomevalonate kinase and isopentenyl-diphosphate delta-isomerase 1, that are involved in synthesis of cholesterol which is a precursor for bufadienolides biosynthesis. STRING protein-protein interaction network predicted for identified proteins showed that most proteins are related to metabolic processes, particularly glycolysis, stress response and DNA repair and replication. The results of GO enrichment and KEGG analyses are also consistent with these findings.ConclusionThis finding indicates that cholesterol may be synthesized in parotoids, and not only in the liver from which is then transferred through the bloodstream to the parotoid macroglands. Presence of proteins that regulate cell cycle, cell division, aging and apoptosis may indicate a high epithelial cell turnover in parotoids. Proteins protecting skin cells from DNA damage may help to minimize the harmful effects of UV radiation. Thus, our work extends our knowledge with new and important functions of parotoids, major glands involved in the bufonid chemical defence.

Identification and functional analysis of isopentenyl pyrophosphate isomerase genes in the whiteflies Bemisia tabaci (Hemiptera: Aleyrodidae).

The juvenile hormone (JH) plays a vital role in the regulation of a number of physiological processes, including development, reproduction, and ovarian maturation. Isopentenyl pyrophosphate isomerase (IPPI) is a key enzyme in the biosynthetic pathway of JH. In this study, we identified an isopentenyl pyrophosphate isomerase protein from Bemisia tabaci and named it BtabIPPI. The open reading frame (ORF) of BtabIPPI is 768 bp and encodes a protein of 255 amino acids that contains a conserved domain of the Nudix family. The temporal and spatial expression profiles showed that BtabIPPI was highly expressed in the female adults.RNA interference (RNAi)-mediated silencing of BtabIPPI reduced JH titers and the relative expression of vitellogenin receptor (VgR) and JH signaling pathway genes, resulting in a dramatic reduction in fecundity and hatchability. These results indicate that the BtabIPPI gene plays an important role in the female fecundity of B. tabaci. This study will broaden our understanding of the function of IPPI in regulating insect reproduction and provide a theoretical basis for targeting IPPI for pest control in the future.

Identification and Functional Analysis of Two Novel Genes-Geranylgeranyl Pyrophosphate Synthase Gene (AlGGPPS) and Isopentenyl Pyrophosphate Isomerase Gene (AlIDI)-from Aurantiochytrium limacinum Significantly Enhance De Novo β-Carotene Biosynthesis in Escherichia coli.

Precursor regulation has been an effective strategy to improve carotenoid production and the availability of novel precursor synthases facilitates engineering improvements. In this work, the putative geranylgeranyl pyrophosphate synthase encoding gene (AlGGPPS) and isopentenyl pyrophosphate isomerase encoding gene (AlIDI) from Aurantiochytrium limacinum MYA-1381 were isolated. We applied the excavated AlGGPPS and AlIDI to the de novo β-carotene biosynthetic pathway in Escherichia coli for functional identification and engineering application. Results showed that the two novel genes both functioned in the synthesis of β-carotene. Furthermore, AlGGPPS and AlIDI performed better than the original or endogenous one, with 39.7% and 80.9% increases in β-carotene production, respectively. Due to the coordinated expression of the 2 functional genes, β-carotene content of the modified carotenoid-producing E. coli accumulated a 2.99-fold yield of the initial EBIY strain in 12 h, reaching 10.99 mg/L in flask culture. This study helped to broaden current understanding of the carotenoid biosynthetic pathway in Aurantiochytrium and provided novel functional elements for carotenoid engineering improvements.

Full-length transcriptome, proteomics and metabolite analysis reveal candidate genes involved triterpenoid saponin biosynthesis in Dipsacus asperoides.

Dipsacus asperoides is a traditional medicinal herb widely used in inflammation and fracture in Asia. Triterpenoid saponins from D. asperoides are the main composition with pharmacological activity. However, the biosynthesis pathway of triterpenoid saponins has not been completely resolved in D. asperoides. Here, the types and contents of triterpenoid saponins were discovered with different distributions in five tissues (root, leaf, flower, stem, and fibrous root tissue) from D. asperoides by UPLC-Q-TOF-MS analysis. The discrepancy between five tissues in D. asperoides at the transcriptional level was studied by combining single-molecule real-time sequencing and next- generation sequencing. Meanwhile, key genes involved in the biosynthesis of saponin were further verified by proteomics. In MEP and MVA pathways, 48 differentially expressed genes were identified through co-expression analysis of transcriptome and saponin contents, including two isopentenyl pyrophosphate isomerase and two 2,3-oxidosqualene β-amyrin cyclase, etc. In the analysis of WGCNA, 6 cytochrome P450s and 24 UDP- glycosyltransferases related to the biosynthesis of triterpenoid saponins were discovered with high transcriptome expression. This study will provide profound insights to demonstrate essential genes in the biosynthesis pathway of saponins in D. asperoides and support for the biosynthetic of natural active ingredients in the future.

Enhancement of linalool production in Saccharomyces cerevisiae by utilizing isopentenol utilization pathway

BackgroundLinalool is a monoterpenoid, also a vital silvichemical with commercial applications in cosmetics, flavoring ingredients, and medicines. Regulation of mevalonate (MVA) pathway metabolic flux is a common strategy to engineer Saccharomyces cerevisiae for efficient linalool production. However, metabolic regulation of the MVA pathway is complex and involves competition for central carbon metabolism, resulting in limited contents of target metabolites.ResultsIn this study, first, a truncated linalool synthase (t26AaLS1) from Actinidia arguta was selected for the production of linalool in S. cerevisiae. To simplify the complexity of the metabolic regulation of the MVA pathway and increase the flux of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), we introduced the two-step isopentenyl utilization pathway (IUP) into S. cerevisiae, which could produce large amounts of IPP/DMAPP. Further, the S. cerevisiae IDI1 (ecoding isopentenyl diphosphate delta-isomerase) and ERG20F96W−N127W (encoding farnesyl diphosphate synthase) genes were integrated into the yeast genome, combined with the strategies of copy number variation of the t26AaLS1 and ERG20F96W−N127W genes to increase the metabolic flux of the downstream IPP, as well as optimization of isoprenol and prenol concentrations, resulting in a 4.8-fold increase in the linalool titer. Eventually, under the optimization of carbon sources and Mg2+ addition, a maximum linalool titer of 142.88 mg/L was obtained in the two-phase extractive shake flask fermentation.ConclusionsThe results show that the efficient synthesis of linalool in S. cerevisiae could be achieved through a two-step pathway, gene expression adjustment, and optimization of culture conditions. The study may provide a valuable reference for the other monoterpenoid production in S. cerevisiae.

Evaluation of the Fecal Proteome in Healthy and Diseased Cheetahs (Acinonyx jubatus) Suffering from Gastrointestinal Disorders.

Simple SummaryThe cheetah (genus Acinonyx), which is considered vulnerable by the IUCN Red List, suffers quite frequently from chronic gastrointestinal (GI) diseases, often associated with Helicobacter spp., with a significant mortality, especially in captive animals. The study of the fecal proteome aims to investigate possible new markers of disease that could be useful in diagnosing and/or monitoring, when performed in feces, gastrointestinal disorders. Considering the invasiveness of some diagnostic investigations (e.g., GI endoscopy) in general and the difficulties of carrying out even simple blood sampling in animals such as the cheetah, the possibility of deriving clinical-diagnostic information from fecal samples is extremely interesting. Herein, a fecal proteomic evaluation from healthy and diseased cheetahs suffering from GI disorders is performed.Fecal proteomics allows for the identification of proteins and peptides present in stools and is useful in finding possible new biomarkers for diagnosing and/or monitoring gastrointestinal (GI) disorders. In the present study, we investigated the fecal proteome in healthy and diseased cheetahs (Acinonyx jubatus). Captive individuals of this species frequently show gastrointestinal disorders characterized by recurrent episodes of diarrhea, rare episodes of vomiting and weight loss, associated with Helicobacter spp. infection. Fecal proteomic evaluation has been performed by two-dimensional electrophoresis followed by liquid chromatography-tandem mass spectrometry. In healthy cheetahs, the results showed the presence of the following proteins: collagen alpha-1 (II) chain, transthyretin, IgG Fc-binding protein, titin, dystonin, isopentenyl-diphosphate Delta-isomerase 1, sodium/potassium-transporting ATPase subunit alpha-1 and protein disulfide-isomerase A6. The presence of albumin isoforms was found only in diseased cheetahs. The present paper reports the study of the fecal proteome in the cheetah, evidences some differences between healthy and diseased patients and confirms, once again, the potential of fecal proteomics for the study of the GI environment, with promising developments regarding the identification of new diagnostic/monitoring markers.

Transcriptome Analysis of Dendrobine Biosynthesis in Trichoderma longibrachiatum MD33.

Dendrobine is a representative component of Dendrobium nobile, and its pharmacological effects have been extensively studied. Trichoderma longibrachiatum MD33 was isolated from the stem of Dendrobium nobile which can produce dendrobine. In order to understand the effect of Methyl Jasmonate (MeJA) on the production of dendrobine, transcriptome analysis was performed after MeJA treatment in the MD33 and control groups. The dendrobine production of MeJA (20 μmol/L) treatment group was 44.6% higher than that of control. In this study, the RNA sequencing technology was applied, a total of 444 differentially expressed genes (DEGs) in the control and MeJA treatment groups, including 226 up-regulated genes and 218 down-regulated genes. The Kyoto Encyclopedia of Genes and Genomes annotation showed that numbers of DEGs were associated with the putative alkaloid biosynthetic pathway in T Trichoderma longibrachiatum MD33. Several MVA pathway enzyme-coding genes (isopentenyl-diphosphate Delta-isomerase, iphosphomevalonate decarboxylase and farnesyl diphosphate synthase) were found to be differentially expressed, suggesting an active precursor supply for alkaloid biosynthesis after MeJA treatment, in other wise, dendrobine may synthesis through the MVA pathway in MD33. Numerous MeJA-induced P450 family genes, aminotransferase genes and methyltransferase genes were identified, providing several important candidates to further elucidate the dendrobine biosynthetic pathway of T. longibrachiatum MD33. Furthermore, several MeJA-induced transcription factors (TFs) encoding genes were identified, suggesting a complex genetic network affecting the dendrobine in T. longibrachiatum MD33. These findings reveal the regulation mechanism underlying the MeJA-induced accumulation of dendrobine in T. longibrachiatum MD33.

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis

BackgroundMenaquinone-7 (MK-7), which is associated with complex and tightly regulated pathways and redox imbalances, is produced at low titres in Bacillus subtilis. Synthetic biology provides a rational engineering principle for the transcriptional optimisation of key enzymes and the artificial creation of cofactor regeneration systems without regulatory interference. This holds great promise for alleviating pathway bottlenecks and improving the efficiency of carbon and energy utilisation.ResultsWe used a bottom-up synthetic biology approach for the synthetic redesign of central carbon and to improve the adaptability between material and energy metabolism in MK-7 synthesis pathways. First, the rate-limiting enzymes, 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl-diphosphate delta-isomerase (Fni), 1-deoxyxylulose-5-phosphate reductase (DXR), isochorismate synthase (MenF), and 3-deoxy-7-phosphoheptulonate synthase (AroA) in the MK-7 pathway were sequentially overexpressed. Promoter engineering and fusion tags were used to overexpress the key enzyme MenA, and the titre of MK-7 was 39.01 mg/L. Finally, after stoichiometric calculation and optimisation of the cofactor regeneration pathway, we constructed two NADPH regeneration systems, enhanced the endogenous cofactor regeneration pathway, and introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis. The strain expressing pos5P was more efficient in converting NADH to NADPH and had excellent MK-7 synthesis ability. Following three Design-Build-Test-Learn cycles, the titre of MK-7 after flask fermentation reached 53.07 mg/L, which was 4.52 times that of B. subtilis 168. Additionally, the artificially constructed cofactor regeneration system reduced the amount of NADH-dependent by-product lactate in the fermentation broth by 9.15%. This resulted in decreased energy loss and improved carbon conversion.ConclusionsIn summary, a "high-efficiency, low-carbon, cofactor-recycling" MK-7 synthetic strain was constructed, and the strategy used in this study can be generally applied for constructing high-efficiency synthesis platforms for other terpenoids, laying the foundation for the large-scale production of high-value MK-7 as well as terpenoids.

Isolation and characterization of AaZFP1, a C2H2 zinc finger protein that regulates the AaIPPI1 gene involved in artemisinin biosynthesis in Artemisia annua.

AaZFP1, a C2H2-type transcription factor, was found to bind the AGT-N1-10-AGT box of AaIPPI1proand activate the expression of AaIPPI1 involved in artemisinin biosynthesis. Artemisinin, an endoperoxide sesquiterpene lactone, is a widely used antimalarial drug isolated from Artemisia annuaL. Isopentenyl pyrophosphate isomerase (AaIPPI1) catalyzes the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate and is the key gene involved in the biosynthesis of artemisinin. However, the AaIPPI1 gene regulation network remains largely unknown. Here, we isolated the AaIPPI1 promoter (AaIPPI1pro) and predicted that it contains cis-elements involved in stress responses, including the TGACG motif (a methyl jasmonate-responsive element), GARE motif (a gibberellin-responsive element), ABRE (an abscisic acid-responsive element), TC-rich repeats (a stress-responsive element), and the AGT-N1-10-AGT box, which is the binding site of Cys/His2 zinc finger protein (C2H2 ZFP). The C2H2 ZFP gene AaZFP1 was discovered by screening a cDNA library using AaIPPI1pro as bait in yeast. AaZFP1 contains two conserved C2H2 regions, a nuclear localization domain (B box), a Leu-rich domain (L box), and a conserved DLN sequence (DLN box) close to its C terminus. A subcellular localization assay indicated that AaZFP1 protein is localized in the nucleus and cytoplasm. An electrophoretic mobility shift assay demonstrated that AaZFP1 binds to the AGT-N1-10-AGT box of AaIPPI1pro. A dual-luciferase assay indicated that AaZFP1 enhanced the promoter activity of AaIPPI1 in vivo. Transient overexpression of AaZFP1 in A. annua increased the expression of AaIPPI1 and the content of artemisinin. Our data demonstrated that AaZFP1 functions as a transcriptional activator that regulates the expression of AaIPPI1 by directly binding to its promoter. The present study provides insights into the transcriptional regulation of genes involved in artemisinin biosynthesis in A. annua.

Juvenile Hormone Synthesis Pathway Gene SfIPPI Regulates Sogatella furcifera Reproduction.

Simple SummaryThe juvenile hormone is essential for insect growth, development, and reproduction. Isopentenyl pyrophosphate isomerase is a key isomerase involved in the synthesis of the juvenile hormone. This study evaluates the effect of the targeted silencing of the SfIPPI gene on the reproduction of Sogatella furcifera (white-backed planthopper). We found that SfIPPI silencing significantly inhibits the ovarian development and egg production in female adults of S. furcifera and significantly inhibits the transcription of downstream genes in the juvenile hormone synthesis pathway. Our data provide insight into the function of juvenile hormone biosynthetic pathway genes in insect reproduction, which could be a potential target to control and prevent agricultural pests.The juvenile hormone (JH) is crucial for insect reproduction, and isopentenyl pyrophosphate isomerase (IPPI) is a key enzyme in the JH synthesis pathway. However, few studies have investigated how IPPI regulates insect reproduction. This study identifies and characterizes the IPPI gene (SfIPPI) from the important agricultural pest Sogatella furcifera. A phylogenetic analysis reveals a high homology of SfIPPI with the IPPI amino acid sequences of Laodelphax striatellus and Nilaparvata lugens (Stål). Furthermore, SfIPPI is expressed at various developmental stages and in various tissues of S. furcifera, and is significantly higher on the 5th day of adult emergence and in integument tissue, while lower levels are found on the 3rd day of adult emergence and in fat body and gut tissue. After silencing SfIPPI using RNA interference, the ovarian development is significantly inhibited and the fecundity is significantly reduced when compared with the control group. Additionally, SfIPPI silencing significantly decreases the expression levels of downstream JH signal transduction pathway genes (SfJHAMT, SfFAMeT, and SfKr-h1) and SfVg. Our findings are helpful in elucidating the molecular mechanism underlying the regulation of insect reproduction through genes in the JH synthesis pathway, and they provide a theoretical basis for the development of pest control treatments targeting SfIPPI.

Identification of the diterpenoid biosynthesis genes and their expression status in relation to oleoresin yield of masson pine

Conifer oleoresin is a highly precious raw material for industrial sectors and a precursor for biofuels. Diterpenoid, particularly diterpenoid resin acids (DRAs), is one of the most abundant oleoresin components. Masson pine (Pinus massoniana L.) is one of the most important native species for oleoresin yield in China. To better understand the biosynthesis of diterpenoid in high oleoresin-yield masson pine and develop methods for its production in needle and trunk xylem, we used the combination of SMRT full-length transcriptome and Illumina RNA sequencing technologies to establish a transcriptome dataset. Approximately 20,560 and 17,950 full-length non-redundant transcripts were assembled in trunk xylem and needle, respectively. Of those, 29 and 20 DEGs were identified involving in the Terpenoid Backbone Biosynthesis pathway (TBB) (KO 00900) and the Diterpenoid Biosynthesis pathway (DB) (KO 00904) in trunk xylem, as well as 35 and 12 DEGs in the TBB and the DB in the needle, respectively. Compared with the low oleoresin-yield germplasm, 13 up-regulated DEGs with complete open reading frame (ORF) sequence were obtained by Venn analysis in trunk xylem and needle of a high oleoresin-yield clone. Subsequently, the expression patterns of the 16 diterpenoid biosynthesis-related genes (including three types of DXS genes) were validated by quantitative real-time PCR in both 12-year-old and 1-year-old high and low oleoresin-yield masson pine clones. The results showed that the 13 genes in the higher yield oleoresin trees were proved to be higher expression in comparison with the lower ones irrespectively of the tree age; as well as MCS (2-C-methyl-d-erythritol 2, 4-cyclodiphosphate synthase), HDS (1-hydroxy-2-methyl-2-(E)-butenyl4-diphosphate synthase), and IDI (Isopentenyl-diphosphate Delta-isomerase) of the methylerythritol 4-phosphate (MEP) pathway demonstrated a higher expression compared to that of other TBB genes. Both cytosol-localized MVA and plastid-localized MEP pathways gave significant contributions to diterpenoid accumulation, and the latter played a more dominant role. To clarify the potential relationships between oleoresin yield and the expression patterns of 16 genes from the TBB and DB pathways, the Pearson correlation analysis was performed using three tissues (young needle, old needle, and trunk xylem) from the 60-year-old tree. Remarkably, HMGR (Hydroxy methylglutaryl-CoA reductase) and ent-CDS (Ent-copalyl diphosphate synthase) were positive correlation with oleoresin yield in the new needle, whereas FPPS (Farnesyl pyrophosphate synthase) was a negative correlation with that in the old needle; furthermore, HMGS (Hydroxy methylglutaryl-CoA synthase), IDI and CDS (Copalyl diphosphate synthase) were positively correlated with oleoresin synthesis in the trunk xylem. Moreover, the Pearson correlation analysis was also carried out to determine the potential relationships between the 13 genes of the TBB pathways and ent-CDS, CDS, and LDS ((13E)-labda-7 13-dien-15-ol synthase) from DB pathways within three tissues of the 60-year-old masson pine. Furthermore, the diterpenoid and diterpene resin acids (DRAs) demonstrated highly positive correlations with oleoresin yield, and DRAs were detected as the most abundant diterpenoids in oleoresin. Promisingly, HMGR, MCS, HDS, IDI, ent-CDS, and CDS may be used as the key target genes of the molecular-assisted selection for high-oleoresin-yielding germplasm in this species.

Transcriptome analysis for molecular landscaping of genes controlling diterpene andrographolide biosynthesis in Andrographis paniculata (Burm . f.) Nees.

Kalmegh [Andrographis paniculata (Burm. f.) Nees.] is one of the essential medicinal plants due to an important terpenoid, i.e. andrographolide possesses immense therapeutic and pharmacological uses. The experiment was performed to elucidate the expression of candidate genes associated with andrographolide biosynthesis. Based on results obtained in chromatography for andrographolide content analysis of six genotypes, two contrast genotypes, i.e. IC-520361 (maximum andrographolide content—2.33%) and Anand Local (lowest andrographolide content—1.01%) were selected for the transcriptome analysis. A total of 1.04 Gb of raw data were produced using MiSeq Illumina platform, in which IC 520361 generated 645 million base pairs sequence along with 4,524,251 raw reads and Anand Local produced 419 million base pairs sequence along with 3,021,316 raw reads. The combined assembly of high quality reads generated for both the samples had 33,247,454 bp of total assembled bases and 38,292 of transcripts. The GC percent of assembled transcripts was 44.79%, an average read length was 800 bp and N50 value was 1186 bp. Species-specific distribution using BLAST X (Nr), showed the highest Blast hits with Sesamum indicum. Out of 23,346 transcripts, 87% of transcripts annotated in UniProt KB (Universal Protein Resource KnowledgeBase) database and only 0.21% of transcripts were annotated in TAIR (The Arabidopsis Information Resources). Biological processes gene ontology classified based on Blast2GO showed, out of 6853 transcripts, 1370 of transcripts were represented by terpenoid biosynthetic pathway, which involved in secondary metabolite andrographolide biosynthesis. The heat map showed 1016 transcripts were differentially expressed between two kalmegh genotypes, in which nine important differentially expressed transcripts related to MEP (2C methyl-d-erythritol 4-phosphate) and MVA (Mevalonic acid) andrographolide biosynthesis pathways such as, geranyl diphosphate synthase small subunit, Isopentenyl-diphosphate delta-isomerase i-like, 4, 13-hydroxy-3-methylglutaryl-coenzyme a reductase etc. were upregulated in IC 520361 as compared to Anand Local, which were validated through RT-qPCR. The highest expression of gene 13-hydroxy-3-methylglutaryl-coenzyme a reductase (HMGR) was reported, which is responsible for accumulation of andrographolide in leaf. This comparative transcriptome analysis confirmed the expression level of genes were higher in accession IC 520361 as compare to Anand Local related to andrographolide biosynthesis pathways i.e. MEP and MVA. These up-regulated genes could be over-expressed to enhance the andrographolide content using genetic engineering of these metabolic pathways. It will also give an idea to the breeder for development of molecular markers for direct screening of the genotypes.Electronic supplementary materialThe online version of this article (10.1007/s13205-020-02511-y) contains supplementary material, which is available to authorized users.

Melatonergic systems of AANAT, melatonin, and its receptor MT2 in the corpus luteum are essential for reproductive success in mammals†

Corpus luteum (CL) plays a critical role in mammalian reproductive physiology. Its dysfunction will lead to infertility or habitual abortion. In the current study, by use of melatonin specific membrane receptor 2 (MT2) knocking out (KO) mice model combined with RNA-Seq, immunohistochemistry, and immunofluorescence analyses, the genes of melatonin synthetic enzyme arylalkylamine N-acetyltransferase (AANAT) and MT2 were identified to strongly express in the CL of sows and mice. KO MT2 significantly impaired the reproductive performance in mice indicated by the reduced litter sizes. Melatonin treatment elevated the progesterone production in sows suggesting the improved CL function. Mechanistic analysis showed that melatonin upregulated a set of progesterone synthesis-related genes including cytochrome P450 family 11 subfamily A member 1 (Cyp11a1), aldo-keto reductase family 1, member C18 (Akr1c18), isopentenyl-diphosphate delta isomerase 1 (Idi1), and luteinizing hormone/choriogonadotropin receptor (Lhcgr). The upregulation of these genes directly related to the increased progesterone production. The regulatory effects of melatonin on these gene expressions were mediated by MT2 and MT2KO diminished the effects of melatonin in this respect. Thus, the presence of melatonergic system of AANAT, melatonin, and its receptor MT2 in CL is essential for reproductive success in mammals.

Improved cis-Abienol production through increasing precursor supply in Escherichia coli

cis-Abienol, a natural diterpene-diol isolated from balsam fir (Abies balsamea), can be employed as precursors for the semi-synthesis of amber compounds, which are sustainable replacement for ambergris and widely used in the fragmented industry. This study combinatorially co-expressed geranyl diphosphate synthase, geranylgeranyl diphosphate synthase, Labda-13-en-8-ol diphosphate synthase and diterpene synthase, with the best combination achieving ~ 0.3 mg/L of cis-abienol. An additional enhancement of cis-abienol production (up to 8.6 mg/L) was achieved by introducing an exogenous mevalonate pathway which was divided into the upper pathway containing acetyl-CoA acetyltransferase/HMG-CoA reductase and HMG-CoA synthase and the lower pathway containing mevalonate kinase, phosphomevalonate kinase, pyrophosphate mevalonate decarboxylase and isopentenyl pyrophosphate isomerase. The genetically modified strain carrying chromosomal copy of low genes of the mevalonate with the trc promoter accumulated cis-abienol up to 9.2 mg/L in shake flask. Finally, cis-abienol titers of ~ 220 mg/L could be achieved directly from glucose using this de novo cis-abienol-producing E. coli in high-cell-density fermentation. This study demonstrates a microbial process to apply the E. coli cell factory in the biosynthesis of cis-abienol.

<p>Potential mevalonate pathway precursors for enhanced production of gymnemic acid</p>

Gymnema sylvestre, locally called Gurmar (meaning sugar destroyer), is well known in Indian medicine for treatment of diabetes which is attributed to its triterpenoid saponin content gymnemic acid. Quantity of the gymnemic acid in is influenced by geographical and seasonal variations. Use of precursors enhances production of gymnemic acid in vitro. Here, we explored three different precursors, namely isopentenyl pyrophosphate (IPP), squalene, and 2,3-oxidosqualene for their effect on gymnemic acid production in in vitro cultures of Gymnema sylvestre. Gymnemic acid production in shoot cultures treated with 2,3-oxidosqualene (23.31 mg g-1 DW) was highest among all three precursors studied, followed by squalene (18.12 mg g-1 DW) and IPP (15.94 mg g-1 DW). Addition of these precursors might enhance the activity of respective enzymes, such as IPP isomerase, squalene synthase (SS), squalene epoxidase (SE), and β-amyrin synthase (β-AS) which are the key enzymes in terpenoid production. Higher concentration and higher harvest duration of all three precursors reduced the production of gymnemic acid. Results have shown that addition of these precursors is effective in enhancement of gymnemic acid production and can be successfully utilized for commercial application.

Genes ptz and idi, Coding for Cytokinin Biosynthesis Enzymes, Are Essential for Tumorigenesis and In Planta Growth by P. syringae pv. savastanoi NCPPB 3335.

The phytopathogenic bacterium Pseudomonas syringae pv. savastanoi elicits aerial tumors on olive plants and is also able to synthesize large amounts of auxins and cytokinins. The auxin indoleacetic acid was shown to be required for tumorigenesis, but there is only correlational evidence suggesting a role for cytokinins. The model strain NCPPB 3335 contains two plasmid-borne genes coding for cytokinin biosynthesis enzymes: ptz, for an isopentenyl transferase and idi, for an isopentenyl-diphosphate delta-isomerase. Phylogenetic analyses showed that carriage of ptz and idi is not strictly associated with tumorigenic bacteria, that both genes were linked when first acquired by P. syringae, and that a different allele of ptz has been independently acquired by P. syringae pv. savastanoi and closely related bacteria. We generated mutant derivatives of NCPPB 3335 cured of virulence plasmids or with site-specific deletions of genes ptz and/or idi and evaluated their virulence in lignified and micropropagated olive plants. Strains lacking ptz, idi, or both produced tumors with average volumes up to 29 times smaller and reached populations up to two orders of magnitude lower than those induced by strain NCPPB 3335; these phenotypes reverted by complementation with the cloned genes. Trans-zeatin was the most abundant cytokinin in culture filtrates of NCPPB 3335. Deletion of gene ptz abolished biosynthesis of trans-zeatin and dihydrozeatin, whereas a reduced but significant amount of isopentenyladenine was still detected in the medium, suggesting the existence of other genes contributing to cytokinin biosynthesis in P. syringae. Conversely, extracts from strains lacking gene idi contained significantly higher amounts of trans-zeatin than extracts from the wild-type strain but similar amounts of the other cytokinins. This suggests that Idi might promote tumorigenesis by ensuring the biosynthesis of the most active cytokinin forms, their correct balance in planta, or by regulating the expression of other virulence genes. Therefore, gene ptz, but not gene idi, is essential for the biosynthesis of high amounts of cytokinins in culture; however, both ptz and idi are individually essential for the adequate development of tumors on olive plants by Psv NCPPB 3335.

Terpenoid Cyclization by SAM-Dependent C-Methyl Transferase

Serratia plymuthica 4Rx13 emits the unique hydrocarbon sodorifen (C16H26) in which every carbon of the bicyclo[3.2.1]octane skeleton carries a methyl unit. Biosynthesis of this homosesquiterpene depends on a gene cluster comprising four genes, an isopentenyl pyrophosphate (PP) isomerase, a deoxyxylulose-5-phosphate synthase, a methyltransferase (MT), and a terpene synthase. The latter two enzymes are required and sufficient for the noncanonical biosynthesis of sodorifen.

Myclobutanil developmental toxicity, bioconcentration and sex specific response in cholesterol in zebrafish (Denio rerio).

The fungicide myclobutanil (MYC) is a common contaminant found in surface water. The aim of this study was to determine the acute toxicity, developmental effects, bioconcentration factor (BCF) and potential bio-molecular mechanisms of MYC toxicity in zebrafish. Susceptibility to MYC toxicity was life-stage dependent with adult fish being the most sensitive (96 h-LC50, 6.34 mg/L) followed by 72 h post-hatch (hph) larvae (8.90 mg/L), 12 hph larvae (20.53 mg/L) and embryos (42.54 mg/L). Zebrafish embryos and larvae (12 hph) responded with decreased hatching, heartbeat and growth, as well as abnormal spontaneous movement and development. BCFs were calculated by quantifying MYC concentrations from different tissues of adult zebrafish exposed to MYC for up to 11 days. Highest BCFs were obtained from gills (18.25 ± 0.07), followed by viscera (16.78 ± 0.04), head (13.13 ± 0.08) and muscle (8.96 ± 0.10). MYC (0.5 mg/L) inhibited gene expression related to cholesterol synthesis pathway, including 24-dehydrocholesterol reductase (DHCR24), 7-dehydrocholesterol reductase (DHCR7), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCRa), HMGCRb, farnesyl-diphosphate farnesyltransferase 1(FDFT1), squa-lene epoxidase (SQLE), isopentenyl-diphosphate delta isomerase 1 (IDI1) and CYP51, while no cholesterol changes were observed in the MYC treated group. These results will contribute to the literature assessing the environmental risk of MYC in aquatic environment.

Abstract 4667: A synergistic combination strategy for optimal inhibition of colon cancer stem cells: Simultaneous inhibition of Insulin-like growth factor-1 receptor-AKT-mammalian target of rapamycin axis and the mevalonate-isoprenoid biosynthetic pathway

Abstract Introduction: Targeting cancer stem cells (CSCs) is an attractive new paradigm to achieve long-term remission/cure. The mevalonate-isoprenoid biosynthetic (MIB) pathway is critical in the regulation of colonic CSCs. However, feedback activation of SREBP2 following inhibition of the MIB pathway reduces the efficiency of the latter strategy. We described that Insulin-like growth factor-1 receptor-AKT-mammalian target of rapamycin (InAT) axis inhibits CSCs via inhibition of the MIB pathway at a level distinct from the target of statin - HMGCR, while simultaneously inhibiting SREBP2. Hence, we hypothesized that combining a MIB pathway inhibitor with the InAT axis inhibition will result in optimal inhibition of CSCs. Methods: Four possible combinations with the MIB pathway inhibitors (Simvastatin or zoledronate) and an InAT axis inhibitors (OSI-906) or Everolimus were tested against spheroids from 12 colorectal cancer cells. Synergistic effect of the combinations was determined using Chou Talalay method (Calcusyn). Most promising combinations were examined in an in vivo model of Dual high CSCs (CD133+/CXCR4+) induced xenografts to assess tumor volume, and ex vivo CSC phenotype (spheroid formation and CSC marker expression) in xenograft-derived cells. Results: OSI-906 + Simvastatin, as well as Everolimus + simvastatin, showed a robust synergistic effect (Combination index <0.75) in a majority of colon cancer cells which was independent of mutation in PI3K/mTOR or RAS pathways. In fact, both the combinations showed a significantly higher inhibition of Dual high CSCs-induced xenograft volume as well as ex-vivo spheroid formation (2°→4°) and CSC markers (CD133, DCLK1, LGR etc.) than either agents alone or vehicle controls in two model of colon cancer HCT-116 (MSI high, KRAS mut), HT29 (MSS, KRAS mut). Importantly, Simvastatin treatment failed to inhibit SREBP2 levels. However, InAT axis inhibitors caused a significant inhibition of SREBP2 and the MIB pathway genes. In fact, inhibition of the MIB pathway at the level of isopentenyl-diphosphate delta isomerase 1 (IDI1) was critical for simultaneous inhibition of the MIB pathway and InAT axis to attenuate CSC self-renewal as supplementation with DMAPP, and FPP, but not mevalonate or IPP, reversed the effect of the combination on CSCs. Conclusion: Orally deliverable and combinations of the FDA-approved drugs targeting the MIB pathway and the InAT axis cause optimum inhibition of colonic CSCs through downregulation of SREBP2, achieving maximal inhibition of the MIB pathway. Note: This abstract was not presented at the meeting. Citation Format: Chetna Sharon, Rio Boothello, Alberto Vera, Bhaumik B. Patel. A synergistic combination strategy for optimal inhibition of colon cancer stem cells: Simultaneous inhibition of Insulin-like growth factor-1 receptor-AKT-mammalian target of rapamycin axis and the mevalonate-isoprenoid biosynthetic pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4667.