Novel Systems for the Functional Characterization of Genes Related to Paclitaxel Metabolism in Taxus Cell Cultures

Human society has benefited greatly from plant secondary metabolites, often utilizing a variety of compounds as dyes, food additives, and drugs. In particular, pharmaceutical development has benefited greatly from plant secondary metabolites. One example of this utility is paclitaxel, a highly substituted diterpene approved in the treatment of breast cancer, ovarian cancer, non-small cell lung cancer, and the AIDSrelated Kaposi’s sarcoma. Demand of paclitaxel is likely to increase, due to the current examination of paclitaxel in numerous clinical trials against a variety of other cancers. Taxus cell culture represents a production source of paclitaxel to meet future demand. However, paclitaxel production through Taxus cell culture is often variable and low. Targeted metabolic engineering of Taxus to produce superior paclitaxelaccumulating lines is a viable strategy to address variable and low yields. To facilitate the production of genetically engineered Taxus cell lines, stable transformation is required to examine the long-term effect of gene expression in vitro. Additionally, suitable transient transformation systems are necessary to characterize novel Taxus genes related to paclitaxel accumulation. A transient particle bombardment-mediated transformation protocol was developed to introduce transgenes into Taxus cells in vitro. Additionally, agroinfiltration in Nicotiana benthamiana was examined as a system to express genes related to paclitaxel biosynthesis and lead to the accumulation of the first dedicated taxane, taxa- 4(5), 11(12)-diene. In regard to stable transformation, an Agrobacterium-mediated transformation protocol was developed, though this method requires further optimization for reliability and increased transformation efficiency. These transformation technologies will aid in the creation of elite paclitaxel-accumulating Taxus cell lines

Investigation of Taxol biosynthetic genes for the production of novel taxanes in heterologous plant systems

The diterpenoid paclitaxel (TaxolTM) is one of the most effective anticancer drugs, used against a wide range of cancers. It is produced as a secondary metabolite in the vascular cambial region of the bark of Taxus brevifolia from which it was first extracted in 1971. Taxol also accumulates in low concentrations in several other Taxus species. As the demands for Taxol greatly exceeded its supply, alternative routes for producing the drug and its related taxanes were developed. Taxol is presently manufactured by semisynthesis from its precursors baccatin III and 10-deacetylbaccatin III found in Taxus needles. The biosynthesis of Taxol mostly occurs via the 2-C-methyl D-erythritol 4- phosphate pathway and requires at least 19 enzymatic steps from the precursor geranylgeranyl diphosphate. This study set out to heterologously express the early genes of the Taxol biosynthetic pathway in Nicotiana tabacum for the subsequent redirection of this precursor for the synthesis of novel taxanes. The first five genes of the Taxol biosynthetic pathway, namely taxadien-5-α-hydroxylase, taxadien-5α-acetyltransferase taxoids 10β, 13α- and 7β-hydroxylase, were isolated from Taxus baccata mRNA. Individual transgenic tobacco lines were generated expressing each of the first three enzymes of the biosynthetic pathway. These lines were crossed with each other in order to obtain all three transgenes expressed together in individual transgenic lines. Progenies from the crosses, expressing the first three transgenes were analysed, however, GS-MS analysis failed to detect the compound taxadiene-5α-ol and its acetylated compound taxadiene-5α-yl acetate. The expression of the Taxol biosynthetic genes in transgenic tobacco plants were accompanied by phenotypic effects, including dwarfism and low fertility of the transgenic plants. To circumvent these sterility issues which made crossing of the plants difficult, a construct was prepared carrying the first two genes of the Taxol biosynthetic pathway, to be transformed in yellow flesh tomato mutant at a later stage. The localisation of taxadiene synthase, 5α-hydroxylase and taxadien-5α acetyltransferase was investigated by making translational fusions to fluorescent protein tags. Confocal microscopy was used to detect the fluorescent proteins GFP, YFP and CFP in Arabiodopsis thaliana roots and tobacco leaf and root cells. Taxadiene synthase was found to be localised to the plastids, taxadien-5-α-hydroxylase spatially positioned on the plastid envelope and the endoplasmic reticulum membrane and taxadien-5α acetyltransferase was localised to the endoplasmic reticulum

Linking Genotype and Phenotype of Saccharomyces cerevisiae Strains Reveals Metabolic Engineering Targets and Leads to Triterpene Hyper-Producers

Background: Metabolic engineering is an attractive approach in order to improve the microbial production of drugs. Triterpenes is a chemically diverse class of compounds and many among them are of interest from a human health perspective. A systematic experimental or computational survey of all feasible gene modifications to determine the genotype yielding the optimal triterpene production phenotype is a laborious and time-consuming process. Methodology/Principal Findings: Based on the recent genome-wide sequencing of Saccharomyces cerevisiae CEN.PK 113-7D and its phenotypic differences with the S288C strain, we implemented a strategy for the construction of a beta-amyrin production platform. The genes Erg8, Erg9 and HFA1 contained non-silent SNPs that were computationally analyzed to evaluate the changes that cause in the respective protein structures. Subsequently, Erg8, Erg9 and HFA1 were correlated with the increased levels of ergosterol and fatty acids in CEN.PK 113-7D and single, double, and triple gene over-expression strains were constructed. Conclusions: The six out of seven gene over-expression constructs had a considerable impact on both ergosterol and beta-amyrin production. In the case of beta-amyrin formation the triple over-expression construct exhibited a nearly 500% increase over the control strain making our metabolic engineering strategy the most successful design of triterpene microbial producers

ENGINEERING NOVEL TERPENE PRODUCTION PLATFORMS IN THE YEAST SACCHAROMYCES CEREVISIAE

The chemical diversity and biological activities of terpene and terpenoids have served in the development of new flavors, fragrances, medicines and pesticides. While terpenes are made predominantly by plants and microbes in small amounts and as components of complex mixtures, chemical synthesis of terpenes remains technically challenging, costly and inefficient. In this dissertation, methods to create new yeast lines possessing a dispensable mevalonate biosynthetic pathway wherein carbon flux can be diverted to build any chemical class of terpene product are described. The ability of this line to generate diterpenes was next investigated. Using a 5.5 L fed bath fermentation system, about 569 mg/L kaurene and approximately 207 mg/L abietadiene plus 136 mg/L additional isomers were achieved. To engineer more highly modified diterpenes might have greater industrial, agricultural or medicinal applications, kaurenoic acid production reached 514 mg/L with byproduct kaurene and kaurenal at 71.7mg/L and 20.1mg/L, respectively, in fed batch fermentation conditions. Furthermore, ZXM lines for engineer monoterpene and ZXB lines for engineer triterpene were generated by additional specific genomic modification, 84.76 ±13.2 mg/L linalool, 20.54±3.8 mg/L nerolidol and 297.7mg/L squalene were accumulate in ZXM144 line ana ZXB line, respectively, in shake flask conditions

Altered ovarian cancer metabolism increases neuronal n-acetylaspartate to promote tumor growth

Background: Altered metabolism is a well-established trait in many cancers, and is an emerging hallmark of cancer. Recent resurgence of cancer metabolism studies has identified dysregulated metabolic pathways that produce novel oncometabolites in various cancers. However, large scale studies of dysregualted high grade serous epithelial ovarian cancers (HGSOC) are unknown. Materials and Methods: Following IRB approval, metabolic profiling of 101 HGSOC patients and 15 normal ovaries were obtained using GC/LC mass spectrometry from 2 U.S. academic centers to identify highly up-regulated metabolites. Samples from a cohort of 135 and 208 patients from a single institution were evaluated for gene expression and protein expression of NAT8L, respectively. Gene expression of NAT8L and clinical outcomes were further investigated from publicly available databases from the cancer genomics atlas (TCGA) using www.cbioportal.org, and two previously published melanoma gene expression profiles. Reverse Phase Protein Array (RPPA) and gene expression array were evaluated in HeyA8 ovarian cancer cell lines to investigate the protein and gene expression changes associated with NAT8L siRNA. In vitro and in vivo v experiments of NAT8L siRNA were investigated to evaluate its effects on cancer proliferation, apoptosis, cell cycle, and invasion/migration. Results: A total of 313 metabolites were identified between these two groups, of which 172 were significantly altered (p HeyA8 and A2780 cell lines showed that NAT8L siRNA significantly increased total apoptosis compared to control (NT) siRNA by 38.53% (p Genomic analysis of HEYA8 cells transfected with NAT8L siRNA compared to NT siRNA showed 1961 significantly different gene expression data (p Conclusion: HGSOC metabolic profiling revealed highly altered metabolism compared to the normal ovary. NAA is one of the most up-regulated metabolites in HGSOC. High levels of NAA are associated with worse overall survival in HGSOC. Furthermore, high expression of its biosynthetic gene (NAT8L) is associated with vii worse overall survival in HGSOC, invasive breast, lung squamous, colon, uterine, melanoma and renal cell cancers. Inhibiting NAA production decreases tumor growth, and tilts the cancer cell to a more catabolic steady state. Therefore, our data indicate that targeting cancer’s NAA production maybe an effective therapeutic approach

Biochemical and genetic insights into asukamycin biosynthesis

Asukamycin, a member of the manumycin family metabolites, is an antimicrobial and potential antitumor agent isolated from Streptomyces nodosus subsp. asukaensis. The entire asukamycin biosynthetic gene cluster was cloned, assembled and expressed heterologously in Streptomyces lividans. Bioinformatic analysis and mutagenesis studies elucidated the biosynthetic pathway at the genetic and biochemical level. Four gene sets, asuA-D, govern the formation and assembly of the asukamycin building blocks, a 3-amino-4-hydroxybenzoic acid (3,4-AHBA) core component, a cyclohexane ring, two triene polyketide chains and a 2-amino-3-hydroxycyclopent-2-enone (C5N) moiety to form the intermediate protoasukamycin. AsuE1 and AsuE2 catalyze the conversion of protoasukamycin to 4-hydroxyprotoasukamycin, which is epoxidized at C5-C6 by AsuE3 to the final product, asukamycin. Branched acyl CoA starter units, derived from Val, Leu and Ile, can be incorporated by the actions of the polyketide synthase KSIII AsuC3/C4 as well as the cellular fatty acid synthase FabH to produce the asukamycin congeners A2-A7. In addition, the type II thioesterase AsuC15 limits the cellular level of ù-cyclohexyl fatty acids and likely maintains homeostasis of the cellular membrane

Endophytes as alternative paclitaxel sources : chemistry and genetics of Taxomyces andreanae and the endophytic flora of Wollemia nobilis

Whether suffering a pathogenic attack, basking in symbiotic comfort, or seemingly symptomless, plants constantly participate in molecular interplay with various classes of microbial organisms. One of the means of interorganismal communication in this dynamic continuum are secondary metabolites. The chemical diversity bearing pharmaceutical potential thus implied reaches beyond the plant kingdom and offers an expended view promising to transform glimpses of reductionist research of the past years to snapshots of an exuberant world of systems biology. Endophytes seem to fit perfectly into this natural ‘warehouse’, only a small part of which we have been able to tap into so far. The introductory section of the hereby presented thesis (chapter 2) provides an elaborate overview on the current state of knowledge about endophytic organisms – microbes colonizing internal tissues of all plant species, creating a huge biodiversity with yet unknown novel natural products presumed to push forward the frontiers of drug discovery (Staniek et al., 2008). Paclitaxel, the world’s first billion dollar anticancer blockbuster, was primarily obtained from Taxus brevifolia. While the search for alternative sources of the powerful antineoplastic agent brought an array of reports on paclitaxel producing endophytes, causing quite a controversy over the past two decades, the world’s market still relies on yew-derived supply of the valuable diterpene.

Metabolic Modeling of Secondary Metabolism in Plant Systems

In the first part of this research, we constructed a Genome scale Metabolic Model (GEM) of Taxus cuspidata, a medicinal plant used to produce paclitaxel (Taxol®). The construction of the T. cuspidata GEM was predicated on recent acquisition of a transcriptome of T. cuspidata metabolism under methyl jasmonate (MJ) elicited conditions (when paclitaxel is produced) and unelicited conditions (when paclitaxel is not produced). Construction of the draft model, in which transcriptomic data from elicited and unelicited conditions were included, utilized tools including the ModelSEED developed by Argonne National Laboratory. Although a model was successfully created and gapfilled by ModelSEED using their software, we were not able to reproduce their results using COBRA, a widely accepted FBA software package. Further work needs to be done to figure out how to run ModelSEED models on commonly available software. In the second part of this research, we modeled the MJ elicited/defense response phenotype in Arabidopsis thaliana. Previously published models of A. thaliana were tested for suitability in modeling the MJ elicited phenotype using publicly available computation tools. MJ elicited and unelicited datasets were compared to ascertain differences in metabolism between these two phenotypes. The MJ elicited and unelicited datasets were significantly different in many respects, including the expression levels of many genes associated with secondary metabolism. However, it was found that the expression of genes related to growth and central metabolism were not generally significantly different for the MJ+ and MJ- datasets, the pathways associated with secondary metabolism were incomplete and could not be modeled, and FBA methods did not show the difference in growth that was expected. These results suggest that behavior associated with the MJ+ phenotype such as slow growth and secondary metabolite production may be controlled by factors not easily modeled with transcriptome data alone. Additional research was performed in the area of cryosectioning and immunostaining of fixed Taxus aggregates. Protocols developed for this work can be found in Appendix B

Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 2.0

This book, "Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity", presents five papers on human cytochrome P450 (CYP) and P450 reductase, three reviews on the role of CYPs in humans and their use as biomarkers, six papers on CYPs in microorganisms, and one study on CYP in insects. The first paper reports the in silico modeling of human CYP3A4 access channels. The second uses structural methods to explain the mechanism-based inactivation of CYP3A4 by mibefradil, 6,7-dihydroxy-bergamottin, and azamulin. The third article compares electron transfer in CYP2C9 and CYP2C19 using structural and biochemical methods, and the fourth uses kinetic methods to study electron transfer to CYP2C8 allelic mutants. The fifth article characterizes electron transfer between the reductase and CYP using in silico and in vitro methods, focusing on the conformations of the reductase. Then, two reviews describe clinical implications in cardiology and oncology and the role of fatty acid metabolism in cardiology and skin diseases. The second review is on the potential use of circulating extracellular vesicles as biomarkers. Five papers analyze the CYPomes of diverse microorganisms: the Bacillus genus, Mycobacteria, the fungi Tremellomycetes, Cyanobacteria, and Streptomyces. The sixth focuses on a specific Mycobacterium CYP, CYP128, and its importance in M. tuberculosis. The subject of the last paper is CYP in Sogatella furcifera, a plant pest, and its resistance to the insecticide sulfoxaflor