scholarly journals Engineering yeast for de-novo synthesis of the insect repellent - nepetalactone

2021 ◽  
Author(s):  
Meghan E Davies ◽  
Daniel Tsyplenkov ◽  
Vincent J. J. Martin
Keyword(s):  
Cytochrome P450 ◽  
De Novo ◽  
Copy Number Variations ◽  
Insect Repellent ◽  
In Planta ◽  
Microtiter Plates ◽  
Pathway Optimization ◽  
Commercial Viability ◽  
Simple Sugar ◽  
Scalable Production

ABSTRACTWhile nepetalactone, the active ingredient in catnip, is a potent insect repellent, its low in planta accumulation limits its commercial viability as an alternative repellent. Here we describe a platform for de novo nepetalactone production in Saccharomyces cerevisiae, enabling sustainable and scalable production. Nepetalactone production required introduction of eight exogenous genes including the cytochrome P450 geraniol-8-hydroxylase, which represented the bottleneck of the heterologous pathway. Combinatorial assessment of geraniol-8-hydroxylase and cytochrome P450 reductase variants, as well as copy-number variations were used to overcome this bottleneck. We found that several reductases improved hydroxylation activity, with a higher geraniol-8-hydroxylase ratio further increasing 8-hydroxygeraniol titers. Another roadblock was the accumulation of an unwanted metabolite that implied inefficient channeling of carbon through the pathway. With the native yeast old yellow enzymes previously shown to use monoterpene intermediates as substrates, both homologs were deleted. These deletions increased 8-hydroxygeraniol yield, resulting in a final de novo accumulation of 3.10 mg/L/OD600 of nepetalactone from simple sugar in microtiter plates. Our pathway optimization will aid in the development of high yielding monoterpene S. cerevisiae strains.

scholarly journals Engineering of new-to-nature halogenated indigo precursors in plants

2018 ◽  
Author(s):  
Sabine Frabel ◽  
Bastian Wagner ◽  
Markus Krischke ◽  
Volker Schmidts ◽  
Christina M Thiele ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
De Novo ◽  
Dry Weight ◽  
Cytochrome P450 Monooxygenase ◽  
Blue Dye ◽  
In Planta ◽  
P450 Monooxygenase ◽  
Catalytic Systems ◽  
Modular Assembly ◽  
Human Cytochrome

Plants are versatile chemists producing a tremendous variety of specialized compounds. Here, we describe the engineering of entirely novel metabolic pathways in planta enabling generation of halogenated indigo precursors as non-natural plant products. Indican (indolyl-beta-D-glucopyranoside) is a secondary metabolite characteristic of a number of dyers plants. Its deglucosylation and subsequent oxidative dimerization leads to the blue dye, indigo. Halogenated indican derivatives are commonly used as detection reagents in histochemical and molecular biology applications; their production, however, relies largely on chemical synthesis. To attain the de novo biosynthesis in a plant-based system devoid of indican, we employed a sequence of enzymes from diverse sources, including three microbial tryptophan halogenases substituting the amino acid at either C5, C6, or C7 of the indole moiety. Subsequent processing of the halotryptophan by bacterial tryptophanase TnaA in concert with a mutant of the human cytochrome P450 monooxygenase 2A6 and glycosylation of the resulting indoxyl derivatives by an endogenous tobacco glucosyltransferase yielded corresponding haloindican variants in transiently transformed Nicotiana benthamiana plants. Accumulation levels were highest when the 5-halogenase PyrH was utilized, reaching 0.93 +/-0.089 mg/g dry weight of 5-chloroindican. The identity of the latter was unambiguously confirmed by NMR analysis. Moreover, our combinatorial approach, facilitated by the modular assembly capabilities of the GoldenBraid cloning system and inspired by the unique compartmentation of plant cells, afforded testing a number of alternative subcellular localizations for pathway design. In consequence, chloroplasts were validated as functional biosynthetic venues for haloindican, with the requisite reducing augmentation of the halogenases as well as the cytochrome P450 monooxygenase fulfilled by catalytic systems native to the organelle. Thus, our study puts forward a viable alternative production platform for halogenated fine chemicals, eschewing reliance on fossil fuel resources and toxic chemicals. We further contend that in planta generation of halogenated indigoid precursors previously unknown to nature offers an extended view on and, indeed, pushes forward the established frontiers of biosynthetic capacity of plants.

scholarly journals Efficient In Planta Detection and Dissection of De Novo Mutation Events in theArabidopsis thalianaDisease Resistance GeneUNI

2016 ◽  
Vol 57 (6) ◽  
pp. 1123-1132
Author(s):  
Tomohiko Ogawa ◽  
Akiko Mori ◽  
Kadunari Igari ◽  
Miyo Terao Morita ◽  
Masao Tasaka ◽  
...  
Keyword(s):  
De Novo ◽  
De Novo Mutation ◽  
In Planta

scholarly journals Frequency of Important CYP450 Enzyme Gene Polymorphisms in the Iranian Population in Comparison with Other Major Populations: A Comprehensive Review of the Human Data

2021 ◽  
Vol 11 (8) ◽  
pp. 804
Author(s):  
Navid Neyshaburinezhad ◽  
Hengameh Ghasim ◽  
Mohammadreza Rouini ◽  
Youssef Daali ◽  
Yalda H. Ardakani
Keyword(s):  
Cytochrome P450 ◽  
Meta Analysis ◽  
Clinical Importance ◽  
Copy Number Variations ◽  
Iranian Population ◽  
Human Populations ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Drug Dosing ◽  
Linkage Information

Genetic polymorphisms in cytochrome P450 genes can cause alteration in metabolic activity of clinically important medicines. Thus, single nucleotide variants (SNVs) and copy number variations (CNVs) in CYP genes are leading factors of drug pharmacokinetics and toxicity and form pharmacogenetics biomarkers for drug dosing, efficacy, and safety. The distribution of cytochrome P450 alleles differs significantly between populations with important implications for personalized drug therapy and healthcare programs. To provide a meta-analysis of CYP allele polymorphisms with clinical importance, we brought together whole-genome and exome sequencing data from 800 unrelated individuals of Iranian population (100 subjects from 8 major ethnics of Iran) and 63,269 unrelated individuals of five major human populations (EUR, AMR, AFR, EAS and SAS). By integrating these datasets with population-specific linkage information, we evolved the frequencies of 140 CYP haplotypes related to 9 important CYP450 isoenzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) giving a large resource for major genetic determinants of drug metabolism. Furthermore, we evaluated the more frequent Iranian alleles and compared the dataset with the Caucasian race. Finally, the similarity of the Iranian population SNVs with other populations was investigated.

scholarly journals Efficient production of vindoline from tabersonine by metabolically engineered Saccharomyces cerevisiae

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Tengfei Liu ◽  
Ying Huang ◽  
Lihong Jiang ◽  
Chang Dong ◽  
Yuanwei Gou ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
De Novo ◽  
Indole Alkaloid ◽  
Functional Expression ◽  
High Yield ◽  
Efficient Production ◽  
Cytochrome P450 Enzymes ◽  
Copy Numbers ◽  
Yield Production ◽  
Engineered Saccharomyces Cerevisiae

AbstractVindoline is a plant derived monoterpene indole alkaloid (MIA) with potential therapeutic applications and more importantly serves as the precursor to vinblastine and vincristine. To obtain a yeast strain for high yield production of vindoline from tabersonine, multiple metabolic engineering strategies were employed via the CRISPR/Cas9 mediated multiplex genome integration technology in the present study. Through increasing and tuning the copy numbers of the pathway genes, pairing cytochrome P450 enzymes (CYPs) with appropriate cytochrome P450 reductases (CPRs), engineering the microenvironment for functional expression of CYPs, enhancing cofactor supply, and optimizing fermentation conditions, the production of vindoline was increased to a final titer as high as ∼16.5 mg/L, which is more than 3,800,000-fold higher than the parent strain and the highest tabersonine to vindoline conversion yield ever reported. This work represents a key step of the engineering efforts to establish de novo biosynthetic pathways for vindoline, vinblastine, and vincristine.

Congenital cerebral palsy: genetic cause and nosological integrity

2021 ◽  
Vol 15 (3-4) ◽  
pp. 65-77
Author(s):  
P. I. Sokolov ◽  
N. V. Chebanenko ◽  
V. P. Zykov ◽  
I. V. Kanivets ◽  
A. G. Prityko ◽  
...  
Keyword(s):  
Cerebral Palsy ◽  
De Novo ◽  
Muscle Tone ◽  
Copy Number Variations ◽  
Genetic Determinism ◽  
Nucleotide Polymorphisms ◽  
Reproductive Ability ◽  
Genetically Determined ◽  
Genomic Anomalies

The review provides an analysis of 73 full-text articles, the source of which was the Medline, OMIM, NCBI, Pubmed, Scopus, eLibrary.ru databases. The data of studies of the main pathogenetic mechanisms of the formation of the cerebral palsy (CP) phenotype, such as chromosomal aberrations, copy number variations, single nucleotide polymorphisms, associated with the development of the CP phenotype, are reviewed and analyzed. Epigenetic effects on the genome, as well as the effects of the genome on the mechanisms of epigenomic regulation, are examined in detail. The data on the genetic determinism of concomitant pathology and reactivity to therapeutic tactics are presented. Based on the study of data from numerous studies, the authors draw the following conclusions:1) the pathogenesis of the phenotype of CP includes a large number of genes that determine violations of cellular metabolism, neuroontogenesis, brain resistance to hypoxia, etc;2) genes whose abnormalities form a syndromic pathology are involved in the pathogenesis of CP;3) the multidirectionality and breadth of the effects of the gene pool with the outcome in a syndrome-specific distinctive picture of the CP allows us to propose the concept of a neurotropic genome;4) the mechanisms of gene involvement can vary from aberrations to epigenetic imbalances;5) different groups of genes can differentially influence the formation of individual syndromes in the phenotype of CP;6) there are data indicating a genetic determinism of the tendency to contracture, pharmacoreactivity to drugs that reduce muscle tone, reactivity to habilitation effects;7) genomic-epigenomic interactions normally ensure the body’s adaptation to environmental conditions, and with pathology, they increase the likelihood of regulatory breakdowns that lead to the formation of a CP phenotype;8) the exclusion from the diagnosis of CP of genetically determined cases of phenotype development is incorrect.The authors present two anthropogenic reasons for the increase in the frequency of occurrence of de novo identified gene abnormalities:1) anthropogenic impact on the environment, increasing the number of anomalies of the genome de novo; 2) iatrogenic effects of technologies for preserving life, vitality and reproductive ability of carriers of genomic anomalies. This effect leads to the fixation of anomalies in the genome of the population.A paradox is formulated, according to which, in the presence of technologies capable of preserving the life of carriers of genomic anomalies, in vivo technologies for genome correction are only just beginning to be put into practice. Based on this, it is concluded that it is necessary to intensify the development of methods for prenatal diagnosis and gene therapy of CP.

scholarly journals Identification of client iron–sulfur proteins of the chloroplastic NFU2 transfer protein in Arabidopsis thaliana

2020 ◽  
Vol 71 (14) ◽  
pp. 4171-4187 ◽  
Author(s):  
Nathalie Berger ◽  
Florence Vignols ◽  
Jonathan Przybyla-Toscano ◽  
Mélanie Roland ◽  
Valérie Rofidal ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
De Novo ◽  
Bimolecular Fluorescence Complementation ◽  
In Planta ◽  
Chlorophyll Metabolism ◽  
Transfer Protein ◽  
Iron Sulfur ◽  
Bimolecular Fluorescence Complementation Assay ◽  
Client Proteins ◽  
S Proteins

Abstract Iron–sulfur (Fe-S) proteins have critical functions in plastids, notably participating in photosynthetic electron transfer, sulfur and nitrogen assimilation, chlorophyll metabolism, and vitamin or amino acid biosynthesis. Their maturation relies on the so-called SUF (sulfur mobilization) assembly machinery. Fe-S clusters are synthesized de novo on a scaffold protein complex and then delivered to client proteins via several transfer proteins. However, the maturation pathways of most client proteins and their specificities for transfer proteins are mostly unknown. In order to decipher the proteins interacting with the Fe-S cluster transfer protein NFU2, one of the three plastidial representatives found in Arabidopsis thaliana, we performed a quantitative proteomic analysis of shoots, roots, and seedlings of nfu2 plants, combined with NFU2 co-immunoprecipitation and binary yeast two-hybrid experiments. We identified 14 new targets, among which nine were validated in planta using a binary bimolecular fluorescence complementation assay. These analyses also revealed a possible role for NFU2 in the plant response to desiccation. Altogether, this study better delineates the maturation pathways of many chloroplast Fe-S proteins, considerably extending the number of NFU2 clients. It also helps to clarify the respective roles of the three NFU paralogs NFU1, NFU2, and NFU3.

Impact of MYC and BCL2 structural variants in tumors of DLBCL morphology and mechanisms of false-negative MYC IHC

Blood ◽  
2020 ◽  
Author(s):  
Brett J Collinge ◽  
Susana Ben-Neriah ◽  
Lauren C. Chong ◽  
Merrill Boyle ◽  
Aixiang Jiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
De Novo ◽  
Cell Lymphoma ◽  
False Negative ◽  
Gene Expression Signature ◽  
B Cell Lymphoma ◽  
Copy Number Variations ◽  
Similar Gene Expression Profile ◽  
Double Hit

When the WHO defined high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) as a clinical category, rearrangements were the only structural variant (SV) incorporated. An "atypical double-hit" entity has been proposed, encompassing tumors with concurrent MYC and BCL2 SVs other than co-occurring translocations - i.e. copy number variations (CNVs). While the identification of a gene expression signature (DHITsig) shared among tumors harboring MYC and BCL2 rearrangements (HGBL-DH/TH-BCL2) has confirmed a shared underlying biology, the biological implication of MYC and BCL2 CNVs requires further elucidation. We performed a comprehensive analysis of MYC and BCL2 SVs, as determined by fluorescent in situ hybridization (FISH), in a cohort of 802 de novo tumors with diffuse large B-cell lymphoma (DLBCL) morphology. While BCL2 CNVs were associated with increased expression, MYC CNVs were not. Furthermore, MYC and BCL2 CNVs, in the context of atypical double-hit, did not confer a similar gene expression profile as HGBL-DH/TH-BCL2. Finally, while MYC IHC has been proposed as a screening tool for FISH testing, two mechanisms were observed that uncoupled MYC rearrangement from IHC positivity. 1) low MYC mRNA expression and 2) false-negative immunohistochemistry (IHC) staining mediated by a single nucleotide polymorphism resulting in an asparagine to serine substitution at the 11th amino acid residue of MYC (MYC-N11S). Taken together, these results support the current exclusion of MYC and BCL2 CNVs from HGBL-DH/TH and highlight the ability of a molecular based classification system to identify tumors with shared biology that FISH and IHC fail to fully capture.

scholarly journals Lessons Learned from CNV Analysis of Major Birth Defects

2020 ◽  
Vol 21 (21) ◽  
pp. 8247
Author(s):  
Alina Christine Hilger ◽  
Gabriel Clemens Dworschak ◽  
Heiko Martin Reutter
Keyword(s):  
Birth Defects ◽  
Copy Number ◽  
Congenital Malformations ◽  
De Novo ◽  
Copy Number Variations ◽  
Lessons Learned ◽  
Organ System ◽  
Molecular Karyotyping ◽  
The Past ◽  
Single Organ

The treatment of major birth defects are key concerns for child health. Hitherto, for the majority of birth defects, the underlying cause remains unknown, likely to be heterogeneous. The implicated mortality and/or reduced fecundity in major birth defects suggest a significant fraction of mutational de novo events among the affected individuals. With the advent of systematic array-based molecular karyotyping, larger cohorts of affected individuals have been screened over the past decade. This review discusses the identification of disease-causing copy-number variations (CNVs) among individuals with different congenital malformations. It highlights the differences in findings depending on the respective congenital malformation. It looks at the differences in findings of CNV analysis in non-isolated complex congenital malformations, associated with central nervous system malformations or intellectual disabilities, compared to isolated single organ-system malformations. We propose that the more complex an organ system is, and the more genes involved during embryonic development, the more likely it is that mutational de novo events, comprising CNVs, will confer to the expression of birth defects of this organ system.

scholarly journals A Case With 4 de Novo Copy Number Variations With Clinical Features That Overlap 1q43q44 Microdeletion and 3q29 Microduplication Syndromes

2018 ◽  
Vol 5 ◽  
pp. 2329048X1879820
Author(s):  
Miriam Kessi ◽  
Jing Peng ◽  
Lifen Yang ◽  
Haolin Duan ◽  
Yulin Tang ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
Corpus Callosum ◽  
Copy Number ◽  
De Novo ◽  
Copy Number Variations ◽  
Language Delay ◽  
Global Developmental Delay ◽  
Recurrent Infections ◽  
Dental Anomalies ◽  
Microdeletion Syndrome

1q43q44 microdeletion syndrome is characterized by intellectual disability/global developmental delay, epilepsy, dysmorphic facies, stereotypic movement, language delay, recurrent infections, dental anomalies, and hand and foot anomalies. Microcephaly and corpus callosum dysplasia are present in some cases depending on gene content. 3q29 microduplication syndrome is characterized by intellectual disability, language delay, microcephaly, and dental anomalies. We report the first case with 4 de novo copy number variations with clinical features which overlap 1q43q44 microdeletion and 3q29 microduplication syndromes. Our case presented with global developmental delay, epilepsy, recurrent infections, stereotypic movements, speech delay, microcephaly, facial dysmorphism, bilateral clinodactyly, and small puffy feet with metatarsus varus; however, she had no corpus callosum dysplasia. Our case highlights the role of multiple copy number variations in the occurrence of a certain phenotype. Moreover, it supports the theory that the loss of HNRNPU gene function cannot explain the occurrence of microcephaly and abnormalities of the corpus callosum in 1q43q44 microdeletion syndrome.

Sign in / Sign up

Export Citation Format

Share Document