scholarly journals Comparative Analysis of NADPH-Cytochrome P450 Reductases From Legumes for Heterologous Production of Triterpenoids in Transgenic Saccharomyces cerevisiae

2021 ◽  
Vol 12 ◽  
Author(s):  
Pramesti Istiandari ◽  
Shuhei Yasumoto ◽  
Pisanee Srisawat ◽  
Keita Tamura ◽  
Ayaka Chikugo ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Higher Plants ◽  
Lotus Japonicus ◽  
Class Ii ◽  
Expression Level ◽  
Class I ◽  
Cytochrome P450 Monooxygenases ◽  
Heterologous Production ◽  
Nadph Cytochrome ◽  
Transgenic Yeast

Triterpenoids are plant specialized metabolites with various pharmacological activities. They are widely distributed in higher plants, such as legumes. Because of their low accumulation in plants, there is a need for improving triterpenoid production. Cytochrome P450 monooxygenases (CYPs) play critical roles in the structural diversification of triterpenoids. To perform site-specific oxidations, CYPs require the electrons that are transferred by NADPH-cytochrome P450 reductase (CPR). Plants possess two main CPR classes, class I and class II. CPR classes I and II have been reported to be responsible for primary and specialized (secondary) metabolism, respectively. In this study, we first analyzed the CPR expression level of three legumes species, Medicago truncatula, Lotus japonicus, and Glycyrrhiza uralensis, showing that the expression level of CPR class I was lower and more stable, while that of CPR class II was higher in almost all the samples. We then co-expressed different combinations of CYP716As and CYP72As with different CPR classes from these three legumes in transgenic yeast. We found that CYP716As worked better with CPR-I from the same species, while CYP72As worked better with any CPR-IIs. Using engineered yeast strains, CYP88D6 paired with class II GuCPR produced the highest level of 11-oxo-β-amyrin, the important precursor of high-value metabolites glycyrrhizin. This study provides insight into co-expressing genes from legumes for heterologous production of triterpenoids in yeast.

scholarly journals Furanoditerpenoid biosynthesis in the bioenergy crop switchgrass is catalyzed by an alternate metabolic pathway

2021 ◽  
Author(s):  
Andrew Muchlinski ◽  
Meirong Jia ◽  
Kira Tiedge ◽  
Jason S. Fell ◽  
Kyle A. Pelot ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Panicum Virgatum ◽  
Furan Ring ◽  
Class Ii ◽  
Class I ◽  
Cytochrome P450 Monooxygenases ◽  
Bioenergy Crop ◽  
Stress Resilience ◽  
P450 Monooxygenases ◽  
Diterpene Synthases

AbstractSpecialized diterpenoid metabolites are important mediators of stress resilience in monocot crops. A deeper understanding of how species-specific diterpenoid-metabolic pathways and functions contribute to plant chemical defenses can enable crop improvement strategies. Here, we report the genomics-enabled discovery of five cytochrome P450 monooxygenases (CYP71Z25-29) that form previously unknown furanoditerpenoids in the monocot bioenergy crop switchgrass (Panicum virgatum). Combinatorial pathway reconstruction showed that CYP71Z25-29 catalyze furan ring addition to diterpene alcohol intermediates derived from distinct class II diterpene synthases, thus bypassing the canonical role of class I diterpene synthases in plant diterpenoid metabolism. Transcriptional co-expression patterns and presence of select diterpenoids in droughted switchgrass roots support possible roles of CYP71Z25-29 in abiotic stress responses. Integrating molecular dynamics, structural analysis, and targeted mutagenesis, identified active site determinants controlling distinct CYP71Z25-29 catalytic specificities and, combined with broad substrate promiscuity for native and non-native diterpenoids, highlights the potential of these P450s for natural product engineering.Significance StatementDiterpenoids play important roles in stress resilience and chemically mediated interactions in many plant species, including major food and bioenergy crops. Enzymes of the cytochrome P450 monooxygenase family catalyze the various functional decorations of core diterpene scaffolds that determine the large diversity of biologically active diterpenoids. This study describes the identification and mechanistic analysis of an unusual group of cytochrome P450 monooxygenases, CYP71Z25-29, from the bioenergy crop switchgrass (Panicum virgatum). These enzymes catalyze the furan ring addition directly to class II diterpene synthase products, thus bypassing the conserved pairwise reaction of class II and class I diterpene synthases in labdane diterpenoid metabolism. Insight into the distinct substrate-specificity of CYP71Z25-29 offers opportunity for engineering of furanoditerpenoid bioproducts.

scholarly journals Integration of a multi-step heterologous pathway in Saccharomyces cerevisiae for the production of abscisic acid

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Maximilian Otto ◽  
Paulo Gonçalves Teixeira ◽  
Maria Isabel Vizcaino ◽  
Florian David ◽  
Verena Siewers
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome P450 ◽  
Abscisic Acid ◽  
Stress Responses ◽  
Higher Plants ◽  
Cytochrome P450 Monooxygenases ◽  
Cell Factory ◽  
Cytochrome P450 Reductase ◽  
Biotechnological Production ◽  
P450 Reductase

Abstract Background The sesquiterpenoid abscisic acid (ABA) is mostly known for regulating developmental processes and abiotic stress responses in higher plants. Recent studies show that ABA also exhibits a variety of pharmacological activities. Affordable and sustainable production will be required to utilize the compound in agriculture and as a potential pharmaceutical. Saccharomyces cerevisiae is an established workhorse for the biotechnological production of chemicals. In this study, we constructed and characterised an ABA-producing S. cerevisiae strain using the ABA biosynthetic pathway from Botrytis cinerea. Results Expression of the B. cinerea genes bcaba1, bcaba2, bcaba3 and bcaba4 was sufficient to establish ABA production in the heterologous host. We characterised the ABA-producing strain further by monitoring ABA production over time and, since the pathway contains two cytochrome P450 enzymes, by investigating the effects of overexpressing the native S. cerevisiae or the B. cinerea cytochrome P450 reductase. Both, overexpression of the native or heterologous cytochrome P450 reductase, led to increased ABA titres. We were able to show that ABA production was not affected by precursor or NADPH supply, which suggested that the heterologous enzymes were limiting the flux towards the product. The B. cinerea cytochrome P450 monooxygenases BcABA1 and BcABA2 were identified as pathway bottlenecks and balancing the expression levels of the pathway enzymes resulted in 4.1-fold increased ABA titres while reducing by-product formation. Conclusion This work represents the first step towards a heterologous ABA cell factory for the commercially relevant sesquiterpenoid.

scholarly journals Tobacco Cutworm (Spodoptera Litura) Larvae Silenced in the NADPH-Cytochrome P450 Reductase Gene Show Increased Susceptibility to Phoxim

2019 ◽  
Vol 20 (15) ◽  
pp. 3839 ◽  
Author(s):  
Hong-Yi Ji ◽  
Christian Staehelin ◽  
Yan-Ping Jiang ◽  
Shi-Wei Liu ◽  
Zhi-Hui Ma ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Amino Acid ◽  
Spodoptera Litura ◽  
Fat Body ◽  
Larval Mortality ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Amino Acid Sequences ◽  
Cytochrome P450 Monooxygenases ◽  
Expression Levels ◽  
Nadph Cytochrome

Nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductases (CPRs) function as redox partners of cytochrome P450 monooxygenases (P450s). CPRs and P450s in insects have been found to participate in insecticide resistance. However, the CPR of the moth Spodoptera litura has not been well characterized yet. Based on previously obtained transcriptome information, a full-length CPR cDNA of S. litura (SlCPR) was PCR-cloned. The deduced amino acid sequence contains domains and residues predicted to be essential for CPR function. Phylogenetic analysis with insect CPR amino acid sequences showed that SlCPR is closely related to CPRs of Lepidoptera. Quantitative reverse transcriptase PCR (RT-qPCR) was used to determine expression levels of SlCPR in different developmental stages and tissues of S. litura. SlCPR expression was strongest at the sixth-instar larvae stage and fifth-instar larvae showed highest expression in the midgut. Expression of SlCPR in the midgut and fat body was strongly upregulated when fifth-instar larvae were exposed to phoxim at LC15 (4 μg/mL) and LC50 (20 μg/mL) doses. RNA interference (RNAi) mediated silencing of SlCPR increased larval mortality by 34.6% (LC15 dose) and 53.5% (LC50 dose). Our results provide key information on the SlCPR gene and indicate that SlCPR expression levels in S. litura larvae influence their susceptibility to phoxim and possibly other insecticides.

scholarly journals The 3D nuclear conformation of the major histocompatibility complex changes upon cell activation both in porcine and human macrophages

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Florence Mompart ◽  
Alain Kamgoué ◽  
Yvette Lahbib-Mansais ◽  
David Robelin ◽  
Agnès Bonnet ◽  
...  
Keyword(s):  
Major Histocompatibility Complex ◽  
Nuclear Organization ◽  
Class Ii ◽  
Expression Level ◽  
Class I ◽  
Activation Process ◽  
Major Histocompatibility ◽  
Class Iii ◽  
Histocompatibility Complex ◽  
The Impact

Abstract Background The crucial role of the major histocompatibility complex (MHC) for the immune response to infectious diseases is well-known, but no information is available on the 3D nuclear organization of this gene-dense region in immune cells, whereas nuclear architecture is known to play an essential role on genome function regulation. We analyzed the spatial arrangement of the three MHC regions (class I, III and II) in macrophages using 3D-FISH. Since this complex presents major differences in humans and pigs with, notably, the presence of the centromere between class III and class II regions in pigs, the analysis was implemented in both species to determine the impact of this organization on the 3D conformation of the MHC. The expression level of the three genes selected to represent each MHC region was assessed by quantitative real-time PCR. Resting and lipopolysaccharide (LPS)-activated states were investigated to ascertain whether a response to a pathogen modifies their expression level and their 3D organization. Results While the three MHC regions occupy an intermediate radial position in porcine macrophages, the class I region was clearly more peripheral in humans. The BAC center-to-center distances allowed us to propose a 3D nuclear organization of the MHC in each species. LPS/IFNγ activation induces a significant decompaction of the chromatin between class I and class III regions in pigs and between class I and class II regions in humans. We detected a strong overexpression of TNFα (class III region) in both species. Moreover, a single nucleus analysis revealed that the two alleles can have either the same or a different compaction pattern. In addition, macrophage activation leads to an increase in alleles that present a decompacted pattern in humans and pigs. Conclusions The data presented demonstrate that: (i) the MHC harbors a different 3D organization in humans and pigs; (ii) LPS/IFNγ activation induces chromatin decompaction, but it is not the same area affected in the two species. These findings were supported by the application of an original computation method based on the geometrical distribution of the three target genes. Finally, the position of the centromere inside the swine MHC could influence chromatin reorganization during the activation process.

Multiple forms of NADPH-cytochrome P450 reductase in higher plants

1991 ◽  
Vol 177 (1) ◽  
pp. 105-112 ◽  
Author(s):  
Irène Benveniste ◽  
Agnès Lesot ◽  
Marie-Paule Hasenfratz ◽  
Georg Kochs ◽  
Francis Durst
Keyword(s):  
Cytochrome P450 ◽  
Higher Plants ◽  
Cytochrome P450 Reductase ◽  
Multiple Forms ◽  
Nadph Cytochrome ◽  
P450 Reductase ◽  
Nadph Cytochrome P450 Reductase

Electron microscopy analysis of degenerating pancreatic ß cells in transgenic mice expressing the MHC Class I antigen Dd

1990 ◽  
Vol 48 (3) ◽  
pp. 548-549
Author(s):  
T. A. Stewart ◽  
D. Liggitt ◽  
S. Pitts ◽  
L. Martin ◽  
M. Siegel ◽  
...  
Keyword(s):  
Type I Diabetes ◽  
Disease Process ◽  
Class Ii ◽  
Class I ◽  
Type I ◽  
Aberrant Expression ◽  
Mhc Molecules ◽  
Endogenous Insulin ◽  
Class Ii Mhc ◽  
Ifn Γ

Insulin-dependant (Type I) diabetes mellitus (IDDM) is a metabolic disorder resulting from the lack of endogenous insulin secretion. The disease is thought to result from the autoimmune mediated destruction of the insulin producing ß cells within the islets of Langerhans. The disease process is probably triggered by environmental agents, e.g. virus or chemical toxins on a background of genetic susceptibility associated with particular alleles within the major histocompatiblity complex (MHC). The relation between IDDM and the MHC locus has been reinforced by the demonstration of both class I and class II MHC proteins on the surface of ß cells from newly diagnosed patients as well as mounting evidence that IDDM has an autoimmune pathogenesis. In 1984, a series of observations were used to advance a hypothesis, in which it was suggested that aberrant expression of class II MHC molecules, perhaps induced by gamma-interferon (IFN γ) could present self antigens and initiate an autoimmune disease. We have tested some aspects of this model and demonstrated that expression of IFN γ by pancreatic ß cells can initiate an inflammatory destruction of both the islets and pancreas and does lead to IDDM.

Pictish Horse Carvings

1991 ◽  
Vol 17 (1) ◽  
pp. 53-62
Author(s):  
Irene Hughson
Keyword(s):  
Class Ii ◽  
Class I ◽  
Early Historic Period ◽  
High Quality ◽  
Historic Period ◽  
Agricultural Economy ◽  
Different Types ◽  
Early Historic

Summary This paper examines the horse carvings to be found on Class I and Class II Pictish sculptured stones and considers their reliability as evidence of the sort of horses and ponies that would have existed in the Early Historic Period. An attempt is made to show that the availability in Britain of good sized, high quality riding horses during that period is not inconsistent with what is known of the development and distribution of different types of horses in pre-hislory. The importance of horses and ponies in Early Historic societies is stressed and inferences drawn about the agricultural economy that could support horses and the skilled specialists required to look after them.

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