scholarly journals Auxin-mediated protein depletion for metabolic engineering in terpene-producing yeast

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zeyu Lu ◽  
Bingyin Peng ◽  
Birgitta E. Ebert ◽  
Geoff Dumsday ◽  
Claudia E. Vickers
Keyword(s):  
Metabolic Engineering ◽  
Protein Degradation ◽  
Metabolic Flux ◽  
Glucose Repression ◽  
Production Capacity ◽  
Loss Of Function ◽  
Farnesyl Pyrophosphate Synthase ◽  
Glucose Signalling ◽  
Signalling Transduction ◽  
Inducible Protein

AbstractIn metabolic engineering, loss-of-function experiments are used to understand and optimise metabolism. A conditional gene inactivation tool is required when gene deletion is lethal or detrimental to growth. Here, we exploit auxin-inducible protein degradation as a metabolic engineering approach in yeast. We demonstrate its effectiveness using terpenoid production. First, we target an essential prenyl-pyrophosphate metabolism protein, farnesyl pyrophosphate synthase (Erg20p). Degradation successfully redirects metabolic flux toward monoterpene (C10) production. Second, depleting hexokinase-2, a key protein in glucose signalling transduction, lifts glucose repression and boosts production of sesquiterpene (C15) nerolidol to 3.5 g L−1 in flask cultivation. Third, depleting acetyl-CoA carboxylase (Acc1p), another essential protein, delivers growth arrest without diminishing production capacity in nerolidol-producing yeast, providing a strategy to decouple growth and production. These studies demonstrate auxin-mediated protein degradation as an advanced tool for metabolic engineering. It also has potential for broader metabolic perturbation studies to better understand metabolism.

scholarly journals Constraint-based metabolic control analysis for rational strain engineering

2020 ◽  
Author(s):  
Sophia Tsouka ◽  
Meric Ataman ◽  
Tuure Hameri ◽  
Ljubisa Miskovic ◽  
Vassily Hatzimanikatis
Keyword(s):  
Metabolic Engineering ◽  
Genome Editing ◽  
Metabolic Control ◽  
Metabolic Flux ◽  
Strain Engineering ◽  
Metabolic Control Analysis ◽  
Physiological Data ◽  
Response Analysis ◽  
Control Analysis ◽  
Wide Range

AbstractThe advancements in genome editing techniques over the past years have rekindled interest in rational metabolic engineering strategies. While Metabolic Control Analysis (MCA) is a well-established method for quantifying the effects of metabolic engineering interventions on flows in metabolic networks and metabolic concentrations, it fails to account for the physiological limitations of the cellular environment and metabolic engineering design constraints. We report here a constraint-based framework based on MCA, Network Response Analysis (NRA), for the rational genetic strain design that incorporates biologically relevant constraints, as well as genome editing restrictions. The NRA core constraints being similar to the ones of Flux Balance Analysis, allow it to be used for a wide range of optimization criteria and with various physiological constraints. We show how the parametrization and introduction of biological constraints enhance the NRA formulation compared to the classical MCA approach, and we demonstrate its features and its ability to generate multiple alternative optimal strategies given several user-defined boundaries and objectives. In summary, NRA is a sophisticated alternative to classical MCA for rational metabolic engineering that accommodates the incorporation of physiological data at metabolic flux, metabolite concentration, and enzyme expression levels.

Analysis of cell signaling during floral abscission in arabidopsis

2017 ◽  
Author(s):  
◽  
Isaiah Taylor
Keyword(s):  
Protein Degradation ◽  
Future Research ◽  
Loss Of Function ◽  
Kinase Gene ◽  
University Of Missouri ◽  
Gene Map ◽  
Map Kinase Phosphatase ◽  
Floral Abscission ◽  
The University ◽  
The Impact

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The shedding of plant organs is known as abscission. Floral abscission in Arabidopsis is regulated by two related receptor[negation symbol]-like protein kinases (RLKs), HAESA and HAESA[negation symbol-like 2 (HAE/HSL2). Double mutants of HAE/HSL2 are completely defective in abscission and retain sepals, petals, and stamen indefinitely. We have utilized genetic suppressor screens of hae hsl2 mutant to identify additional regulatory mechanisms of floral abscission. We have uncovered a series of gain-of-function alleles of the receptor-like protein kinase gene SERK1, as well as loss of function alleles of the gene MAP-KINASE-PHOSPHATASE-1/MKP1. We further show that mutation of two components of the endoplasmic reticulum-associated protein degradation system can suppress a weak hae hsl2 mutant, suggesting that the weak hae hsl2 mutant receptor proteins undergo ER-associated protein degradation. We further perform a number of experiments to examine the impact of phosphorylation on the activity of HAE. These results provide a number of important mechanistic details to our understanding of floral abscission, and suggest many lines of inquiry for future research.

228. Proteasomal activity during mouse preimplantation development

2008 ◽  
Vol 20 (9) ◽  
pp. 28 ◽  
Author(s):  
K. McCue ◽  
M. Pantaleon ◽  
P. L. Kaye
Keyword(s):  
Cell Proliferation ◽  
Protein Degradation ◽  
Specific Treatment ◽  
Preimplantation Development ◽  
26S Proteasome ◽  
Preimplantation Embryos ◽  
Cell Cycle Regulators ◽  
Loss Of Function ◽  
Cell Stage ◽  
Proteasomal Activity

Function of the 26S proteasome, a proteolytic organelle directed at proteins targeted for turnover by polyubiquitination, in preimplantation embryos is unclear. But it is well known to play a role in regulating meiosis. This paper reports the distribution of the proteasome and assessment of its functional importance in preimplantation development. Embryos from superovulated mice were either paraformaldehyde fixed for immunolabelling with a rabbit polyclonal antibody against the 20S proteasome core or cultured in KSOM medium with and without reversible (MG132) or irreversible (β-lactone) proteasomal inhibitors. Morphology, cell number, apoptosis and proteolysis were measured. Although diffuse throughout embryonic cytoplasm, there were distinct proteasomal concentrations in pronuclei, nuclei and cortical cytoplasm. When β-lactone was used to block blastocyst proteasomal proteolysis, ~25% of protein degradation was found to be proteasome-specific. Treatment of 2-cell embryos for more than 3 h with MG132 blocked blastocyst formation completely, even after washout, whilst both inhibitors reduced cell proliferation over the ensuing 48 h. Two hours exposure to MG132 tripled the proportion of apoptotic cells in expanded blastocysts 96 h post hCG. The nuclear concentration of proteasomes suggests a particular role in nuclear protein degradation possibly including the timed destruction of cell-cycle regulators and anti-apoptotic factors. This is supported by the loss-of-function studies which show that cell proliferation as well as morphogenesis require proteasomal activity at the late 2-cell stage and that without it apoptosis is dramatically increased. The mechanisms involved in the activation of apoptosis as a result of proteasomal inhibition in the early embryo are unknown but may include JNK signalling although this is controversial. More intriguing however is the identity of the proteasomal targets in the 2-cell embryo that must be degraded to permit continued morphogenesis.

scholarly journals Combinatorial Metabolic Engineering in Saccharomyces cerevisiae for the Enhanced Production of the FPP-Derived Sesquiterpene Germacrene

2020 ◽  
Vol 7 (4) ◽  
pp. 135
Author(s):  
Jan Niklas Bröker ◽  
Boje Müller ◽  
Dirk Prüfer ◽  
Christian Schulze Gronover
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Metabolic Flux ◽  
Mevalonate Pathway ◽  
Plant Secondary Metabolites ◽  
Product Formation ◽  
Culture Conditions ◽  
Efficient Production ◽  
Enhanced Production ◽  
Engineering Strategy

Farnesyl diphosphate (FPP)-derived isoprenoids represent a diverse group of plant secondary metabolites with great economic potential. To enable their efficient production in the heterologous host Saccharomyces cerevisiae, we refined a metabolic engineering strategy using the CRISPR/Cas9 system with the aim of increasing the availability of FPP for downstream reactions. The strategy included the overexpression of mevalonate pathway (MVA) genes, the redirection of metabolic flux towards desired product formation and the knockout of genes responsible for competitive reactions. Following the optimisation of culture conditions, the availability of the improved FPP biosynthesis for downstream reactions was demonstrated by the expression of a germacrene synthase from dandelion. Subsequently, biosynthesis of significant amounts of germacrene-A was observed in the most productive strain compared to the wild type. Thus, the presented strategy is an excellent tool to increase FPP-derived isoprenoid biosynthesis in yeast.

scholarly journals Human pancreatic β-cell glucokinase: subcellular localization and glucose repression signalling function in the yeast cell

2008 ◽  
Vol 415 (2) ◽  
pp. 233-239 ◽  
Author(s):  
Alberto Riera ◽  
Deifilia Ahuatzi ◽  
Pilar Herrero ◽  
Maria Adelaida Garcia-Gimeno ◽  
Pascual Sanz ◽  
...  
Keyword(s):  
High Glucose ◽  
Glucose Repression ◽  
Pancreatic Β Cell ◽  
Β Cells ◽  
Β Cell ◽  
Sucrose Fermentation ◽  
Suc2 Gene ◽  
Glucose Signalling ◽  
Low Glucose ◽  
Regulatory Properties

Human GKβ (pancreatic β-cell glucokinase) is the main glucose-phosphorylating enzyme in pancreatic β-cells. It shares several structural, catalytic and regulatory properties with Hxk2 (hexokinase 2) from Saccharomyces cerevisiae. In fact, it has been previously described that expression of GKβ in yeast could replace Hxk2 in the glucose signalling pathway of S. cerevisiae. In the present study we report that GKβ exerts its regulatory role by association with the yeast transcriptional repressor Mig1 (multicopy inhibitor of GAL gene expression 1); the presence of Mig1 allows GKβ to bind to the SUC2 (sucrose fermentation 2) promoter, helping in this way in the maintenance of the repression of the SUC2 gene under high-glucose conditions. Since a similar mechanism has been described for the yeast Hxk2, the findings of the present study suggest that the function of the regulatory domain present in these two proteins has been conserved throughout evolution. In addition, we report that GKβ is enriched in the yeast nucleus of high-glucose growing cells, whereas it shows a mitochondrial localization upon removal of the sugar. However, GKβ does not exit the nucleus in the absence of Mig1, suggesting that Mig1 regulates the nuclear exit of GKβ under low-glucose conditions. We also report that binding of GKβ to Mig1 allows the latter protein to be located at the mitochondrial network under low-glucose conditions.

scholarly journals Tbx1, a 22q11.2-encoded gene, is a link between alterations in fimbria myelination and cognitive speed in mice

2021 ◽  
Author(s):  
Takeshi Hiramoto ◽  
Akira Sumiyoshi ◽  
Takahira Yamauchi ◽  
Kenji Tanigaki ◽  
Qian Shi ◽  
...  
Keyword(s):  
Cognitive Deficits ◽  
Copy Number Variants ◽  
Production Capacity ◽  
Loss Of Function ◽  
Structural Alterations ◽  
Cellular Basis ◽  
Hemizygous Deletion ◽  
Chromosome 22Q11.2 ◽  
Cognitive Speed ◽  
The Brain

Copy number variants (CNVs) have provided a reliable entry point to identify structural correlates of atypical cognitive development. Hemizygous deletion of human chromosome 22q11.2 is associated with impaired cognitive function; however, the mechanisms by which numerous genes encoded in this CNV contribute to cognitive deficits via diverse structural alterations in the brain remain unclear. This study aimed to determine the cellular basis of the link between alterations in brain structure and cognitive functions in a mouse model. The heterozygosity of Tbx1, a 22q11.2 gene, altered the composition of myelinated axons in the fimbria, reduced oligodendrocyte production capacity, and slowed the acquisition of spatial memory and cognitive flexibility. Our findings provide a cellular basis for specific cognitive dysfunctions that occur in patients with loss-of-function TBX1 variants and 22q11.2 hemizygous deletion.

scholarly journals Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor

Antioxidants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 288
Author(s):  
Habiba Kausar ◽  
Ghazala Ambrin ◽  
Mohammad K. Okla ◽  
Walid Soufan ◽  
Abdullah A. Al-Ghamdi ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Real Time ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Regulatory Element ◽  
Flux Analysis ◽  
Bacterial Cells ◽  
Biosynthesis Pathway ◽  
Real Time Monitoring ◽  
E Coli

(+)-Catechin is an important antioxidant of green tea (Camelia sinensis (L.) O. Kuntze). Catechin is known for its positive role in anticancerous activity, extracellular matrix degradation, cell death regulation, diabetes, and other related disorders. As a result of enormous interest in and great demand for catechin, its biosynthesis using metabolic engineering has become the subject of concentrated research with the aim of enhancing (+)-catechin production. Metabolic flux is an essential concept in the practice of metabolic engineering as it helps in the identification of the regulatory element of a biosynthetic pathway. In the present study, an attempt was made to analyze the metabolic flux of the (+)-catechin biosynthesis pathway in order to decipher the regulatory element of this pathway. Firstly, a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor (FLIP-Cat, fluorescence indicator protein for (+)-catechin) was developed for real-time monitoring of (+)-catechin flux. In vitro characterization of the purified protein of the nanosensor showed that the nanosensor was pH stable and (+)-catechin specific. Its calculated Kd was 139 µM. The nanosensor also performed real-time monitoring of (+)-catechin in bacterial cells. In the second step of this study, an entire (+)-catechin biosynthesis pathway was constructed and expressed in E. coli in two sets of plasmid constructs: pET26b-PT7-rbs-PAL-PT7-rbs-4CL-PT7-rbs-CHS-PT7-rbs-CHI and pET26b-T7-rbs-F3H-PT7-rbs- DFR-PT7-rbs-LCR. The E. coli harboring the FLIP-Cat was transformed with these plasmid constructs. The metabolic flux analysis of (+)-catechin was carried out using the FLIP-Cat. The FLIP-Cat successfully monitored the flux of catechin after adding tyrosine, 4-coumaric acid, 4-coumaroyl CoA, naringenin chalcone, naringenin, dihydroquercetin, and leucocyanidin, individually, with the bacterial cells expressing the nanosensor as well as the genes of the (+)-catechin biosynthesis pathway. Dihydroflavonol reductase (DFR) was identified as the main regulatory element of the (+)-catechin biosynthesis pathway. Information about this regulatory element of the (+)-catechin biosynthesis pathway can be used for manipulating the (+)-catechin biosynthesis pathway using a metabolic engineering approach to enhance production of (+)-catechin.

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