scholarly journals Optimizing mevalonate pathway for squalene production in Yarrowia lipolytica

2020 ◽  
Author(s):  
Huan Liu ◽  
Fang Wang ◽  
Li Deng ◽  
Peng Xu
Keyword(s):  
Mevalonate Pathway ◽  
Cell Metabolism ◽  
Fold Increase ◽  
Cell Factory ◽  
Promising Alternative ◽  
Plant Extraction ◽  
Cost Efficient ◽  
Skin Compatibility ◽  
Coa Reductase ◽  
Engineered Strain

AbstractSqualene is the gateway molecule for triterpene-based natural products and steroids-based pharmaceuticals. As a super lubricant, it has been used widely in health care industry due to its skin compatibility and thermostability. Squalene is traditionally sourced from shark-hunting or oil plant extraction, which is cost-prohibitive and not sustainable. Reconstitution of squalene biosynthetic pathway in microbial hosts is considered as a promising alternative for cost-efficient and scalable synthesis of squalene. In this work, we reported the engineering of the oleaginous yeast, Y. lipolytica, as a potential host for squalene production. We systematically identified the bottleneck of the pathway and discovered that the native HMG-CoA reductase led to the highest squalene improvement. With the recycling of NADPH from the mannitol cycle, the engineered strain produced about 180.3 mg/l and 188.2 mg/L squalene from glucose or acetate minimal media, respectively. By optimizing the C/N ratio, controlling the media pH and mitigating the acetyl-CoA flux competition from lipogenesis, the engineered strain produced about 502.7 mg/L squalene in shake flaks, a 28-fold increase compared to the parental strain (17.2 mg/L). We also profiled the metabolic byproducts citric acid and mannitol level and observed that they are reincorporated into cell metabolism at the late stage of fermentation. This work may serve as a baseline to harness Y. lipolytica as an oleaginous cell factory for production of squalene or terpene-based chemicals.

Metabolism of farnesyl diphosphate in tobacco BY-2 cells treated with squalestatin

2000 ◽  
Vol 28 (6) ◽  
pp. 794-796 ◽  
Author(s):  
M.-A. Hartmann ◽  
L. Wentzinger ◽  
A. Hemmerlin ◽  
T. J. Bach
Keyword(s):  
Mevalonate Pathway ◽  
Fold Increase ◽  
Enzymic Activity ◽  
Farnesyl Diphosphate ◽  
Allylic Alcohol ◽  
Central Position ◽  
Mrna Levels ◽  
Ubiquinone Q10 ◽  
Wide Range ◽  
Coa Reductase

Plant isoprenoids represent a large group of compounds with a wide range of physiological functions. In the cytosol, isoprenoids are synthesized via the classical acetate/mevalonate pathway. In this pathway, farnesyl diphosphate (FPP) occupies a central position, from which isoprene units are dispatched to the different classes of isoprenoids, with sterols as the major end products. The present work deals with effects of squalestatin (SQ) on the metabolism of FPP in proliferating and synchronized cultured tobacco cv. Bright Yellow-2 cells. SQ is a potent inhibitor of squalene synthase (SQS), the first committed enzyme in the sterol pathway. At nanomolar concentrations, SQ severely impaired cell growth and sterol biosynthesis, as attested by the rapid decrease in SQS activity. At the same time, it triggered a several-fold increase in both the enzymic activity and mRNA levels of 3-hydroxy-3-methylglutaryl CoA reductase. When SQ was added to cells synchronized by aphidicolin treatment, it was found to block the cell cycle at the end of G1 phase, but no cell death was induced. Tobacco cells were also fed exogenous tritiated trans-trans farnesol, the allylic alcohol derived from FPP, in the presence and absence of SQ. Evidence is presented that this compound was incorporated into sterols and ubiquinone Q10. In the presence of SQ, the sterol pathway was inhibited, but no increase in the radioactivity of ubiquinone was observed, suggesting that this metabolic channel was already saturated under normal conditions.

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

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Tao Cheng ◽  
Guang Zhao ◽  
Mo Xian ◽  
Congxia Xie
Keyword(s):  
De Novo ◽  
Mevalonate Pathway ◽  
High Cell Density ◽  
Abies Balsamea ◽  
Cell Factory ◽  
Geranylgeranyl Diphosphate Synthase ◽  
Geranyl Diphosphate ◽  
E Coli ◽  
Geranyl Diphosphate Synthase ◽  
Coa Reductase

Abstract 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.

Abstract 15384: Laminar Flow Regulates Endothelial Cell Metabolism via Long Non-coding RNA lncflow2 (Best of Basic Science Abstract)

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Larissa Pfisterer ◽  
Teresa Hartung ◽  
Katharina M Michalik ◽  
Wei Chen ◽  
Andreas M Zeiher ◽  
...  
Keyword(s):  
Laminar Flow ◽  
Mevalonate Pathway ◽  
Cell Metabolism ◽  
Acid Oxidation ◽  
Glucose And Lipid Metabolism ◽  
Rna Immunoprecipitation ◽  
Endothelial Adhesion Molecules ◽  
Coa Reductase ◽  
Sprout Formation ◽  
The Impact

Considering that the transcriptome consists largely of non-coding transcripts, we analysed the impact of atheroprotective laminar flow (20 dynes/cm2) on the expression of long non-coding RNAs (lncRNA) in human endothelial cells (EC) by RNA deep sequencing. Among the significantly up-regulated lncRNAs, we specifically confirmed the up-regulation of lncflow2 upon laminar flow (3.4±1.5, p<0.05) and by overexpressing the flow-induced transcription factor KLF2 (1.95+0.2, p<0.05) by qRT-PCR. Lncflow2 is preferentially localized to the nucleus and RNA immunoprecipitation data indicate an association with the chromatin mark H3K27me3 suggesting that lncflow2 might epigenetically control gene expression. Functionally, the reduction of lncflow2 using LNA-Gapmers suppressed spheroid sprout formation of ECs (34.7 % reduction, p<0.05) and increased expression of endothelial adhesion molecules suggesting that lncflow2 improves EC function and reduces endothelial inflammation. Furthermore, gene ontology analysis of exon microarray analysis with RNA of lncflow2-silenced ECs revealed a significant regulation of metabolic pathways, such as the mevalonate pathway, indicated by elevated acetyl-CoA-acetyltransferase-2, HMG-CoA-reductase and HMG-CoA-synthase-1 expression (confirmed by RT-PCR; all p<0.05). In this context, inhibition of the HMG-Co-reductase by atorvastatin rescued the diminished spheroid sprouting upon loss of lncflow2. To elucidate additional effects on endothelial metabolic function, a mass spectrometry metabolome screening revealed significant alterations in amino acid, glucose and lipid metabolism. Seahorse analysis confirmed these data showing an elevated respiration and fatty acid oxidation in lncflow2 silenced ECs. Our data identify lncflow2 as a flow-regulated lncRNA that modifies EC metabolic responses by controlling metabolism-associated genes- particularly of the mevalonate pathway. Thereby, lncflow2 critically modifies EC functions and may play a regulating role in cellular metabolic adaption to stressors.

scholarly journals Arsenic Removal by Advanced Electrocoagulation Processes: The Role of Oxidants Generated and Kinetic Modeling

Catalysts ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 928
Author(s):  
Micah Flor V. Montefalcon ◽  
Meliton R. Chiong ◽  
Augustus C. Resurreccion ◽  
Sergi Garcia-Segura ◽  
Joey D. Ocon
Keyword(s):  
Kinetic Modeling ◽  
Arsenic Removal ◽  
Fold Increase ◽  
Electrochemical Treatment ◽  
Promising Alternative ◽  
Iron Electrodes ◽  
Removal Capacity ◽  
Naturally Occurring ◽  
Treatment Technologies

Arsenic (As) is a naturally occurring element in the environment that poses significant risks to human health. Several treatment technologies have been successfully used in the treatment of As-contaminated waters. However, limited literature has explored advanced electrocoagulation (EC) processes for As removal. The present study evaluates the As removal performance of electrocoagulation, electrochemical peroxidation (ECP), and photo-assisted electrochemical peroxidation (PECP) technologies at circumneutral pH using electroactive iron electrodes. The influence of As speciation and the role of oxidants in As removal were investigated. We have identified the ECP process to be a promising alternative for the conventional EC with around 4-fold increase in arsenic removal capacity at a competitive cost of 0.0060 $/m3. Results also indicated that the rate of As(III) oxidation at the outset of electrochemical treatment dictates the extent of As removal. Both ECP and PECP processes reached greater than 96% As(III) conversion at 1 C/L and achieved 86% and 96% As removal at 5 C/L, respectively. Finally, the mechanism of As(III) oxidation was evaluated, and results showed that Fe(IV) is the intermediate oxidant generated in advanced EC processes, and the contribution of •OH brought by UV irradiation is insignificant.

scholarly journals A QM/MM Evaluation of the Missing Step in the Reduction Mechanism of HMG-CoA by Human HMG-CoA Reductase

Processes ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 1085
Author(s):  
Paula Mihaljević-Jurič ◽  
Sérgio F. Sousa
Keyword(s):  
Mevalonate Pathway ◽  
Atomic Level ◽  
Amino Acid Residues ◽  
Protonation State ◽  
Primary Target ◽  
Cholesterol Levels ◽  
The Subject ◽  
Electron Reduction ◽  
Dynamics Simulations ◽  
Coa Reductase

Statins are important drugs in the regulation of cholesterol levels in the human body that have as a primary target the enzyme β-hydroxy-β-methylglutaryl-CoA reductase (HMGR). This enzyme plays a crucial role in the mevalonate pathway, catalyzing the four-electron reduction of HMG-CoA to mevalonate. A second reduction step of this reaction mechanism has been the subject of much speculation in the literature, with different conflicting theories persisting to the present day. In this study, the different mechanistic hypotheses were evaluated with atomic-level detail through a combination of molecular dynamics simulations (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations. The obtained Gibbs free activation and Gibbs free reaction energy (15 kcal mol−1 and −40 kcal mol−1) show that this hydride step takes place with the involvement of a cationic His405 and Lys639, and a neutral Glu98, while Asp715 remains in an anionic state. The results provide an atomic-level portrait of this step, clearly demonstrating the nature and protonation state of the amino acid residues involved, the energetics associated, and the structure and charge of the key participating atoms in the several intermediate states, finally elucidating this missing step.

Inhibition of protein geranylgeranylation induces apoptosis in myeloma plasma cells by reducing Mcl-1 protein levels

Blood ◽  
2003 ◽  
Vol 102 (9) ◽  
pp. 3354-3362 ◽  
Author(s):  
Niels W. C. J. van de Donk ◽  
Marloes M. J. Kamphuis ◽  
Berris van Kessel ◽  
Henk M. Lokhorst ◽  
Andries C. Bloem
Keyword(s):  
Multiple Myeloma ◽  
Plasma Cells ◽  
Mevalonate Pathway ◽  
Cytochrome C Release ◽  
Hmg Coa Reductase ◽  
Myeloma Cells ◽  
Protein Levels ◽  
Geranylgeranyl Transferase ◽  
Induced Apoptosis ◽  
Coa Reductase

AbstractHMG-CoA reductase is the rate-limiting enzyme of the mevalonate pathway leading to the formation of cholesterol and isoprenoids such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). The inhibition of HMG-CoA reductase by lovastatin induced apoptosis in plasma cell lines and tumor cells from patients with multiple myeloma. Here we show that cotreatment with mevalonate or geranylgeranyl moieties, but not farnesyl groups, rescued myeloma cells from lovastatin-induced apoptosis. In addition, the inhibition of geranylgeranylation by specific inhibition of geranylgeranyl transferase I (GGTase I) induced the apoptosis of myeloma cells. Apoptosis triggered by the inhibition of geranylgeranylation was associated with reduction of Mcl-1 protein expression, collapse of the mitochondrial transmembrane potential, expression of the mitochondrial membrane protein 7A6, cytochrome c release from mitochondria into the cytosol, and stimulation of caspase-3 activity. These results imply that protein geranylgeranylation is critical for regulating myeloma tumor cell survival, possibly through regulating Mcl-1 expression. Our results show that pharmacologic agents such as lovastatin or GGTase inhibitors may be useful in the treatment of multiple myeloma.

scholarly journals Metabolic Engineering of Non-carotenoid-Producing Yeast Yarrowia lipolytica for the Biosynthesis of Zeaxanthin

2021 ◽  
Vol 12 ◽  
Author(s):  
Yuxiao Xie ◽  
Shulin Chen ◽  
Xiaochao Xiong
Keyword(s):  
Yarrowia Lipolytica ◽  
Fold Increase ◽  
Genetically Engineered ◽  
Cell Factory ◽  
Yeast Nitrogen Base ◽  
Nitrogen Base ◽  
Yeast Extract Peptone Dextrose ◽  
Dry Cell Weight ◽  
A Cell ◽  
Β Carotene

Zeaxanthin is vital to human health; thus, its production has received much attention, and it is also an essential precursor for the biosynthesis of other critical carotenoids such as astaxanthin and crocetin. Yarrowia lipolytica is one of the most intensively studied non-conventional yeasts and has been genetically engineered as a cell factory to produce carotenoids such as lycopene and β-carotene. However, zeaxanthin production by Y. lipolytica has not been well investigated. To fill this gap, β-carotene biosynthesis pathway has been first constructed in this study by the expression of genes, including crtE, crtB, crtI, and carRP. Three crtZ genes encoding β-carotene hydroxylase from different organisms were individually introduced into β-carotene-producing Y. lipolytica to evaluate their performance for producing zeaxanthin. The expression of crtZ from the bacterium Pantoea ananatis (formerly Erwinia uredovora, Eu-crtZ) resulted in the highest zeaxanthin titer and content on the basis of dry cell weight (DCW). After verifying the function of Eu-crtZ for producing zeaxanthin, the high-copy-number integration into the ribosomal DNA of Y. lipolytica led to a 4.02-fold increase in the titer of zeaxanthin and a 721% increase in the content of zeaxanthin. The highest zeaxanthin titer achieved 21.98 ± 1.80 mg/L by the strain grown on a yeast extract peptone dextrose (YPD)–rich medium. In contrast, the highest content of DCW reached 3.20 ± 0.11 mg/g using a synthetic yeast nitrogen base (YNB) medium to culture the cells. Over 18.0 g/L of citric acid was detected in the supernatant of the YPD medium at the end of cultivation. Furthermore, the zeaxanthin-producing strains still accumulated a large amount of lycopene and β-carotene. The results demonstrated the potential of a cell factory for zeaxanthin biosynthesis and opened up an avenue to engineer this host for the overproduction of carotenoids.

Production of Brucella melitensis Omp16 protein fused to the human interleukin 2 in Lactococcus lactis MG1363 toward developing a Lactococcus-based vaccine against brucellosis

2020 ◽  
Vol 66 (1) ◽  
pp. 39-45
Author(s):  
Marzieh Rezaei ◽  
Mohammad Rabbani Khorasgani ◽  
Sayyed Hamid Zarkesh Esfahani ◽  
Rahman Emamzadeh ◽  
Hamid Abtahi
Keyword(s):  
Fusion Protein ◽  
Lactococcus Lactis ◽  
Fusion Gene ◽  
Brucella Melitensis ◽  
Expression System ◽  
Interleukin 2 ◽  
Cell Factory ◽  
Promising Alternative ◽  
Alternative Expression ◽  
Restriction Sites

The use of the food-grade bacterium Lactococcus lactis as a new cell factory is a promising alternative expression system for producing a desired protein. The Omp16-IL2 fusion protein antigen was cloned, expressed, and purified in this study. The Omp16-IL2 fusion gene was designed and cloned in pGH plasmid with appropriate restriction sites and subcloned in pAMJ2008 expression vector digested with the same enzymes. The purified recombinant constructed pAMJ-rOmp-IL2 was introduced into L. lactis subsp. cremoris MG1363 by electrotransformation. Finally, the expression and purification of Omp16-IL2 fusion protein was investigated. This study reports the construction of a recombinant L. lactis expressing the Omp16-IL2 fusion protein as an oral Lactococcus-based vaccine, as compared with commonly used live attenuated vaccines, for future studies against brucellosis.

Altering potato isoprenoid metabolism increases biomass and induces early flowering

2020 ◽  
Vol 71 (14) ◽  
pp. 4109-4124
Author(s):  
Moehninsi ◽  
Iris Lange ◽  
B Markus Lange ◽  
Duroy A Navarre
Keyword(s):  
Metabolic Regulation ◽  
Transcriptional Control ◽  
Mevalonate Pathway ◽  
Early Flowering ◽  
Bioenergy Crops ◽  
Isoprenoid Metabolism ◽  
Transgenic Lines ◽  
Coa Reductase ◽  
Rate Limiting

Abstract Isoprenoids constitute the largest class of plant natural products and have diverse biological functions including in plant growth and development. In potato (Solanum tuberosum), the regulatory mechanism underlying the biosynthesis of isoprenoids through the mevalonate pathway is unclear. We assessed the role of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) homologs in potato development and in the metabolic regulation of isoprenoid biosynthesis by generating transgenic lines with down-regulated expression (RNAi-hmgr) or overexpression (OE) of one (StHMGR1 or StHMGR3) or two genes, HMGR and farnesyl diphosphate synthase (FPS; StHMGR1/StFPS1 or StHMGR3/StFPS1). Levels of sterols, steroidal glycoalkaloids (SGAs), and plastidial isoprenoids were elevated in the OE-HMGR1, OE-HMGR1/FPS1, and OE-HMGR3/FPS1 lines, and these plants exhibited early flowering, increased stem height, increased biomass, and increased total tuber weight. However, OE-HMGR3 lines showed dwarfism and had the highest sterol amounts, but without an increase in SGA levels, supporting a rate-limiting role for HMGR3 in the accumulation of sterols. Potato RNAi-hmgr lines showed inhibited growth and reduced cytosolic isoprenoid levels. We also determined the relative importance of transcriptional control at regulatory points of isoprenoid precursor biosynthesis by assessing gene–metabolite correlations. These findings provide novel insights into specific end-products of the sterol pathway and could be important for crop yield and bioenergy crops.

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