cholesterol oxidase
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2022 ◽  
Author(s):  
Amir Akhgari ◽  
Bikash Baral ◽  
Arina Koroleva ◽  
Vilja Siitonen ◽  
David P Fewer ◽  
...  

Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying high-performing individual cells from a mutant population directly from liquid cultures. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l-1 in a second round of mutant selection. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5-fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g-1 in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products.


Author(s):  
Olabisi Ojo ◽  
Diana L. Williams ◽  
Linda B. Adams ◽  
Ramanuj Lahiri

Mycobacterium leprae, the causative agent of leprosy, is an obligate intracellular pathogen primarily residing within host macrophages and Schwann cells. Whole genome sequencing predicts a highly degraded genome with approximately one third of the coding capacity resulting in the loss of many catabolic pathways. Therefore, it can be assumed that M. leprae obtains many of the necessary metabolites for intracellular survival and growth from the host cells. In this study, global transcriptomic analyses were done on freshly harvested M. leprae growing in athymic mouse footpads for five months (MFP5) and compared to those held in axenic medium for 48 (ML48) and 96 (ML96) hours. Results show that all of the genes and pseudogenes were transcribed under both in vivo and in vitro conditions. 24% and 33% of gene transcript levels were significantly altered in ML48 and ML96 respectively, compared to MFP5. Approximately 45% (39/86) of lipid metabolism genes were significantly downregulated in ML96 compared to MFP5, majority of which are in the β-oxidation pathway. Cholesterol oxidase, acyl-CoA dehydrogenase, and coenzyme F420-dependent oxidoreductase, were significantly upregulated in both ML48 and ML96 compared to MFP5. 30% of cell wall and cell processes functional category genes had altered gene transcription at 96hr compared to MFP5. 40% of 57 genes associated with mycobacterial virulence showed significantly altered transcript levels with 52% significantly downregulated in ML96, including most of the Pro-Glu/Pro-Pro-Glu genes. All 111 hypothetical protein genes with unknown function were expressed. Adenosine triphosphate (ATP) synthesis in M. leprae appears to be significantly downregulated under ex vivo conditions. This is the first study comparing M. leprae global gene expression during in vivo growth and ex vivo stationery phase in axenic medium confirming that during the growth phase in the footpads of experimentally infected mice, M. leprae is metabolically active and its primary source of energy production is probably lipids.


Author(s):  
PRISKA ERNESTINA TENDA ◽  
ELEONORA MARYETA TOYO ◽  
ARBA PRAMUNDITA RAMADANI

Objectives: To evaluate the effect of extract and its fraction (n-hexane, ethyl acetate, and water) of the mulberry leaf (Morus australis Poir.) in reducing triglyceride and total cholesterol levels on HFD and PTU induced rats. Methods: In this study, mulberry leaves were collected from East Nusa Tenggara-Indonesia, extracted using maceration method, and fractionated with various solvents. The thin layer chromatography with silica gel 60 F254 was utilized to identify the active compound on both extract and fraction. Furthermore, using a quasi-experimental with control group design, this study employed 40 healthy male Wistar rats and divided into 8 groups, namely: normal and negative control, positive controls (simvastatin and gemfibrozil), ethanolic extract, and fractions (n-hexane, ethyl acetate, and water). All groups were fed with a high cholesterol diet and 0.01% PTU, except for the normal group. During the study, the blood was drawn at day 0, 28, 35, and 42. The triglyceride level was observed using Glycerophosphate-Oxidase Phenol Aminoantipyrine (GPO-PAP) method, while the total cholesterol was measured by Cholesterol Oxidase Phenol Aminoantipyrine (CHOD-PAP). Results: On the 42nd day, ethyl fraction of mulberry leaf (Morus australis Poir.) showed the most significant result of reducing the level of triglyceride and total cholesterol compared to the high-fat diet and PTU induced rat on day 0: triglyceride of 104.89±2.70 mg/dl vs 64.76±1.97 mg/dl and total cholesterol of 96.70±2.45 vs 88.02±1.38 mg/dl. Ethyl acetate fraction was similar to simvastatin in lowering the level of total cholesterol. TLC identification also showed that mulberry leaf contains flavonoids and polyphenols that act as anti-hyperlipidemic. Conclusion: Ethyl acetate fraction of mulberry leaf (Morus australis Poir.) showed best activity on lowering both triglyceride and total cholesterol.


2021 ◽  
Vol 9 (12) ◽  
pp. 2554
Author(s):  
Tatyana Lobastova ◽  
Victoria Fokina ◽  
Sergey Tarlachkov ◽  
Andrey Shutov ◽  
Eugeny Bragin ◽  
...  

The application of thermophilic microorganisms opens new prospects in steroid biotechnology, but little is known to date on steroid catabolism by thermophilic strains. The thermophilic strain Saccharopolyspora hirsuta VKM Ac-666T has been shown to convert various steroids and to fully degrade cholesterol. Cholest-4-en-3-one, cholesta-1,4-dien-3-one, 26-hydroxycholest-4-en-3-one, 3-oxo-cholest-4-en-26-oic acid, 3-oxo-cholesta-1,4-dien-26-oic acid, 26-hydroxycholesterol, 3β-hydroxy-cholest-5-en-26-oic acid were identified as intermediates in cholesterol oxidation. The structures were confirmed by 1H and 13C-NMR analyses. Aliphatic side chain hydroxylation at C26 and the A-ring modification at C3, which are putatively catalyzed by cytochrome P450 monooxygenase CYP125 and cholesterol oxidase, respectively, occur simultaneously in the strain and are followed by cascade reactions of aliphatic sidechain degradation and steroid core destruction via the known 9(10)-seco-pathway. The genes putatively related to the sterol and bile acid degradation pathways form three major clusters in the S. hirsuta genome. The sets of the genes include the orthologs of those involved in steroid catabolism in Mycobacterium tuberculosis H37Rv and Rhodococcus jostii RHA1 and related actinobacteria. Bioinformatics analysis of 52 publicly available genomes of thermophilic bacteria revealed only seven candidate strains that possess the key genes related to the 9(10)-seco pathway of steroid degradation, thus demonstrating that the ability to degrade steroids is not widespread among thermophilic bacteria.


2021 ◽  
pp. 106028
Author(s):  
Aliakbar Fazaeli ◽  
Saeed Ebrahimi Fana ◽  
Abolfazl Golestani ◽  
Mahdi Aminian

2021 ◽  
Author(s):  
Bin Du ◽  
Mei Zheng ◽  
Jingshu Huang ◽  
Qingqing Jiao ◽  
Yimeng Bai ◽  
...  

Abstract Multidrug resistance is still a major obstacle to cancer treatment. The most studies are to inhibit the activity of the drug transporter P-glycoprotein (P-gp), but the effect is not ideal. Herein, a nanosystem was built based on cascade catalytic consumption of cholesterol. Cholesterol oxidase (natural enzyme, COD) was immobilized on the carrier (NH2-MIL-88B, MOF) through amide reaction, COD catalyzed the consumption of cholesterol, the reaction product H2O2 was further produced by the MOF with its peroxidase-like activity to produce hydroxyl radicals (•OH) with killing effect. Due to the high expression of CD44 receptor on the surface of tumor cells, we encapsulated chondroitin sulfate gel shell (CS-shell) with CD44 targeting and apoptosis promoting effect on the surface of [email protected] nanoparticles, which can accurately and efficiently deliver the drugs to the tumor site and improve the effect of reversing drug resistance. Taking drug-resistant cell membrane as "breakthrough", this paper will provide a new idea for reversing multidrug resistance of tumor.


2021 ◽  
Author(s):  
Tatyana Gennadyevna Lobastova ◽  
Victoria V. Fokina ◽  
Sergey V. Tarlachkov ◽  
Andrey A. Shutov ◽  
Eugeny Yu. Bragin ◽  
...  

Abstract Application of thermophile microorganisms opens new prospects in steroid biotechnology, however little is known on steroid catabolism by the thermophile strains.The thermophilic Saccharopolyspora hirsuta subsp. hirsuta strain VKM Ac-666T is capable of structural modification of different steroids, and fully degrades cholesterol. The intermediates of the cholesterol degradation pathway were identified as cholest-4-en-3-one, cholesta-1,4-dien-3-one, 26-hydroxycholest-4-en-3-one, 3-oxo-cholest-4-en-26-oic acid, 3-oxo-cholesta-1,4-dien-26-oic acid, 26-hydroxycholesterol, 3β-hydroxy-cholest-5-en-26-oic acid by MS, and H1- and C13-NMR analyses. The data evidence sterol degradation by the strain occurs simultaneously through the aliphatic side chain hydroxylation at C26 and the A-ring modification that are putatively catalyzed by cytochrome P450 monooxygenase CYP125 and cholesterol oxidase, respectively.The genes orthologous to those related to the sterol side chain degradation, steroid core rings A/B and C/D disruption and the steroid uptake were revealed. Most of the genes related to steroid degradation are grouped in three clusters. The sets of the genes putatively involved in steroid catabolism and peculiarities of their organization in S. hirsuta are discussed.Despite steroids abundancy in the environments, the ability to degrade them is not widespread among thermophilic bacteria as follows from the bioinformatic analysis of 52 publicly available genomes. Only seven candidate strains were revealed to possess the key genes related to the only known 9(10)-seco pathway of steroid degradation.The results contribute to the knowledge on diversity of microbial steroid degraders, the features of sterol catabolism by thermophilic actinobacteria and could be useful for application in the pharmaceutical and environmental biotechnology.


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