The Basal-Level Expression of the Cytochrome P450 BM-1 Gene Is Negatively Affected by the bm1P1 Gene of Bacillus megaterium

2000 ◽  
Vol 40 (1) ◽  
pp. 47-50 ◽  
Author(s):  
Gwo-Chyuan Shaw ◽  
Yi-Huang Hsueh ◽  
Hsun-Sheng Kao
Keyword(s):  
Cytochrome P450 ◽  
Bacillus Megaterium ◽  
Basal Level Expression

Construction and Functional Analysis of Mycolicibacterium smegmatis Recombinant Strains Carrying the Genes of Bacillary Cytochromes CYP106A1 and CYP106A2

2021 ◽  
Vol 37 (6) ◽  
pp. 34-47
Author(s):  
V.M. Nikolaeva ◽  
V.V. Fokina ◽  
A.A. Shutov ◽  
A.V. Kazantsev ◽  
N.I. Strizhov ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Heterologous Expression ◽  
Bacillus Megaterium ◽  
Cytochromes P450 ◽  
Maximum Level ◽  
13C Nmr Spectroscopy ◽  
Cytochrome P450 Monooxygenases ◽  
Russian Science ◽  
Steroid Nucleus ◽  
Recombinant Strains

Mycolicibacterium smegmatis mc2155 has been genetically modified to be used as a platform for the expression of foreign cytochrome P450 monooxygenases by introducing deletions in the kshB and kstD genes that encode key stages of the enzymatic destruction of the steroid nucleus. Three sets of genetic constructs have been created for heterologous expression of the genes of cytochromes P450 CYP106A1 from Bacillus megaterium DSM319 and CYP106A2 from Bacillus megaterium ATCC13368 in Mycolicibacterium smegmatis mc2155 (ΔkshBΔkstD) cells. The recombinant plasmids contained monocistronic expression cassettes of cytochrome genes (NS31 and pNS32), or tricistronic cassettes of cytochrome genes together with cDNA copies of adrenodoxin and andrenodoxin reductase genes of the bovine adrenal cortex (pNS33 and pNS34), or monocistronic gene cassettes of chimeric cytochromes fused with the DNA sequence encoding the CYP116B2 reductase domain from the soil bacterium Rhodococcus sp. NCIMB 9784 (pNS35 and pNS36). The recombinant strains of mycolicibacteria were shown to selectively monohydroxylate androstenedione (AD) under growth conditions. The product was identified as 15-hydroxyandrostenedione (15-OH-AD) by mass spectrometry and 1H and 13C NMR spectroscopy. The maximum level of 15-OH-AD production (17.3 ± 1.5 mg/L) was observed when using the recombinant M. smegmatis mc2155 (ΔkshBΔkstD) (pNS32) strain, which expresses a single cyp106A2 gene from B. megaterium ATCC13368. Host proteins of M. smegmatis mc2155 were shown to be capable of supplying electrons to heterologous cytochromes to support their hydroxylating activity. The results are of priority character, expand the understanding of the hydroxylation of steroid compounds by bacterial cytochromes CYP106A1/A2 and are important for the creation of microbial strains producing valuable hydroxysteroids. cyp106A1, cyp106A2, cytochrome P450, heterologous expression, Bacillus megaterium, Mycolicibacterium smegmatis, 15β-hydroxylation, bioconversion, steroids This work was supported by the Russian Science Foundation (project No. 21-64-00024).

scholarly journals P450BM3-Catalyzed Oxidations Employing Dual Functional Small Molecules

Catalysts ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 567 ◽  
Author(s):  
Willot ◽  
Tieves ◽  
Girhard ◽  
Urlacher ◽  
Hollmann ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hydrogen Peroxide ◽  
Cytochrome P450 ◽  
Small Molecules ◽  
Bacillus Megaterium ◽  
Cytochrome P450 Monooxygenase ◽  
Oxidation Reactions ◽  
P450 Monooxygenase ◽  
Dual Functional ◽  
Catalytic Epoxidation

A set of dual functional small molecules (DFSMs) containing different amino acids has been synthesized and employed together with three different variants of the cytochrome P450 monooxygenase P450BM3 from Bacillus megaterium in H2O2-dependent oxidation reactions. These DFSMs enhance P450BM3 activity with hydrogen peroxide as an oxidant, converting these enzymes into formal peroxygenases. This system has been employed for the catalytic epoxidation of styrene and in the sulfoxidation of thioanisole. Various P450BM3 variants have been evaluated in terms of activity and selectivity of the peroxygenase reactions.

Cloning, Expression and Characterization of a Monooxygenase P450BM3 from Bacillus megaterium ALA2

2012 ◽  
Vol 518-523 ◽  
pp. 5533-5538
Author(s):  
Ting Wang ◽  
Liang Liang Wang ◽  
Xun Li
Keyword(s):  
Escherichia Coli ◽  
Cytochrome P450 ◽  
Linoleic Acid ◽  
Bacillus Megaterium ◽  
Cytochrome P450 Monooxygenases ◽  
P450 Monooxygenases ◽  
Nadph Oxidation ◽  
First Time ◽  
Optimal Ph

Cytochrome P450 monooxygenases are enzymes which are capable of oxidising saturated and unsaturated substrates. P450BM3 from Bacillus megaterium is one of this family. For the first time, the cyp gene for coding P450BM3 from B. megaterium ALA2 has been cloned and expressed in Escherichia coli. The recombinant enzyme is 120 kDa, containing 1049 aa. The highest activity of purified enzyme is 14.8 U/mg towards palmitic acid by monitoring the NADPH oxidation. The optimal pH and temperature were 9.0 and 40°C. The enzyme has higher activity towards linoleic acid, and 2-Methyl-7-octadecene can also be catalyzed which is a precursor of displar.

Function and engineering of the 15β-hydroxylase CYP106A2

2006 ◽  
Vol 34 (6) ◽  
pp. 1215-1218 ◽  
Author(s):  
C. Virus ◽  
M. Lisurek ◽  
B. Simgen ◽  
F. Hannemann ◽  
R. Bernhardt
Keyword(s):  
Escherichia Coli ◽  
Cytochrome P450 ◽  
Steroid Hormones ◽  
Bacillus Megaterium ◽  
Crystallization Process ◽  
High Yield ◽  
Recent Developments ◽  
Adrenodoxin Reductase ◽  
Hydroxylation Activity

CYP106A2 from Bacillus megaterium ATCC 13368 is a bacterial cytochrome P450 that is capable of transforming steroid hormones. It can be easily expressed in Escherichia coli with a high yield. Its activity in vitro can be achieved by using the adrenal redox proteins adrenodoxin and adrenodoxin reductase. So far, it was not possible to crystallize CYP106A2 because of degradation during the crystallization process. Nevertheless, CYP106A2 is an interesting enzyme for biotechnological use. It hydroxylates pharmaceutically important steroids such as progesterone and 11-deoxycortisol. However, it will be necessary for efficient application of CYP106A2 in biotechnology to improve the hydroxylation activity and manipulate the regiospecificity. The present paper gives an overview of recent developments in protein engineering of CYP106A2.

Characterization of cytochrome P450 CYP109E1 from Bacillus megaterium as a novel vitamin D3 hydroxylase

2017 ◽  
Vol 243 ◽  
pp. 38-47 ◽  
Author(s):  
Ammar Abdulmughni ◽  
Ilona K. Jóźwik ◽  
Natalia Putkaradze ◽  
Elisa Brill ◽  
Josef Zapp ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Bacillus Megaterium ◽  
Vitamin D3
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