scholarly journals Improving the Production of 9,21-di​hydroxy-​20-​methyl-pregna-​4-​en-​3-​one from Phytosterols in Mycobacterium Neoaurum by Modifying Multiple Genes and Improving the Intracellular Environment

2021 ◽  
Author(s):  
Chen-Yang Yuan ◽  
Zhi-Guo Ma ◽  
Jing-Xian Zhang ◽  
Xiang-Cen Liu ◽  
Gui-Lin Du ◽  
...  
Keyword(s):  
Nadh Oxidase ◽  
Side Chain ◽  
Bacterial Strains ◽  
Synthetic Process ◽  
Mycobacterium Neoaurum ◽  
Intracellular Environment ◽  
Steroid Drug ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Combined Strategy ◽  
Steroid Structure

Abstract BackgroundSteroid drugs are particularly important for disease prevention and clinical treatment. However, traditional chemical methods are rarely implemented during the whole synthetic process to generate steroid intermediates due to the intricate steroid structure. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9-OH-4-HP is a potential steroid drug precursor for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain.ResultsA Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5 encoding an acyl-COA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains could accumulate 0.59 g L-1 and 0.47 g L-1 9-OH-4-HP from 1 g L-1 phytosterols. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 double-deficient strain was 11.9% higher than that of the Hsd4A -deficient strain and 40.4% higher than that of the strain with FadA5 deficiency, and its selectivity reached 94.9%. Subsequently, the catalase katE from Mycobacterium and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment. Ultimately, 9-OH-4-HP production reached 3.58 g L-1 from 5 g L-1 phytosterols, and the selectivity of 9-OH-4-HP improved to 97%.Conclusionhsd4A and fadA5 are key enzymes in the C19 pathway for phytosterol side chain degradation. Deletion of hsd4A and fadA5 could almost entirely block the C19 pathway. Improving the intracellular environment of Mycobacterium during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

scholarly journals Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Chen-Yang Yuan ◽  
Zhi-Guo Ma ◽  
Jing-Xian Zhang ◽  
Xiang-Cen Liu ◽  
Gui-Lin Du ◽  
...  
Keyword(s):  
Nadh Oxidase ◽  
Bacterial Strains ◽  
Synthetic Process ◽  
Molar Yield ◽  
Mycobacterium Neoaurum ◽  
Intracellular Environment ◽  
Double Deletion ◽  
Steroid Drug ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Combined Strategy

Abstract Background Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. Results The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L−1 and 0.47 g L−1 9-OH-4-HP from 1 g L−1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L−1 from 5 g L−1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. Conclusion KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

scholarly journals Intracellular Environment Improvement of Mycobacterium neoaurum for Enhancing Androst-1,4-Diene-3,17-Dione Production by Manipulating NADH and Reactive Oxygen Species Levels

Molecules ◽  
2019 ◽  
Vol 24 (21) ◽  
pp. 3841
Author(s):  
Minglong Shao ◽  
Youxi Zhao ◽  
Yu Liu ◽  
Taowei Yang ◽  
Meijuan Xu ◽  
...  
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Parent Strain ◽  
Transformation Efficiency ◽  
Nadh Oxidase ◽  
Steroid Hormone ◽  
Microbial Transformation ◽  
Major Product ◽  
Oxygen Species ◽  
Mycobacterium Neoaurum ◽  
Intracellular Environment

As one of the most significant steroid hormone precursors, androst-1,4-diene-3,17-dione (ADD) could be used to synthesize many valuable hormone drugs. The microbial transformation of sterols to ADD has received extensive attention in recent years. In a previous study, Mycobacterium neoaurum JC-12 was isolated and converted sterols to the major product, ADD. In this work, we enhanced ADD yield by improving the cell intracellular environment. First, we introduced a nicotinamide adenine dinucleotide (NADH) oxidase from Bacillus subtilis to balance the intracellular NAD+ availability in order to strengthen the ADD yield. Then, the catalase gene from M. neoaurum was also over-expressed to simultaneously scavenge the generated H2O2 and eliminate its toxic effects on cell growth and sterol transformation. Finally, using a 5 L fermentor, the recombinant strain JC-12yodC-katA produced 9.66 g/L ADD, which increased by 80% when compared with the parent strain. This work shows a promising way to increase the sterol transformation efficiency by regulating the intracellular environment.

scholarly journals Synthesis, Tyrosinase Inhibiting Activity and Molecular Docking of Fluorinated Pyrazole Aldehydes as Phosphodiesterase Inhibitors

2019 ◽  
Vol 9 (8) ◽  
pp. 1704
Author(s):  
Vesna Rastija ◽  
Harshad Brahmbhatt ◽  
Maja Molnar ◽  
Melita Lončarić ◽  
Ivica Strelec ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Antibacterial Properties ◽  
Phosphodiesterase Inhibitors ◽  
Pyrazole Ring ◽  
Binding Mode ◽  
Van Der Waals Interactions ◽  
Side Chain ◽  
Bacterial Strains ◽  
1H And 13C Nmr ◽  
Monophenolase Activity

A series of fluorinated 4,5-dihydro-1H-pyrazole derivatives were synthesized in the reaction of corresponding acetophenone and different aldehydes followed by the second step synthesis of desired compounds from synthesized chalcone, hydrazine hydrate, and formic acid. Structures of all compounds were confirmed by both 1H and 13C NMR and mass spectrometry. Antibacterial properties of compounds were tested on four bacterial strains, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. Among synthesized compounds, the strongest inhibitor of monophenolase activity of mushroom tyrosinase (32.07 ± 3.39%) was found to be 5-(2-chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole-1-carbaldehyde. The PASS program has predicted the highest probable activity for the phosphodiesterase inhibition. To shed light on molecular interactions between the synthesized compounds and phosphodiesterase, all compounds were docked into the active binding site. The obtained results showed that the compound with the dimethoxyphenyl ring could be potent as an inhibitor of phosphodiesterase, which interacts in PDE5 catalytic domain of the enzyme. Key interactions are bidentate hydrogen bond (H-bond) with the side-chain of Gln817 and van der Waals interactions of the dimethoxyphenyl ring and pyrazole ring with hydrophobic clamp, which contains residuals, Val782, Phe820, and Tyr612. Interactions are similar to the binding mode of the inhibitor sildenafil, the first oral medicine for the treatment of male erectile dysfunction.

Na+ responses of two marine bacteria that did not appear to require Na+ for growth

1993 ◽  
Vol 39 (3) ◽  
pp. 297-303 ◽  
Author(s):  
Michelle F. Manuel ◽  
Gesine A. Wisse ◽  
Robert A. MacLeod
Keyword(s):  
Carbon Source ◽  
Liquid Medium ◽  
Marine Bacteria ◽  
Binding Sites ◽  
Initial Rate ◽  
Nadh Oxidase ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Limited Extent ◽  
Bacterial Strains

Two Gram-negative heterotrophic marine bacterial strains had been reported not to require Na+ when grown on a chemically defined medium solidifed with purified agar and prepared without added Na+. When these strains were tested in a chemically defined liquid medium they required at least 3 mM Na + for growth. The agar used in the plating medium was found to contribute 3.3 mM Na+. Increasing the concentrations of Na+ in the liquid medium above 3 mM increased the rate and extent of growth of both organisms and decreased the lag periods. Optimal Na+ concentrations for growth varied from 100 to 500 mM depending on the organism and the carbon source in the medium. Na+ was also required for the transport of the carbon source into the cells. For the maximal rate of transport of L-glutamate, one organism required only 10 mM Na +, the other, 50 mM. For acetate and succinate transport the optimal Na+ concentrations varied from 30 to 200 mM depending on the substrate and the organism. When the initial rate of transport of glutamate into one of the organisms was plotted against Na+ concentration the reponse curve was sigmoid and a Hill plot of the data indicated that the transport protein may possess three binding sites for Na+. Evidence was obtained indicating that both organisms possess a Na+-stimulated NADH oxidase. The results indicate that there are marine bacteria that grow to a limited extent at appreciably lower concentrations of Na+ than have been realized previously and for these a much more definitive examination of the requirement for Na+ is necessary.Key words: marine bacteria, Na+ requirement, growth, membrane transport, NADH oxidase.

scholarly journals Identification and characterization of the 2-enoyl-CoA hydratases involved in peroxisomal β-oxidation in rat liver

1997 ◽  
Vol 321 (1) ◽  
pp. 253-259 ◽  
Author(s):  
Martine DIEUAIDE-NOUBHANI ◽  
Dmitry NOVIKOV ◽  
Joël VANDEKERCKHOVE ◽  
Paul P. Van VELDHOVEN ◽  
Guy P. MANNAERTS
Keyword(s):  
Rat Liver ◽  
Hydroxysteroid Dehydrogenase ◽  
Acid Synthesis ◽  
Bile Acid Synthesis ◽  
Side Chain ◽  
Multifunctional Protein ◽  
Hydratase Activity ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Enoyl Coa Hydratase

In this study we attempted to determine the number of 2-enoyl-CoA hydratases involved in peroxisomal β-oxidation. We therefore separated peroxisomal proteins from rat liver on several chromatographic columns and measured hydratase activities on the eluates with different substrates. The results indicate that rat liver peroxisomes contain two hydratase activities: (1) a hydratase activity associated with multifunctional protein 1 (MFP-1) (2-enoyl-CoA hydratase/Δ3,Δ2-enoyl-CoA isomerase/l-3-hydroxyacyl-CoA dehydrogenase) and (2) a hydratase activity associated with MFP-2 (17β-hydroxysteroid dehydrogenase/d-3-hydroxyacyl-CoA dehydrogenase/2-enoyl-CoA hydratase). MFP-1 forms and dehydrogenates l-3-hydroxyacyl-CoA species, whereas MFP-2 forms and dehydrogenates d-3-hydroxyacyl-CoA species. A portion of MFP-2 is proteolytically cleaved, most probably in the peroxisome, into a 34 kDa 17β-hydroxysteroid dehydrogenase/d-3-hydroxyacyl-CoA dehydrogenase and a 45 kDa d-specific 2-enoyl-CoA hydratase. Finally, the results confirm that MFP-1 is involved in the degradation of straight-chain fatty acids, whereas MFP-2 and its cleavage products seem to be involved in the degradation of the side chain of cholesterol (bile acid synthesis)

The role of α-methylacyl-CoA racemase in bile acid synthesis

2002 ◽  
Vol 363 (3) ◽  
pp. 801-807 ◽  
Author(s):  
Dean A. CUEBAS ◽  
Christopher PHILLIPS ◽  
Werner SCHMITZ ◽  
Ernst CONZELMANN ◽  
Dmitry K. NOVIKOV
Keyword(s):  
Fatty Acids ◽  
Bile Acid ◽  
Alternative Pathway ◽  
Acid Synthesis ◽  
Bile Acid Synthesis ◽  
Side Chain ◽  
Multifunctional Enzyme ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Dehydrogenation Reactions ◽  
Enoyl Coa Hydratase

According to current views, the second peroxisomal β-oxidation pathway is responsible for the degradation of the side chain of bile acid intermediates. Peroxisomal multifunctional enzyme type 2 [peroxisomal multifunctional 2-enoyl-CoA hydratase/(R)-3-hydroxyacyl-CoA dehydrogenase; MFE-2] catalyses the second (hydration) and third (dehydrogenation) reactions of the pathway. Deficiency of MFE-2 leads to accumulation of very-long-chain fatty acids, 2-methyl-branched fatty acids and C27 bile acid intermediates in plasma, but bile acid synthesis is not blocked completely. In this study we describe an alternative pathway, which allows MFE-2 deficiency to be overcome. The alternative pathway consists of α-methylacyl-CoA racemase and peroxisomal multifunctional enzyme type 1 [peroxisomal multifunctional 2-enoyl-CoA hydratase/(S)-3-hydroxyacyl-CoA dehydrogenase; MFE-1]. (24E)-3α,7α,12α-Trihydroxy-5β-cholest-24-enoyl-CoA, the presumed physiological isomer, is hydrated by MFE-1 with the formation of (24S,25S)-3α,7α,12α,24-tetrahydroxy-5β-cholestanoyl-CoA [(24S,25S)-24-OH-THCA-CoA], which after conversion by a α-methylacyl-CoA racemase into the (24S,25R) isomer can again be dehydrogenated by MFE-1 to 24-keto-3α,7α,12α-trihydroxycholestanoyl-CoA, a physiological intermediate in cholic acid synthesis. The discovery of the alternative pathway of cholesterol side-chain oxidation will improve diagnosis of peroxisomal deficiencies by identification of serum 24-OH-THCA-CoA diastereomer profiles.

scholarly journals Evidence that multifunctional protein 2, and not multifunctional protein 1, is involved in the peroxisomal β-oxidation of pristanic acid

1997 ◽  
Vol 325 (2) ◽  
pp. 367-373 ◽  
Author(s):  
Martine DIEUAIDE-NOUBHANI ◽  
Stanny ASSELBERGHS ◽  
Guy P. MANNAERTS ◽  
Paul P. VAN VELDHOVEN
Keyword(s):  
Fatty Acids ◽  
Bile Acid ◽  
Acid Synthesis ◽  
Bile Acid Synthesis ◽  
Side Chain ◽  
Acid Oxidation ◽  
Synthetic Analogue ◽  
Pristanic Acid ◽  
Multifunctional Protein ◽  
Hydroxyacyl Coa Dehydrogenase

The second (enoyl-CoA hydratase) and third (3-hydroxyacyl-CoA dehydrogenase) steps of peroxisomal β-oxidation are catalysed by two separate multifunctional proteins (MFPs), MFP-1 being involved in the degradation of straight-chain fatty acids and MFP-2 in the β-oxidation of the side chain of cholesterol (bile acid synthesis). In the present study we determined which of the two MFPs is involved in the peroxisomal degradation of pristanic acid by using the synthetic analogue 2-methylpalmitic acid. The four stereoisomers of 3-hydroxy-2-methylpalmitoyl-CoA were separated by gas chromatography after hydrolysis, methylation and derivatization of the hydroxy group with (S)-2-phenylpropionic acid, and the stereoisomers were designated I–IV according to their order of elution from the column. Purified MFP-1 dehydrated stereoisomer IV but dehydrogenated stereoisomer III, so by itself MFP-1 is not capable of converting a branched enoyl-CoA into a 3-ketoacyl-CoA. In contrast, MFP-2 dehydrated and dehydrogenated the same stereoisomer (II), so it is highly probable that MFP-2 is involved in the peroxisomal degradation of branched fatty acids and that stereoisomer II is the physiological intermediate in branched fatty acid oxidation. By analogy with the results obtained with the four stereoisomers of the bile acid intermediate varanoyl-CoA, stereoisomer II can be assigned the 3R-hydroxy, 2R-methyl configuration.

scholarly journals Complete Biodegradation of 4-Fluorocinnamic Acid by a Consortium ComprisingArthrobactersp. Strain G1 andRalstoniasp. Strain H1

2010 ◽  
Vol 77 (2) ◽  
pp. 572-579 ◽  
Author(s):  
Syed A. Hasan ◽  
Maria Isabel M. Ferreira ◽  
Martijn J. Koetsier ◽  
Muhammad I. Arif ◽  
Dick B. Janssen
Keyword(s):  
Oxidation Mechanism ◽  
Cell Extract ◽  
Side Product ◽  
Side Chain ◽  
Bacterial Strains ◽  
Aerobic Conditions ◽  
Coa Ligase ◽  
Dead End ◽  
Carbon Side Chain ◽  
Complete Mineralization

ABSTRACTA consortium of the newly isolated bacterial strainsArthrobactersp. strain G1 andRalstoniasp. strain H1 utilized 4-fluorocinnamic acid for growth under aerobic conditions. Strain G1 converted 4-fluorocinnamic acid into 4-fluorobenzoic acid and used the two-carbon side chain for growth, with some formation of 4-fluoroacetophenone as a dead-end side product. In the presence of strain H1, complete mineralization of 4-fluorocinnamic acid and release of fluoride were obtained. Degradation of 4-fluorocinnamic acid by strain G1 occurred through a β-oxidation mechanism and started with the formation of 4-fluorocinnamoyl-coenzyme A (CoA), as indicated by the presence of 4-fluorocinnamoyl-CoA ligase. Enzymes for further transformation were detected in cell extract, i.e., 4-fluorocinnamoyl-CoA hydratase, 4-fluorophenyl-β-hydroxy propionyl-CoA dehydrogenase, and 4-fluorophenyl-β-keto propionyl-CoA thiolase. Degradation of 4-fluorobenzoic acid by strain H1 proceeded via 4-fluorocatechol, which was converted by anortho-cleavage pathway.

scholarly journals Antimicrobial activity of some novel 2-(2-iodophenylimino)-5-arylidenethiazolidin-4-one derivatives

2018 ◽  
Vol 11 (5) ◽  
pp. 405-412 ◽  
Author(s):  
Duy Toan Pham ◽  
Thi My Huong Vo ◽  
Phuong Truong ◽  
Phuoc Tinh Ho ◽  
Manh Quan Nguyen
Keyword(s):  
Antimicrobial Activity ◽  
Aldol Condensation ◽  
Antibacterial Activities ◽  
Multidrug Resistant ◽  
Therapeutic Agents ◽  
Antifungal Drugs ◽  
Resistant Bacteria ◽  
Bacterial Strains ◽  
Synthetic Process ◽  
New Compounds

AbstractBackgroundInfectious diseases, especially those caused by multidrug-resistant bacteria, are becoming a serious problem worldwide because of the lack of effective therapeutic agents. Moreover, most antifungal drugs exhibit low efficacy and high toxicity because of the similarity between fungal and human cells. These issues warrant the search for potential new agents.ObjectivesTo synthesize potent 2-(2-iodophenylimino)-5-arylidenethiazolidin-4-one derivatives, improve the synthetic process, elucidate their structures, and determine their antimicrobial activity.Methods2-Iodoaniline was used as an initial reactant in a 3-step process for the synthesis of 2-(2-iodophenylimino)-5-arylidenethiazolidin-4-one derivatives, including an acylation reaction, a cyclization reaction, and aldol condensation reactions. The structures of the obtained derivatives were investigated and elucidated using spectral methods. Antimicrobial activity toward 5 bacterial strains and 2 fungal strains was determined using Kirby–Bauer and agar dilution methods.ResultsWe successfully synthesized 12 novel compounds and elucidated their structures. The derivatives had no antifungal activities. By contrast, they showed remarkable antibacterial activities. Some of them with minimum inhibitory concentrations (MICs) ≤8 μg/mL in both Staphylococcus aureus and methicillin-resistant S. aureus.ConclusionsA simple and flexible way to synthesize new compounds with a thiazolidin-4-one ring was determined. Some of these new compounds have outstanding effects with low MICs for bacteria. Their further investigation as therapeutic agents is warranted.

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