Biosynthesis of vitamin B12: the preparative multi-enzyme synthesis of precorrin-3A and 20-methylsirohydrochlorin (a 2,7,20-trimethylisobacteriochlorin)

The Bacillus subtilis genes hemB, hemC and hemD, encoding respectively the enzymes porphobilinogen synthase, hydroxymethylbilane synthase and uroporphyrinogen III synthase, have been expressed in Escherichia coli using a single plasmid construct. An enzyme preparation from this source converts 5-aminolaevulinic acid (ALA) preparatively and in high yield into uroporphyrinogen III. The Pseudomonas denitrificans genes cobA and cobI, encoding respectively the enzymes S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) and S-adenosyl-L-methionine:precorrin-2 methyltransferase (SP2MT), were also expressed in E. coli. When SUMT was combined with the coupled-enzyme system that produces uroporphyrinogen III, precorrin-2 was synthesized from ALA, and when SP2MT was also added the product from the coupling of five enzymes was precorrin-3A. Both of these products are precursors of vitamin B12, and they can be used directly for biosynthetic experiments or isolated as their didehydro octamethyl esters in > 40% overall yield. The enzyme system which produces precorrin-3A is sufficiently stable to allow long incubations on a large scale, affording substantial quantities (15-20 mg) of product.

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Revealing the promoting effect of betaine on vitamin B12 biosynthetic pathway of Pseudomonas denitrificans by using a proteomics analysis

Current Pharmaceutical Biotechnology ◽

10.2174/1389201022666210531120935 ◽

2021 ◽

Vol 22 ◽

Author(s):

Kun-tai Li ◽

Yong Yang ◽

Xin Cheng

Keyword(s):

Vitamin B12 ◽

Biosynthetic Pathway ◽

Aminolevulinic Acid ◽

Fermentation Medium ◽

Promoting Effect ◽

Pseudomonas Denitrificans ◽

Methyl Group ◽

Integral Effect ◽

Comparative Metabolomics ◽

Porphobilinogen Synthase

Background: Our previous comparative metabolomics research revealed that betaine (N,N,N-trimethylglycine, a typically essential methyl-group donor for vitamin B12 biosynthesis) had a powerful promoting effect on the generation of vitamin B12 precursors and intermediates in vitamin B12-producing Pseudomonas denitrificans. However, the integral effect of betaine on the vitamin B12 biosynthetic pathway is still unclear. Objective: Considering the vitamin B12 biosynthetic pathway of P. denitrificans as a whole, this work aimed to reveal the biological function of betaine on the vitamin B12 biosynthetic pathway in P. denitrificans, which would sharpen and expand the understanding of betaine as the methyl-group donor for vitamin B12 biosynthesis. Materials and Methods: By using a proteomics method based on the iTRAQ technique, the present study compared and analyzed the differential expression of proteins involved in vitamin B12 biosynthetic pathway under 10 g/L betaine addition to P. denitrificans fermentation medium. Results: The results showed that betaine could significantly up-regulate the expression of proteins related to the vitamin B12 biosynthetic pathway, which was mainly reflected in the following three aspects: 1) the δ-aminolevulinic acid (ALA) synthase and porphobilinogen synthase that were responsible for the formation of the committed precursors for tetrapyrrole-derived macrocycle in vitamin B12 molecule; 2) the C-methylation-related enzymes (such as precorrin-4 C(11)-methyltransferase, Precorrin-2 C(20)-methyltransferase, Precorrin-8X methylmutase, and Precorrin-6Y C5,15-methyltransferase) and methionine synthase that were crucial to the C-methylation reactions for vitamin B12 biosynthesis; 3) the late-stage key enzymes (Cobaltochelatase, and Cob(I)yrinic acid a,c-diamide adenosyltransferase) that were related to cobalt chelation of vitamin B12 molecule. Conclusions: The present study clearly demonstrated that betaine could significantly promote the expression of the integral enzymes involved in the vitamin B12 biosynthetic pathway of P. denitrificans, thus promoting vitamin B12 biosynthesis.

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Improved large-scale production of vitamin B12 by Pseudomonas denitrificans with betaine feeding

Bioresource Technology ◽

10.1016/j.biortech.2008.03.023 ◽

2008 ◽

Vol 99 (17) ◽

pp. 8516-8520 ◽

Cited By ~ 31

Author(s):

Kun-Tai Li ◽

Dong-Hong Liu ◽

Yong-Liang Li ◽

Ju Chu ◽

Yong-Hong Wang ◽

...

Keyword(s):

Vitamin B12 ◽

Large Scale ◽

Scale Production ◽

Pseudomonas Denitrificans ◽

Large Scale Production

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Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128900 ◽

1987 ◽

Vol 45 ◽

pp. 924-925

Author(s):

F. A. Durum ◽

R. G. Goldman ◽

T. J. Bolling ◽

M. F. Miller

Keyword(s):

Inclusion Bodies ◽

Large Scale ◽

Recombinant Dna ◽

Test System ◽

Cell Protein ◽

Gram Negative Bacteria ◽

Cytoplasmic Inclusions ◽

Isolation And Purification ◽

E Coli ◽

Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

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Purification of Urokinase from Complex Mixtures Using Immobilized Monoclonal Antibody Against Urokinase Light Chain

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657308 ◽

1983 ◽

Vol 49 (01) ◽

pp. 024-027 ◽

Cited By ~ 20

Author(s):

David Vetterlein ◽

Gary J Calton

Keyword(s):

Monoclonal Antibody ◽

Light Chain ◽

Culture Media ◽

Biological Fluids ◽

Single Step ◽

High Yield ◽

Step Method ◽

Commercial Preparation ◽

E Coli ◽

Tissue Culture Media

SummaryThe preparation of a monoclonal antibody (MAB) against high molecular weight (HMW) urokinase light chain (20,000 Mr) is described. This MAB was immobilized and the resulting immunosorbent was used to isolate urokinase starting with an impure commercial preparation, fresh urine, spent tissue culture media, or E. coli broth without preliminary dialysis or concentration steps. Monospecific antibodies appear to provide a rapid single step method of purifying urokinase, in high yield, from a variety of biological fluids.

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Identification of N-Substituted Triazolo-azetidines as Novel Antibacterials using pDualrep2 HTS Platform

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207322666190412165316 ◽

2019 ◽

Vol 22 (5) ◽

pp. 346-354

Author(s):

Yan A. Ivanenkov ◽

Renat S. Yamidanov ◽

Ilya A. Osterman ◽

Petr V. Sergiev ◽

Vladimir A. Aladinskiy ◽

...

Keyword(s):

Antibacterial Activity ◽

Large Scale ◽

Underlying Mechanism ◽

Luciferase Assay ◽

Reporter System ◽

E Coli ◽

Starting Point ◽

High Concentrations ◽

Mic Value

Aim and Objective: Antibiotic resistance is a serious constraint to the development of new effective antibacterials. Therefore, the discovery of the new antibacterials remains one of the main challenges in modern medicinal chemistry. This study was undertaken to identify novel molecules with antibacterial activity. Materials and Methods: Using our unique double-reporter system, in-house large-scale HTS campaign was conducted for the identification of antibacterial potency of small-molecule compounds. The construction allows us to visually assess the underlying mechanism of action. After the initial HTS and rescreen procedure, luciferase assay, C14-test, determination of MIC value and PrestoBlue test were carried out. Results: HTS rounds and rescreen campaign have revealed the antibacterial activity of a series of Nsubstituted triazolo-azetidines and their isosteric derivatives that has not been reported previously. Primary hit-molecule demonstrated a MIC value of 12.5 µg/mL against E. coli Δ tolC with signs of translation blockage and no SOS-response. Translation inhibition (26%, luciferase assay) was achieved at high concentrations up to 160 µg/mL, while no activity was found using C14-test. The compound did not demonstrate cytotoxicity in the PrestoBlue assay against a panel of eukaryotic cells. Within a series of direct structural analogues bearing the same or bioisosteric scaffold, compound 2 was found to have an improved antibacterial potency (MIC=6.25 µg/mL) close to Erythromycin (MIC=2.5-5 µg/mL) against the same strain. In contrast to the parent hit, this compound was more active and selective, and provided a robust IP position. Conclusion: N-substituted triazolo-azetidine scaffold may be used as a versatile starting point for the development of novel active and selective antibacterial compounds.

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Large-scale High-throughput Screening Revealed 5'-(carbonylamino)-2,3'- bithiophene-4'-carboxylate as Novel Template for Antibacterial Agents

Current Drug Discovery Technologies ◽

10.2174/1570163816666190603095521 ◽

2020 ◽

Vol 17 (5) ◽

pp. 716-724

Author(s):

Yan A. Ivanenkov ◽

Renat S. Yamidanov ◽

Ilya A. Osterman ◽

Petr V. Sergiev ◽

Vladimir A. Aladinskiy ◽

...

Keyword(s):

Antibacterial Activity ◽

High Throughput ◽

High Throughput Screening ◽

Large Scale ◽

Antibacterial Agents ◽

Sos Response ◽

Luciferase Assay ◽

Translational Machinery ◽

E Coli ◽

New Class

Background: The key issue in the development of novel antimicrobials is a rapid expansion of new bacterial strains resistant to current antibiotics. Indeed, World Health Organization has reported that bacteria commonly causing infections in hospitals and in the community, e.g. E. Coli, K. pneumoniae and S. aureus, have high resistance vs the last generations of cephalosporins, carbapenems and fluoroquinolones. During the past decades, only few successful efforts to develop and launch new antibacterial medications have been performed. This study aims to identify new class of antibacterial agents using novel high-throughput screening technique. Methods: We have designed library containing 125K compounds not similar in structure (Tanimoto coeff.< 0.7) to that published previously as antibiotics. The HTS platform based on double reporter system pDualrep2 was used to distinguish between molecules able to block translational machinery or induce SOS-response in a model E. coli system. MICs for most active chemicals in LB and M9 medium were determined using broth microdilution assay. Results: In an attempt to discover novel classes of antibacterials, we performed HTS of a large-scale small molecule library using our unique screening platform. This approach permitted us to quickly and robustly evaluate a lot of compounds as well as to determine the mechanism of action in the case of compounds being either translational machinery inhibitors or DNA-damaging agents/replication blockers. HTS has resulted in several new structural classes of molecules exhibiting an attractive antibacterial activity. Herein, we report as promising antibacterials. Two most active compounds from this series showed MIC value of 1.2 (5) and 1.8 μg/mL (6) and good selectivity index. Compound 6 caused RFP induction and low SOS response. In vitro luciferase assay has revealed that it is able to slightly inhibit protein biosynthesis. Compound 5 was tested on several archival strains and exhibited slight activity against gram-negative bacteria and outstanding activity against S. aureus. The key structural requirements for antibacterial potency were also explored. We found, that the unsubstituted carboxylic group is crucial for antibacterial activity as well as the presence of bulky hydrophobic substituents at phenyl fragment. Conclusion: The obtained results provide a solid background for further characterization of the 5'- (carbonylamino)-2,3'-bithiophene-4'-carboxylate derivatives discussed herein as new class of antibacterials and their optimization campaign.

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A Simple Synthesis of the Anticancer Drug Altretamine

Letters in Organic Chemistry ◽

10.2174/1570178617666200210111041 ◽

2020 ◽

Vol 17 (8) ◽

pp. 628-630

Author(s):

Vu Binh Duong ◽

Pham Van Hien ◽

Tran Thai Ngoc ◽

Phan Dinh Chau ◽

Tran Khac Vu

Keyword(s):

Aqueous Solution ◽

Anticancer Drug ◽

Potassium Hydroxide ◽

Large Scale ◽

Synthesis Method ◽

Practical Method ◽

Cyanuric Chloride ◽

Simple Synthesis ◽

High Yield ◽

One Step

A simple and practical method for the synthesis on a large scale of altretamine (1), a wellknown antitumor drug, has been successfully developed. The synthesis method involves the conversion of cyanuric chloride (2) into altretamine (1) by dimethylamination of 2 with an aqueous solution of 40% dimethylamine and potassium hydroxide in 1, -dioxan 4in one step to give altretamine (1) in high yield.

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Quality Improvement of the DNA extracted by boiling method in Gram negative bacteria

International Journal of Bioassays ◽

10.21746/ijbio.2017.04.004 ◽

2017 ◽

Vol 6 (04) ◽

pp. 5347 ◽

Cited By ~ 3

Author(s):

Omar B. Ahmed* ◽

Anas S. Dablool

Keyword(s):

Dna Extraction ◽

Control Method ◽

Extraction Procedure ◽

Cost Effective ◽

High Yield ◽

Gram Negative Bacteria ◽

Bacterial Dna ◽

Gram Negative ◽

E Coli

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.

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