Biosynthesis Of Chlortetracycline Research Articles

Crystal structure determination of the halogenase CtcP from Streptomyces aureofaciens.

Chlortetracycline (CTC), a derivative of tetracycline (TC), is a broadly used antibiotic that inhibits the synthesis of bacterial proteins by competing with the A-site tRNA on ribosomes. A recent study showed that during the biosynthesis of CTC in Streptomyces aureofaciens, the halogenase CtcP catalyzes the final chlorination reaction and transforms TC into CTC. However, the structure of this fundamental enzyme is still lacking. Here, selenomethionine-derivatized CtcP from S. aureofaciens was overexpressed and purified and its structure was determined at 2.7 Å resolution. The structure of CtcP reveals the conserved monooxygenase domain shared by all flavin-dependent halogenases and a unique C-terminal domain. Although FAD was not observed in the structure, the monooxygenase domain has a conserved FAD-binding pocket and active center. The C-terminal domain displays an α-helical bundle fold, which could contribute to substrate specificity. This work provides a molecular basis for enzyme engineering to improve the industrial production of CTC.

CtcS, a MarR family regulator, regulates chlortetracycline biosynthesis

BackgroundChlortetracycline (CTC) is one of the commercially important tetracyclines (TCs) family product and is mainly produced by Streptomyces. CTC is still in a great demand due to its broad-spectrum activity against pathogens. Engineering transcriptional control allows the cell to allocate its valuable resources towards protein production and provides an important method for the build-up of desired metabolites. Despite extensive efforts concerning transcriptional regulation for increasing the productivities of TCs, the regulatory mechanisms of the CTC biosynthesis remain poorly understood.ResultsIn this study, the possible regulatory function of CtcS, a potential member of MarR (multiple antibiotic resistance regulator) family of transcriptional regulators in S. aureofaciens F3, was demonstrated. Knockdown of ctcS altered the transcription of several biosynthesis-related genes and reduced the production of tetracycline (TC) and CTC, without obvious effect on morphological differentiation and cell growth. Especially, CtcS directly repressed the transcription of the adjacent divergent gene ctcR (which encodes a putative TC resistance efflux protein). A CtcS-binding site was identified within the promoter region of ctcR by DNase I footprinting and an inverted repeat (5′-CTTGTC-3′) composed of two 6-nt half sites in the protected region was found. Moreover, both CTC and TC could attenuate the binding activity of CtcS with target DNA.ConclusionctcS regulated the production of TC and CTC in S. aureofaciens F3 and the overexpression of it could be used as a simple approach for the construction of engineering strain with higher productivity. Meanwhile, CtcS was characterized as a TC- and CTC-responsive MarR family regulator. This study provides a previously unrecognized function of CtcS and will benefit the research on the regulatory machinery of the MarR family regulators.

Deciphering and engineering of the final step halogenase for improved chlortetracycline biosynthesis in industrial Streptomyces aureofaciens

Chlortetracycline (CTC) is an important member from antibiotics tetracycline (TC) family, which inhibits protein synthesis in bacteria and is widely involved in clinical therapy, animal feeds and aquaculture. Previous works have reported intricately the biosynthesis of CTC from the intermediates in random mutants of Streptomyces aureofaciens and the crucial chlorination remained unclear. We have developed the genetic manipulation in an industrial producer, in which about 15.0g/l CTC predominated along with 1.2g/l TC, and discovered that chlorination by ctcP (an FADH2-dependent halogenase gene) is the last inefficient step during CTC biosynthesis. Firstly, the ΔctcP strain accumulated about 18.9g/l “clean” TC without KBr addition and abolished the production of CTC. Subsequently, CtcP was identified to exhibit a substrate stereo-specificity to absolute TC (4S) rather than TC (4R), with low kcat of 0.51±0.01min−1, while it could halogenate several TC analogs. Accordingly, we devised a strategy for overexpression of ctcP in S. aureofaciens and improved CTC production to a final titer of 25.9g/l. We anticipate that our work will provide a biotechnological potential of enzymatic evolution and strain engineering towards new TC derivatives in microorganisms.

Identification and cloning of the gene involved in the final step of chlortetracycline biosynthesis in Streptomyces aureofaciens.

For chlortetracycline biosynthesis in Streptomyces aureofaciens, the final reduction step is essential to give an antibiotic activity to its intermediate, which is catalyzed by tetracycline dehydrogenase with 7,8-dedimethyl-8-hydroxy-5-deazariboflavin (FO) as a cofactor. We identified and cloned the gene, which is essential for the biosynthesis of 6-demethyltetracycline and participates in the final step of its biosynthesis, from the genomic DNA of the 6-demethyltetracycline producer S. aureofaciens HP77. DNA sequence analysis revealed that the gene (tchA) had an open reading frame of 455 amino acids with an estimated molecular mass of 48.1 kDa. Southern hybridization analysis revealed that the tchA gene was located external to the chlortetracycline biosynthetic gene cluster in the genome. A conserved domain search of protein sequence databases indicated that TchA showed a similarity to FbiB, which is involved in the modification of FO in Mycobacterium bovis.

Major proteins related to chlortetracycline biosynthesis in a Streptomyces aureofaciens production strain studied by quantitative proteomics

Changes in synthesis and abundance of proteins associated with chlortetracycline (CTC) production in Streptomyces aureofaciens were investigated by two-dimensional polyacrylamide gel electrophoresis of proteins pulse-labelled in vivo with L-[35S]methionine. Eleven individual protein spots were selected as being related to formation of the antibiotic. Expression of these prominent proteins was not observed in the non-producing mutant; moreover, they were overexpressed in cultures grown in the presence of benzyl thiocyanate, a specific stimulator of CTC biosynthesis used in industrial fermentations. The expression kinetics of the selected proteins was assessed using the technique of computer-assisted image analysis with the EQIAS software and the elongation factor Tu as an internal standard. Interestingly, the kinetic profiles were generally not identical. including those of anhydrotetracycline monooxygenase and the 13-kDa subunit of tetracycline dehydrogenase, two enzymes involved, in the terminal sequential steps of the CTC biosynthetic pathway. The presence of more forms of these enzymes with different charge characteristics was observed. The data presented demonstrated how dramatically the industrial microorganism can change its protein repertoire during the production phase; at least five proteins were nearly comparable in level to the most prominent proteins, exemplified by elongation factor Tu.

Protein profiles of Streptomyces aureofaciens producing tetracyclines: Reappraisal of the effect of benzyl thiocyanate

Cell protein profiles of submerged cultures of Streptomyces aureofaciens cultivated in the absence or presence of 12 microM benzyl thiocyanate (BT) were analyzed by one-dimensional SDS polyacrylamide gel electrophoresis. Substantial increase in the intensity of the 13, 35, 37, 60, and 100 kDa protein bands was observed in cultures treated with BT. Similar increase in the 35, 37, and 60 kDa bands was found in a mutant blocked in the last chlortetracycline biosynthesis step. Effect of BT on the solid medium-grown cultures was also observed, with a more intensive substrate mycelium pigmentation and alteration in the spore size and shape as the most characteristic features. Earlier studies of BT effect involving those on the stimulation of chlortetracycline biosynthesis are summarized and a possible signal-transducing mechanism is discussed from the point of view of adaptation of S. aureofaciens to the uncoupling of oxidative phosphorylation.

Biotransformation of benzyl thiocyanate by Streptomyces aureofaciens.

Benzyl thiocyanate, a specific stimulator of chlortetracycline biosynthesis, was transformed into dibenzyl disulphide by Streptomyces aureofaciens. The disulphide stimulated chlortetracycline production to a lesser extent than did benzyl thiocyanate.

Subcellular organization of Streptomyces aureofaciens and overproduction of chlortetracycline

Biosynthesis of chlortetracycline is localized differently under low- and high-production conditions (standard low-production strain and its high-production variant). The experimental evidence was based on the assay of anhydrotetracycline oxygenase in subcellular fractions, ultracytochemical localization and electron-probe X-ray microanalysis of the product in the mycelium. Overproduction of chlortetracycline is closely associated with compartmentation of biosynthetic enzymes and with an efficient export of the antibiotic out of the cell.

Role of phosphatases in the biosynthesis of chlortetracycline in Streptomyces aureofaciens.

ATP diphosphohydrolase activity and inorganic pyrophosphatase reached two maxima during cultivation of the low- and high-producing variant of Streptomyces aureofaciens under conditions of phosphate limitation, i.e. after 30 and 70 h of cultivation. Increased levels of inorganic phosphate in a medium inhibitory to biosynthesis of chlortetracycline markedly decreased the levels of both enzymes. The ATP diphosphohydrolase activity was detected both in the supernatant and membrane fractions of the cell-free preparation of the mycelium.

Role of ATP-glucokinase and polyphosphate glucokinase in Streptomyces aureofaciens.

The activity of ATP-glucokinase and of polyphosphate glucokinase was examined during growth of the actinomycete Streptomyces aureofaciens 8425 under conditions of intense chlortetracycline (CTC) synthesis. ATP-glucokinase was active in the strain only during the logarithmic phase of culture growth; the activity of polyphosphate glucokinase appears only at the end of the logarithmic phase of growth and rises in parallel with the rate of CTC biosynthesis in the stationary phase. During the rise of activity of polyphosphate glucokinase and of CTC biosynthesis the cells accumulate sugar phosphates, mainly glucose-6-phosphate. It appears that the biosynthesis of CTC in Streptomyces aureofaciens takes place at the expense of glycolysis, using up the high-energy phosphate of high-molecular polyphosphates.

Regulation of biosynthesis of secondary metabolites. I. Biosynthesis of chlortetracycline and tricarboxylic acid cycle activity.

AbstractIn the course of submerged cultivation of low‐production and industrial production strains of Streptomyces aureofaciens, the activity of enzymes of the tricurboxylic acid cycle was studied. The activities of citrate synthase (EC 4.1.3.7), aconitate hydratase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), fumarate hydratase (EC 4.2.1.2), and malate dehydrogenase (EC 1.1.1.37) were estimated spectrophotometrically in cell‐free preparations. In the growth phase, mainly the initial reactions of the cycle were active with both strains. In production‐phase, the activities of enzymes in the low‐production strain were 2–5 × higher than in the production strain. Benzylthioeyanate, at a concentration of 5 × l0−5M, stimulated chlortetracycline production of both strains with accompanying decrease in activity of the enzymes of the tricarboxylic acid cycle. The role of the tricarboxylic acid cycle in control of chlortetracycline biosynthesis is discussed.

Chlorination Inhibitors in Streptomyces aureofaciens

IN a Belgian patent1 granted to Spofa (Czechoslovakia) a number of pyrimidines chlorinated in the 2 position were reported to be active in inhibiting halogenation in the biosynthesis of chlortetracycline resulting instead in the production of high concentrations of tetracycline. It was of interest to us to test such compounds in the Streptomyces aureofaciens BC-41 system we have used before2. The two antibiotics were determined spectro-photometrically, taking advantage of their differential degradation rates in acid and alkaline solution. Compared with tetracycline, chlortetracycline is relatively stable to acid and unstable to base. Selected results were confirmed by paper chromatography. In the course of these trials, in which some thirty halogenated pyrimidines and related heterocycles were tested, very few were found to show activity. The most active compound was found to be 2,4-dichloro-5-methyl pyrimidine which, when added to the medium in a concentration of 10 p.p.m., resulted in the production of 80–95 per cent tetracycline in the final broth, confirming in part the work reported in the Belgian patent1. Closely related pyrimidines were either much less or not at all active for our strain BC-41. This is shown in Table 1.

RELATIONSHIP BETWEEN THE CARBOHYDRATE METABOLISM OF STREPTOMYCES AUREOFACIENS AND THE BIOSYNTHESIS OF CHLORTETRACYCLINE. II. THE EFFECT OF BENZYL THIOCYANATE ON THE RESPIRATION OF WASHED MYCELIUM OF STREPTOMYCES AUREOFACIENS.

RELATIONSHIP BETWEEN THE CARBOHYDRATE METABOLISM OF STREPTOMYCES AUREOFACIENS AND THE BIOSYNTHESIS OF CHLORTETRACYCLINE. II. THE EFFECT OF BENZYL THIOCYANATE ON THE RESPIRATION OF WASHED MYCELIUM OF STREPTOMYCES AUREOFACIENS.

RELATIONSHIP BETWEEN THE CARBOHYDRATE METABOLISM OF STREPTOMYCES AUREOFACIENS AND THE BIOSYNTHESIS OF CHYLORTETRACYCLINE.. III. THE EFFECT OF BENZYL THIOCYANATE ON CARBOHYDRATE METABOLISM OF STREPTOMYCES AUREOFACIENS.

RELATIONSHIP BETWEEN THE CARBOHYDRATE METABOLISM OF STREPTOMYCES AUREOFACIENS AND THE BIOSYNTHESIS OF CHYLORTETRACYCLINE.. III. THE EFFECT OF BENZYL THIOCYANATE ON CARBOHYDRATE METABOLISM OF STREPTOMYCES AUREOFACIENS.

RELATIONSHIP BETWEEN THE CARBOHYDRATE METABOLISM OF STREPTOMYCES AUREOFACIENS AND THE BIOSYNTHESIS OF CHLORTETRACYCLINE. I. THE EFFECT OF INTERRUPTED AERATION, INORGANIC PHOSPHATE AND BENZYL THIOCYANATE ON CHLORTETRACYCLINE BIOSYNTHESIS.

RELATIONSHIP BETWEEN THE CARBOHYDRATE METABOLISM OF STREPTOMYCES AUREOFACIENS AND THE BIOSYNTHESIS OF CHLORTETRACYCLINE. I. THE EFFECT OF INTERRUPTED AERATION, INORGANIC PHOSPHATE AND BENZYL THIOCYANATE ON CHLORTETRACYCLINE BIOSYNTHESIS.

The influence of phosphorus on the development of Streptomyces aureofaciens and on its ability to produce chlortetracycline.

SUMMARY: Parallel physiological and cytological investigations of a chlortetra-cycline-producing culture—Streptomyces aureofaciens, strain 536—indicate some intracellular causes of the dependence of chlortetracycline biosynthesis upon the inorganic phosphorus content of the nutrient medium. Increase of inorganic phosphorus in the fermentation medium led to a formation of large nuclear elements containing much DNA (in corn steep medium) or ribonucleo-proteid complexes in the shape of large volutin granules (corn steep liquor ash medium). As compared with the control, the nuclear substance/cytoplasm ratio in the hyphae is increased. The ability of mycelium overloaded with nuclear elements or volutin granules for chlortetracycline biosynthesis is markedly decreased. The iufluence of phosphorus on the microscopical structure of mycelium and biosynthesis of antibiotic depends on the composition of medium. The poor quality of certain batches of corn steep liquor used for the production of chlortetracycline is largely due to their high content of inorganic phosphorus.

Acetylmethylcarbinol production by actinomycetes

The production of acetylmethylcarbinol by various strains of actinomycetes was established. The biosynthesis of acetylmethylcarbinol depends largely on the composition of the fermentation medium and on the presence of arsenite, which regulates the metabolism of pyruvic acid. In the case ofStreptomyces aureofaciens the stimulating effect of phosphate on the production of acetylmethylcarbinol gives a partial explanation of the well-known negative effect of phosphate on the biosynthesis of chlortetracycline.