Production Of Rifamycin Research Articles

Prediction of Transcription Factors and Their Involvement in Regulating Rifamycin Production in Amycolatopsis mediterranei S699.

Amycolatopsis mediterranei S699 produces rifamycin B and successors of this strain are in use for the industrial production of rifamycin B. Semisynthetic derivatives of rifamycin B are used against Mycobacterium tuberculosis that causes tuberculosis. Although the rifamycin biosynthetic gene cluster was characterized two decades ago, the regulation of rifamycin B biosynthesis in Amycolatopsis mediterranei S699 is poorly understood. In this study, we analysed the genome and proteome of Amycolatopsis mediterranei S699 and identified 1102 transcription factors which comprise about 10% of the total genome. Using interactomics approaches we delineated 30 unique transcription factors directly involved in secondary metabolism that regulate rifamycin B biosynthesis. We also predict the role of RifN as hub in controlling the regulation of other genes involved in rifamycin biosynthesis. RifN is important for maintaining the integrity of the rifamycin-network. Thus, these transcription factor can be exploited to improve rifamycin B production in Amycolatopsis mediterranei S699.

GlnR Dominates Rifamycin Biosynthesis by Activating the rif Cluster Genes Transcription Both Directly and Indirectly in Amycolatopsis mediterranei.

Because of the remarkable efficacy in treating mycobacterial infections, rifamycin and its derivatives are still first-line antimycobacterial drugs. It has been intensely studied to increase rifamycin yield from Amycolatopsis mediterranei, and nitrate is found to provide a stable and remarkable stimulating effect on the rifamycin production, a phenomenon known as “nitrate-stimulating effect (NSE)”. Although the NSE has been widely used for the industrial production of rifamycin, its detailed molecular mechanism remains ill-defined. And our previous study has established that the global nitrogen regulator GlnR may participate in the NSE, but the underlying mechanism is still enigmatic. Here, we demonstrate that GlnR directly controls rifamycin biosynthesis in A. mediterranei and thus plays an essential role in the NSE. Firstly, GlnR specifically binds to the upstream region of rifZ, which leads us to uncover that rifZ has its own promoter. As RifZ is a pathway-specific activator for the whole rif cluster, GlnR indirectly upregulates the whole rif cluster transcription by directly activating the rifZ expression. Secondly, GlnR specifically binds to the upstream region of rifK, which is also characterized to have its own promoter. It is well-known that RifK is a 3-amino-5-hydroxybenzoic acid (AHBA, the starter unit of rifamycin) synthase, thus GlnR can promote the supply of the rifamycin precursor by directly activating the rifK transcription. Notably, GlnR and RifZ independently activate the rifK transcription through binding to different sites in rifK promoter region, which suggests that the cells have a sophisticated regulatory mechanism to control the AHBA biosynthesis. Collectively, this study reveals that GlnR activates the rif cluster transcription in both direct (for rifZ and rifK) and indirect (for the whole rif cluster) manners, which well interprets the phenomenon that the NSE doesn’t occur in the glnR null mutant. Furthermore, this study deepens our understanding about the molecular mechanism of the NSE.

OPTIMIZATION OF RIFAMYCIN B USING NOCARDIA MEDITERRANEA (ATCC 13865) BY FED BATCH MODE IN SHAKE FLASKS

The present study was carried out to evaluate the optimum Production of Rifamycin B by using fed- batch mode in the shake flask at the defenit level of different agitation and aeration rate, amongst different carbohydrate in the form of monoshacride. Amongst the various carbohydrate (Arabinose, Fructose, Galactose, Glucose, Glycerol, Maltose, Mannitol, Sorbitol, and Sucrose) sources maximum, the rifamycin (578 µgml -1 ) with 10% glucose level was found as compared to control which was (138 ± 2.70µgml -1 ).The environmental factors such as incubation temperature. The optimum temperature for maximum antibiotic production (525µgml -1 ) was found to be 29°C, initial pH, aeration and agitation rates were also optimized. The optimum rifamycin yield (950 to 1100µgml -1 ) was obtained in 1.0 L fermenter with working volume of 1.5 L at 28 °C with initial pH of 6.2 to 6.5. The agitation and aeration were 300 rpm and 0.5 LL -1 min -1 respectively. Keywords: Nocardia mediterranea ATCC 13865, Agitator, Carbohydrate, Rifamycin B, Fed-batch, Optimization.

LC-MS-based metabolomics study of marine bacterial secondary metabolite and antibiotic production in Salinispora arenicola.

An LC-MS-based metabolomics approach was used to characterise the variation in secondary metabolite production due to changes in the salt content of the growth media as well as across different growth periods (incubation times). We used metabolomics as a tool to investigate the production of rifamycins (antibiotics) and other secondary metabolites in the obligate marine actinobacterial species Salinispora arenicola, isolated from Great Barrier Reef (GBR) sponges, at two defined salt concentrations and over three different incubation periods. The results indicated that a 14 day incubation period is optimal for the maximum production of rifamycin B, whereas rifamycin S and W achieve their maximum concentration at 29 days. A “chemical profile” link between the days of incubation and the salt concentration of the growth medium was shown to exist and reliably represents a critical point for selection of growth medium and harvest time.

Draft Genome Sequence of Amycolatopsis mediterranei DSM 40773, a Tangible Antibiotic Producer.

Amycolatopsis mediterranei DSM 40773 has been of special interest as successors of this strain are in use for the commercial production of rifamycin B. Here we present the draft genome sequence (~10 Mb) of this strain, which contains 108 contigs, 9,198 genes, and has a G+C content of 71.3%.

Discovering the Recondite Secondary Metabolome Spectrum of Salinispora Species: A Study of Inter-Species Diversity

Patterns of inter-species secondary metabolite production by bacteria can provide valuable information relating to species ecology and evolution. The complex nature of this chemical diversity has previously been probed via directed analyses of a small number of compounds, identified through targeted assays rather than more comprehensive biochemical profiling approaches such as metabolomics. Insights into ecological and evolutionary relationships within bacterial genera can be derived through comparative analysis of broader secondary metabolite patterns, and this can also eventually assist biodiscovery search strategies for new natural products. Here, we investigated the species-level chemical diversity of the two marine actinobacterial species Salinispora arenicola and Salinispora pacifica, isolated from sponges distributed across the Great Barrier Reef (GBR), via their secondary metabolite profiles using LC-MS-based metabolomics. The chemical profiles of these two species were obtained by UHPLC-QToF-MS based metabolic profiling. The resultant data were interrogated using multivariate data analysis methods to compare their (bio)chemical profiles. We found a high level of inter-species diversity in strains from these two bacterial species. We also found rifamycins and saliniketals were produced exclusively by S. arenicola species, as the main secondary metabolites differentiating the two species. Furthermore, the discovery of 57 candidate compounds greatly increases the small number of secondary metabolites previously known to be produced by these species. In addition, we report the production of rifamycin O and W, a key group of ansamycin compounds, in S. arenicola for the first time. Species of the marine actinobacteria harbour a much wider spectrum of secondary metabolites than suspected, and this knowledge may prove a rich field for biodiscovery as well as a database for understanding relationships between speciation, evolution and chemical ecology.

Improved Rifamycin B Production by Nocardia mediterranei MTCC 14 under Solid-State Fermentation through Process Optimization.

Optimization of various production parameters using response surface methodology (RSM) was performed to assess maximum yield of rifamycin B from Nocardia mediterranei MTCC 14. Plackett-Burman design test was applied to determine the significant effects of various production parameters such as glucose, maltose, ribose, galactose, beef extract, peanut meal, ammonium chloride, ammonium sulphate, barbital, pH, and moisture content on production of rifamycin B. Among the eleven variables tested, galactose, ribose, glucose, and pH were found to have significant effect on rifamycin B production. Optimum levels of the significant variables were decided by using a central composite design. The most appropriate condition for production of rifamycin B was found to be a single step production at galactose (8% w/w), ribose (3% w/w), glucose (9% w/w), and pH (7.0). At these optimum production parameters, the maximum yield of rifamycin B obtained experimentally (9.87 g/kgds dry sunflower oil cake) was found to be very close to its predicted value of 10.35 g/kgds dry sunflower oil cake. The mathematical model developed was found to fit greatly with the experimental data of rifamycin B production.

Scaling up for the industrial production of rifamycin B; optimization of the process conditions in bench-scale fermentor

Optimization of fermentation process conditions using a gene amplified variant of Amycolatopsis mediterranei (NCH) was carried out. The use of aeration level 1.5vvm increased the yield by 16.6% (from 13.81 to 16.1g/l) upon controlling the temperature at 28°C. Adjustment of the aeration level at 1.5vvm for 3days then controlling the dissolved oxygen (DO) at 30% saturation further increased the yield to 17.8g/l. The optimum pH was 6.5 for 3days then 7 thereafter when a production yield of 16.1g/l was recorded using an aeration rate of 1.5vvm. Controlling the pH at constant value (6.5 or 7) all through the fermentation process decreased the yield by 5–21%. Controlling the temperature at 30°C for 3days then 28°C thereafter slightly increased the yield by 5% upon using an aeration rate of 1vvm while it decreased upon using an aeration rate of 1.5vvm. Integration of the most optimum conditions increased the production yield by 22% from 13.81 to 17.8g/l.

India’s Latent Foe: Multi Drug Resistant Tuberculosis

Tuberculosis (TB), an infectious bacterial disease, is caused by Mycobacterium tuberculosis. An effective drug for the control of tuberculosis is rifamycin which is clinically used in combination with ethambutol, isoniazid and pyrazinamide. Generally, a six month treatment regime is competent enough to cure tuberculosis, but due to inappropriate treatment and a non committal approach of the patients towards the treatment, there has been a drastic rise in the cases of Multi drug resistant (MDR) mycobacterial strains. Such cases, have increased in drastic numbers all across the globe especially in the developing and under developed countries, where severe lack of awareness has fuelled this ominous a phenomena. Besides this, the incidence of TB and HIV infection seem to go hand in hand, as a suppressed immune system makes the patient more amenable to other bacterial infections. India alone harbours around a million patients coinfected with HIV and M. tuberculosis. While several efforts are being made to tackle this problem, nothing of great significance has been brought out. Rifamycin, the classically used drug for curing TB is produced by an actinobacterium Amycolatopsis mediterranei. However, rifamycin and its semisynthetic derivatives are largely becoming ineffective against the drug resistant forms of mycobacteria. Eversince the commercial production of rifamycin in 1962, it has been a boon to the mankind in its ability to control TB. With immense efforts of the researchers, the yield of this wonder drug from A. mediterranei was increased from a meagre 500 mg/L in 1962 to up to 24 g/L through classical strain improvement methods. However, with the incidence of 73,000 cases of MDR-TB in India, and around a million across the world, there is an urgent need to develop rifamycin analogs or completely new drugs effective against MDR-TB strains. So far only six semisynthetic derivatives of rifamycin B could be produced by modifying the naphthoquinone ring at positions C3 and C4, beyond which, modifications are not possible. Alternative to this, could be the combinatorial approach of deleting, swapping or inactivating modules or domains of rifamycin biosynthetic gene cluster (also called polyketide synthase gene cluster). This approach has been successful with erythromycin polyketide gene cluster, in Saccharospora erythraea, but has not been applied to rifamycin polyketide synthase gene cluster, yet. Thus, in future, emphasis should be laid on this approach to tackle the problem of MDR-TB.

Scaling Up for the Industrial Production of Rifamycin B Fed-Batch Production Mode in Shake Flasks and Bench-Scale Fermentor

The production of rifamycin B using the gene amplified variant of Amycolatopsis mediterranei (NCH) was initially optimized in shake flasks through medium modifications and fed-batch addition of uracil. The yield was increased by 21.7% (from 11.7 to 14.3 g/l) when F2m1 medium was used. The production was further verified and optimized in fedbatch- mode in a laboratory fermentor using F2m3 medium and the optimized conditions (agitation 500 rpm, aeration; 1.5 for 3 days then control DO at 30% thereafter, pH; 6.5 for 3 days then 7 thereafter and control temperature at 28°C). Fed-batching of glucose syrup (5% v/v at day 3) and glucose (1% at days 6 and 8) increased the yield from 17.8 to 20.9 g/l (17.3%) at day 10. A yield of upto 20 g/l was recorded when 0.1% uracil was fed-batched at day 2. Integration of the most optimum conditions for fed-batching glucose syrup, glucose and uracil further increased the yield from 17.8 to 24.8 g/l (39%) in 10 days. The overall optimization of rifamycin B production increased the yield almost 2 folds. Statistical analysis revealed that there is a significant increase in rifamycin B production by using One-Way ANOVA at p<0.05 in all the tested fed-batch addition regimes.

Complete genome sequence of the rifamycin SV-producing Amycolatopsis mediterranei U32 revealed its genetic characteristics in phylogeny and metabolism

Amycolatopsis mediterranei is used for industry-scale production of rifamycin, which plays a vital role in antimycobacterial therapy. As the first sequenced genome of the genus Amycolatopsis, the chromosome of strain U32 comprising 10,236,715 base pairs, is one of the largest prokaryotic genomes ever sequenced so far. Unlike the linear topology found in streptomycetes, this chromosome is circular, particularly similar to that of Saccharopolyspora erythraea and Nocardia farcinica, representing their close relationship in phylogeny and taxonomy. Although the predicted 9,228 protein-coding genes in the A. mediterranei genome shared the greatest number of orthologs with those of S. erythraea, it was unexpectedly followed by Streptomyces coelicolor rather than N. farcinica, indicating the distinct metabolic characteristics evolved via adaptation to diverse ecological niches. Besides a core region analogous to that common in streptomycetes, a novel 'quasi-core' with typical core characteristics is defined within the non-core region, where 21 out of the total 26 gene clusters for secondary metabolite production are located. The rifamycin biosynthesis gene cluster located in the core encodes a cytochrome P450 enzyme essential for the conversion of rifamycin SV to B, revealed by comparing to the highly homologous cluster of the rifamycin B-producing strain S699 and further confirmed by genetic complementation. The genomic information of A. mediterranei demonstrates a metabolic network orchestrated not only for extensive utilization of various carbon sources and inorganic nitrogen compounds but also for effective funneling of metabolic intermediates into the secondary antibiotic synthesis process under the control of a seemingly complex regulatory mechanism.

Corn husk as a novel substrate for the production of rifamycin B by isolated Amycolatopsis sp. RSP 3 under SSF

Rifamycin B production by isolated Amycolatopsis sp. RSP 3 was investigated under solid state fermentation (SSF) using agro-industrial waste materials. Corn husk was the most suitable substrate/support material with 4-fold higher production than wheat bran and corn cobs. A two-level (conventional and statistical) methodology was used to optimize fermentation parameters belong to physiological (pH, temperature and aeration), nutritional (carbon and nitrogen sources) and microbial (inoculum level and incubation time). Conventional optimization significantly improved (450%) the rifamycin B production of which two-third was associated with carbon and nitrogen sources. Starch as carbon source showed negative impact. Statistical optimization of suggested potassium nitrate (at individual level), soya bean meal and barbital (at interactive level) were observed to be the most noticeable variables in the maximization of production. At optimized conditions, inorganic nitrogen source played vital role (>59%) compared to all other factors. Overall, more than 920% increase in rifamycin B production was achieved at optimized environment.

Expression of the Bacterial Hemoglobin Gene from Vitreoscilla stercoraria Increases Rifamycin B Production in Amycolatopsis mediterranei

It is well known that the culture for rifamycin B production by Amycolatopsis mediterranei requires high levels of dissolved oxygen, particularly in industrial processes. In this study, we report the construction of a vector for the expression of the bacterial hemoglobin gene (vhb) from Vitreoscilla stercoraria in a rifamycin B-overproducing strain of A. mediterranei. The effect was evaluated in the presence and absence of barbital. The vhb gene was cloned under the control of the PermE promoter, the Amycolatopsis lactamdurans plasmid pULVK2 origin of replication, the kanamycin-resistant gene (Km), the erythromycin-resistant gene (ermE) for selection, and ColE1. Industrial fermentation conditions were simulated in shake-flask cultures. Under low aeration, the transformed A. mediterranei strain with the vhb gene showed a 13.9% higher production of rifamycin B in a culture with barbital compared with the parental strain, and 29.5% higher production under the same conditions without barbital.

Medium optimization for enhanced production of Rifamycin B by Amycolatopsis mediterranei S699: Combining a full factorial design and a statistical approach

This paper describes improved optimization method that combines the Plackett–Burman design, a full factorial design and the response surface method, which were used to optimize the medium for the production of rifamycin B by Amycolatopsis mediterranei S699. The five nutrients used to amend the medium were preliminary selected from 15 medium components based on the results of the Plackett–Burman design experiments and subsequent screening process using the full factorial design experiments was performed. Glucose, (NH 4) 2SO 4, and corn steep liquor were found to be the best nutrients for the optimization of rifamycin B productions, and central composite design experiments indicated that the optimal concentrations of these nutrients were 63 g/L, 7.9 g/L, and 10 g/L, respectively. A bioreactor culture was used to investigate the production characteristics obtained when Amycolatopsis mediterranei S699 was grown in the determined optimum medium. Although cell growth stopped at 88 h, rifamycin B production continued, reaching its maximum value at the end of the experiment (172 h) after the glucose in the medium was completely consumed. The maximum cell concentration and rifamycin B titer were 8.6 g/L and 1260 mg/L, respectively. The combined optimization method described here is new and effective method for screening carbon and nitrogen sources as well as determining their optimum levels.

English

Optimization of the fermentation process using the gene amplified variant of Amycolatopsis mediterranei, NCH, in the fermentation medium F2 was carried out by the application of fed-batch regime. The addition of 12% glucose alone at day 4 or simultaneously with 0.1% yeast extract at day 2 led to an increase in the yield of rifamycin B by 46% and 57%, respectively. The application of fed-batch regime together with replacing (NH4)2SO4&nbsp;with the better yielding inorganic nitrogen sources NH4NO3&nbsp;or KNO3 (F2m1 and F2m2 media, respectively) increased the production of rifamycin B. The use of F2m1 medium alone or with an additional 12% glucose added at day 4 increased the yield by 53% and 120%, respectively. However, further addition of 0.1% yeast extract led to an increase in the yield by only 63%. The addition of 3% soytone or 0.05% NH4NO3&nbsp;to F2m1 at day 3 increased the yield by 72% and 61%, respectively, compared to productivity in F2 medium. The use of F2m2 medium increased the yield by 50%. The addition of 12% glucose at day 4 or of 0.1% yeast extract at day 2 to F2m2 medium led to an increase in the yield by 119 and 55%, respectively, compared to F2 medium. However, when both 12% glucose and 0.1% yeast extract were added at similar scheduled times only 64% increase in the yield occurred. By applying the three most effective optimization regimes determined using variant NCH with F2m2 medium on a standard rifamycin B producing strain, Nocardia mediterranei ATCC 21789, a similar pattern of increase in the antibiotic yield was observed. Thus, the use of F2m2 instead of F2 medium either alone or with an additional 12% glucose added at day 4 increased the yield by 36 and 75%, respectively, whereas the addition of 0.1% yeast extract to F2 medium increased the yield by 15%. In conclusion, the application of fed-batch technique with the optimum modifications of the medium constituents increased rifamycin B production by variant NCH to a maximum of 17.17 g/l compared to a yield of 5.3 g/l by the tested standard strain under the same conditions. The increase in rifamycin B production using the standard strain confirms the usefulness of the tested medium modifications in the improvement of rifamycin B production and its possible application in fermentations using other rifamycin B producer strains and also shows the superiority of variant NCH as a producer, when compared to the standard strain. &nbsp; Key words:&nbsp;Rifamycin B, fermentation, biotechnology, Amycolatopsis mediterranei, optimization, fed-batch and physiological factors.

English

A systematic approach to process optimization for production of rifamycin B was applied to a strain of Amycolatopsis mediterranei . Examination of the growth revealed 6 different morphologically distinct colonies on Bennett's agar medium. Rifamycin B production in shake flasks by the six different colony types ranged between 0.5 and 1.2 g/l. There was a clear correlation between the colony morphology and rifamycin B productivity. The highest yield of rifamycin B (1.03-1.2 g/l) was obtained by using the orange-red colored colonies, rosette shaped, devoid of hollow center and 2-3 mm in diameter. Variability in colony morphology, however, remained and the appropriate colonies had to be picked up for preparing the inoculum of each experiment. Addition of yeast extract to the fermentation medium at different times increased rifamycin B production. The highest antibiotic production was obtained upon the addition of 0.1% yeast extract after 2 days of incubation, where the yield increased from 1.15 to 1.95 g/l (70%). The use of 1.8% KNO 3 in the fermentation medium, instead of 0.96% (NH 4 ) 2 SO 4 , markedly increased rifamycin B production from 1.15 to 2.92 g/l (154%). It was also observed, upon microscopical examination, that KNO 3 decreased branching and fragmentation of the mycelia in the fermentation medium. Keywords: Rifamycin B; fermentation; biotechnology; Amycolatopsis mediterranei; strain selection. African Journal of Biotechnology Vol.3(5) 2004: 266-272

Biochemical Mechanism of the Effect of Barbital on Rifamycin B Biosynthesis by Amycolatopsis mediterranei (M18 Strain)

It is well known that 5,5-diethylbarbituric acid (barbital) in the culture medium can stimulate the production of rifamycin B by Amycolatopsis mediterranei, particularly in industrial processes. However, the mechanism by which barbital exerts this effect is unknown. Results in this work show that the barbital effect is only evident under low aeration conditions (50-ml microfermentors with 7 ml of medium, 0.08 l/h air flow). Under these conditions, cultures with barbital showed similar CO2 production (in relation to a control without barbital), but higher oxygen uptake indicated that the extra O2 consumed was used in the increased rifamycin biosynthesis. Moreover, using a resting cell system where no antibiotic is produced, it was possible to show that barbital inhibits the respiratory chain, since O2 uptake decreased by 30%. Finally, we present biochemical results that suggest that a cytochrome P450-type monoxygenase, which can use atmospheric oxygen, is induced by barbital in an industrial-type strain of A. mediterranei.

Biochemical mechanism of the effect of barbital on rifamycin B biosynthesis by Amycolatopsis mediterranei (M18 strain)

It is well known that 5,5-diethylbarbituric acid (barbital) in the culture medium can stimulate the production of rifamycin B by Amycolatopsis mediterranei, particularly in industrial processes. However, the mechanism by which barbital exerts this effect is unknown. Results in this work show that the barbital effect is only evident under low aeration conditions (50-ml microfermentors with 7 ml of medium, 0.08 l/h air flow). Under these conditions, cultures with barbital showed similar CO 2 production (in relation to a control without barbital), but higher oxygen uptake indicated that the extra O 2 consumed was used in the increased rifamycin biosynthesis. Moreover, using a resting cell system where no antibiotic is produced, it was possible to show that barbital inhibits the respiratory chain, since O 2 uptake decreased by 30%. Finally, we present biochemical results that suggest that a cytochrome P450-type monoxygenase, which can use atmospheric oxygen, is induced by barbital in an industrial-type strain of A. mediterranei.

Thioesterases and the premature termination of polyketide chain elongation in rifamycin B biosynthesis by Amycolatopsis mediterranei S699.

The role of two thioesterase genes in the premature release of polyketide synthase intermediates during rifamycin biosynthesis in the Amycolatopsis mediterranei S699 strain was investigated. Creation of an in-frame deletion in the rifR gene led to a 30 approximately 60% decrease in the production of both rifamycin B by the S699 strain or a series of tetra- to decaketide shunt products of polyketide chain assembly by the rifF strain. Since a similar percentage decrease was seen in both genetic backgrounds, we conclude that the RifR thioesterase 2 is not involved in premature release of the carbon chain assembly intermediates. Similarly, fusion of the Saccharopolyspora erythraea DEBS3 thioesterase I domain to the C-terminus of the RifE PKS subunit did not result in a noticeable increase in the amount of the undecaketide intermediate formed nor in the amounts of the tetra- to decaketide shunt products. Hence, premature release of the carbon chain assembly intermediates is an unusual property of the Rif PKS itself.

Production of Rifamycin using Amycolatopsis mediterranei (MTCC14)

Production of Rifamycin with Amycolatopsis mediterranei (MTCC14) was studied using carbon and locally available cheaper nitrogen sources. A. Mediterranei gave initial yield of 650 mg/l with standard fermentation medium. This was improved to 1400 mg/l by using various Carbon, Nitrogen sources and optimizing various cultural conditions. Glucose, Ammonium sulphate and combination of soya bean and pea nut meals were found to induce more Rifamycin production in 7 days with a pH of 7.2, temperature 28 °C.