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Substandard and falsified medicines have severe public health and socioeconomic effects, especially in low- and middle-income countries. The WHO has emphasized the need for reliable estimates of the prevalence of such medicines to efficiently respond to this problem. In the present study, we used 601 medicine samples collected in Cameroon, the DR Congo, and Malawi to assess the rates of substandard and falsified medicines based on different criteria. Based on the specifications of the U.S. Pharmacopoeia for the amount of the active pharmaceutical ingredients, the rate of out-of-specification medicines was 9.3%. By contrast, this rate ranged from 3.3% up to 35.0% if the tolerance limits of other pharmacopoeias or recently published medicine quality studies were used. This shows an urgent need for harmonization. Principal methods to assess the rate of falsified medicines are packaging analysis, chemical analysis, and authenticity inquiries. In the present study, we carried out an authenticity inquiry for the aforementioned medicine samples, contacting 126 manufacturers and 42 distributors. Response rates were higher for samples stated to be manufactured in Asia (52.4%) or Europe (53.8%) than for samples manufactured in Africa (27.4%; P < 0.001). One sample had been identified as falsified by packaging analysis by the local researchers and two additional ones by chemical analysis. Notably, seven additional falsified samples were identified by the authenticity inquiries. The total rate of falsified medicines resulted as 1.7%. Considerations are discussed for assessing the rates of “substandard” and “falsified” medicines in future medicine quality studies.

Substandard and falsified medicines present a serious threat to public health. Simple, low-cost screening tools are important in the identification of such products in low- and middle-income countries. In the present study, a smartphone-based imaging software was developed for the quantification of thin-layer chromatographic (TLC) analyses. A performance evaluation of this tool in the TLC analysis of 14 active pharmaceutical ingredients according to the procedures of the Global Pharma Health Fund (GPHF) Minilab was carried out, following international guidelines and assessing accuracy, repeatability, intermediate precision, specificity, linearity, range and robustness of the method. Relative standard deviations of 2.79% and 4.46% between individual measurements were observed in the assessments of repeatability and intermediate precision, respectively. Small deliberate variations of the conditions hardly affected the results. A locally producible wooden box was designed which ensures TLC photography under standardized conditions and shielding from ambient light. Photography and image analysis were carried out with a low-cost Android-based smartphone. The app allows to share TLC photos and quantification results using messaging apps, e-mail, cable or Bluetooth connections, or to upload them to a cloud. The app is available free of charge as General Public License (GPL) open-source software, and interested individuals or organizations are welcome to use and/or to further improve this software.

In this paper, we argue that understanding and addressing the problem of poor-quality medical products requires a more interdisciplinary approach than has been evident to date. While prospective studies based on rigorous standardized methodologies are the gold standard for measuring the prevalence of poor-quality medical products and understanding their distribution nationally and internationally, they should be complemented by social science research to unpack the complex set of social, economic, and governance factors that underlie these patterns. In the following sections, we discuss specific examples of prospective quality surveys and of social science studies, highlighting the value of cross-sector partnerships in driving high-quality, policy-relevant research in this area.

Oxytocin is used for the prevention and treatment of postpartum hemorrhage, the leading cause of maternal mortality in low- and middle-income countries. Because of the high instability of oxytocin, most products are labeled for storage at 2–8°C. Some other products are on the market which are labeled for non-refrigerated storage, but independent evaluations of their stability hardly exist. In the present study, seven brands (nine batches) of oxytocin were purchased from wholesalers and medical stores in Malawi and Rwanda and investigated by accelerated stability testing according to the ICH/WHO guidelines. Two oxytocin brands approved by a stringent regulatory authority (SRA) or by the WHO Prequalification of Medicines program and purchased in Europe were used as comparison. All investigated brands which were either produced in countries with SRAs, or were WHO-prequalified products, were labeled for storage at 2–8°C, and all of them passed stability testing with very good results. Even exposure to 25°C or 30°C for several months hardly affected their oxytocin content. However, two other investigated brands were labeled for non-refrigerated storage, and both of them had been produced in countries without SRAs. These two preparations showed not higher but lower stability than the brands labeled for storage at 2–8°C, and, for both of them, noncompliance with pharmacopoeial specifications was found after accelerated stability testing. At 40°C, and in forced degradation studies at 80°C, chlorobutanol showed a remarkable stabilizing effect on oxytocin, which may deserve further investigation. The results of the present study support the policy “Buy Quality Oxytocin, Keep It Cool.”

Falsified and substandard medicines may undermine the progress toward the Sustainable Development Goals. The present study investigated the quality of 13 essential medicines in Cameroon and the Democratic Republic of Congo (DR Congo). Five hundred six medicine samples were collected from the government and faith-based health facilities, private pharmacies, and informal vendors (total 60 facilities). Collected samples were analyzed according to the U.S. Pharmacopeia (USP) for identity, content, and dissolution of their active pharmaceutical ingredients (APIs) and for uniformity of dosage units. Three samples (0.6%) were identified as falsified. Overall, 8.5% of the samples failed USP specifications for the content of the API and 11.7% failed dissolution testing. Medicines from informal vendors showed a higher out-of-specification rate (28.2%) than other types of drug outlets (12.3%; P < 0.0001). All three falsified medicines had been sold by informal vendors. The failure rate of medicines stated to be produced in Europe (5.1%) was lower than that for medicines from Asia (17.7%; P = 0.0049) and Africa (22.2%; P = 0.0042). Medicines against noncommunicable diseases showed a higher failure rate than antibiotics (25.3% versus 12.1%; P = 0.0004). Four hundred fifty-one of the samples were analyzed in Cameroon and the DR Congo with the Global Pharma Health Fund Minilab (thin-layer chromatography and disintegration testing). The three falsified medicines were readily detected in Minilab analysis. However, substandard samples were detected with low sensitivity. A well-enforced ban of medicine sales by informal vendors and increased attention to supplier qualification in the procurement process may reduce the prevalence of substandard and falsified medicines.

Reports that chloroquine and hydroxychloroquine may be effective against COVID-19 have received worldwide attention, increasing the risk of the introduction of falsified versions of these medicines. Five different types of falsified chloroquine tablets were discovered between March 31, 2020 and April 4, 2020, in Cameroon and the Democratic Republic of Congo by locally conducted thin layer chromatographic analysis. Subsequent investigation by liquid chromatography and mass spectrometry in Germany proved the absence of detectable amounts of chloroquine and the presence of undeclared active pharmaceutical ingredients, that is, paracetamol and metronidazole, in four of the samples. The fifth sample contained chloroquine, but only 22% of the declared amount. Such products represent a serious risk to patients. Their occurrence exemplifies that once medicines or vaccines against COVID-19 may be developed, falsified products will enter the market immediately, especially in low- and middle-income countries (LMICs). Timely preparations for the detection of such products are required, including the establishment of appropriate screening technologies in LMICs.

BackgroundAccess to affordable and good quality medicines is a key to meeting Sustainable Development Goal No. 3 by the year 2030. Prices, availability and affordability of essential medicines have been studied in many developing countries, but no such information has been published about Rwanda yet. This study aimed at providing data on prices, availability and affordability of medicines in different health facilities of Rwanda.MethodsA survey was carried out on availability, prices and affordability of 18 medicines in Kigali City and five districts of Rwanda. 44 health facilities were surveyed, including public and faith-based hospitals, public and faith-based health centers and private pharmacies. The standardized methodology developed by WHO and Health Action International (HAI) was used to collect and analyze the data.FindingsPrices for generic medicines in public and faith-based health facilities were remarkably low, with median price ratios (MPRs) of 1.0 in comparison to the international procurement prices published by Management Sciences for Health. In private pharmacies, prices were twice as high (MPR = 1.99 for generics). Availability of medicines fell short of the of 80% target set by WHO, but was better than reported from many other developing countries. Availability of medicines was highest in the private sector (71.3%) and slightly lower in the faith-based (62.8%) and public (59.6%) sectors. The government procurement agency was found to work efficiently, achieving prices 30% below the international procurement price given in the International Medical Product Price Guide. Affordability of medicines was better in the public and faith-based sectors than in the private sector.ConclusionIn Rwanda, medicines are affordable but poorly available in both the public and the faith-based sectors. Further improvements of the availability of medicines in the public and the faith-based health facilities represent the most important key to increase accessibility and affordability of medicines in Rwanda.

Access to safe, effective and affordable medicines of good quality is included into the Sustainable Development Goals of the United Nations. Furthermore, WHO has developed a Global Action Plan with the aim to raise access to essential medicines against non-communicable diseases (NCDs) to 80%, and to improve their affordability. In order to contribute to the monitoring of progress towards these goals, the present study investigated the availability and affordability of seven antibiotics and six medicines against non-communicable diseases in the northeast of the Democratic Republic of Congo and the west of the Republic of Cameroon. Data on availability and prices of these medicines were collected in 60 different sites (34 in the DR Congo, 26 in Cameroon), including government health facilities, church health facilities, private pharmacies and informal vendors, as part of a study on medicine quality. The data were analyzed using a standardized procedure developed by WHO and Health Action International (HAI). Average availability of the investigated antibiotics ranged from 62% to 98% in the different types of facilities in both countries, including the informal vendors. Average availability for medicines against NCDs in the different types of facilities showed a higher variation in both countries, ranging from 11% up to 87%. The average availability of medicines against NCDs in government health facilities was only 33% in Cameroon, and as low as 11% in the DR Congo. In contrast, availability of medicines against NCDs in church health facilities in Cameroon was 70%, not far from the 80% availability goal set by WHO. Medicine prices were clearly higher in Cameroon than in the DR Congo, with median price ratios to an international reference price of 5.69 and 2.17, respectively (p < 0.001). In relation to the daily minimum wages in both countries, treatment courses with five of the seven investigated antibiotics could be considered as affordable, while in each country only one out of the five investigated medicines against NCDs could be considered as affordable. Especially generic medicines provided by government and church health facilities showed reasonable affordability in most cases, while originator medicines offered by private pharmacies were clearly unaffordable to a major part of the population. Despite some encouraging findings on the availability of antibiotics in both countries, the availability and affordability of medicines against NCDs urgently requires further improvements.

BackgroundQuality-assured medicines are a principal means of achieving health-related Sustainable Development Goals. An example of quality assurance/quality control (QA/QC) procedures in drug procurement is provided by the operation of the Global Drug Facility (GDF) of the Stop TB Partnership, the largest provider of tuberculosis (TB) medicines to the public sector worldwide.MethodsProcedures and results of GDF's quality assurance/quality control (QA/QC) over the five-year period 2013-2017 were analysed retrospectively. 13,999 batches of 51 different medicines had been procured and reviewed within this period. 1,388 of these batches had been analysed in the laboratories of GDF's external quality control agent (QCA). Assay and dissolution results determined by the manufacturers and by the external QCA were compared using Bland-Altman analysis.ResultsAll investigated batches of medicines were in specifications at the time of shipment. The costs for QA/QC were 0.8% of purchase costs. The median time required for chemical analysis was 10 working days. Comparison of the medicine quality analysis results showed for the poorly water-soluble compound rifampicin a bias of 4.4%, with the manufacturers reporting higher values than the external QCA, most likely due to different methods employed for the analysis. Overall 95% limits of agreement (LOAs) were -6.7 to +8.0% for assay, and -10.1 to +11.8% for dissolution. In case of kanamycin injections, 95% LOAs for assay reached -14.5 to +13.2%, largely attributable to samples from one manufacturer who had used a microbiological assay while the external QCA had used an HPLC assay.ConclusionsGDF's procedures represent a useful benchmark when evaluating QA/QC procedures of other medicine procurement operations. Inter-laboratory comparison using Bland-Altman plots allows to investigate bias and variability in medicine quality control and should be considered as a routine procedure by drug procurement agencies, to identify priorities for further improvements.

Substandard and falsified antimalarial and antibiotic medicines represent a serious problem for public health, especially in low- and middle-income countries. However, information on the prevalence of poor-quality medicines is limited. In the present study, samples of six antimalarial and six antibiotic medicines were collected from 31 health facilities and drug outlets in southern Malawi. Random sampling was used in the selection of health facilities. For sample collection, an overt approach was used in licensed facilities, and a mystery shopper approach in nonlicensed outlets. One hundred and fifty-five samples were analyzed by visual and physical examination and by rapid prescreening tests, that is, disintegration testing and thin-layer chromatography using the GPHF-Minilab. Fifty-six of the samples were analyzed according to pharmacopeial monographs in a World Health Organization-prequalified quality control laboratory. Seven out-of-specification medicines were identified. One sample was classified as falsified, lacking the declared active ingredients, and containing other active ingredients instead. Three samples were classified as substandard with extreme deviations from the pharmacopeial standards, and three further samples as substandard with nonextreme deviations. Of the substandard medicines, three failed in dissolution testing, two in the assay for the content of the active pharmaceutical ingredient, and one failed in both dissolution testing and assay. Six of the seven out-of-specification medicines were from private facilities. Only one out-of-specification medicine was found within the samples from public and faith-based health facilities. Although the observed presence of substandard and falsified medicines in Malawi requires action, their low prevalence in public and faith-based health facilities is encouraging.

Background Postpartum haemorrhage (PPH) is the leading cause of maternal mortality in low- and middle-income countries (LMICs). Oxytocin and misoprostol are used for the prevention and treatment of PPH. However, both medicines are chemically unstable and sensitive to environmental conditions. Previous studies reported a high prevalence of substandard oxytocin and misoprostol preparations in LMICs. Methods In randomly selected health facilities of four districts of Malawi, the availability of oxytocin and misoprostol was determined, and the knowledge of health workers on storage requirements and use of oxytocics was assessed. Temperature loggers were used to record the storage temperature of oxytocics. Samples of oxytocin injections and misoprostol tablets were collected from the health facilities and from wholesalers. Oxytocin samples were analysed for identity, assay (= quantity of oxytocin) and for pH value according to United States Pharmacopeia 40. Misoprostol samples were analysed for identity, assay, dissolution and related substances according to the International Pharmacopeia 2017. Results All visited hospitals and health centers had oxytocin available. At non-refrigerated storage sites, the recorded mean kinetic temperature exceeded the oxytocic’s storage temperature stated on the labels in 42% of the sites. At refrigerated storage sites, the required temperature of 2–8 °C was exceeded in 33% of the sites. Out of 65 oxytocin samples, 7 (11%) showed moderate deviations from specification, containing 82.2–86.8% of the declared amount of oxytocin. Out of 30 misoprostol samples, 5 (17%) showed extreme deviations, containing only 12.7–30.2% of the declared amount. The extremely substandard misoprostol was reported to the national authorities and to WHO, leading to an immediate recall of the respective brand in Malawi. The UK-based distributor of this brand closed its business shortly thereafter. Conclusion Availability of oxytocin was excellent in Malawi, and its quality was better than reported in previous studies in other LMICs. However, storage conditions at the health facilities often did not meet the requirements. Extremely substandard misoprostol tablets were found, representing a serious risk to maternal health. This shows the need for continued efforts for quality assurance in medicine procurement and registration, as well as for post-marketing surveillance.

This paper describes the artificial induction of secondary metabolite production in transgenic plant cell cultures using a recombinant, inducible plant promoter. The bacterial gene ubiC from Escherichia coli encodes the enzyme chorismate pyruvate lyase (CPL) which catalyses the conversion of chorismate to 4-hydroxybenzoate (4HB). This gene was fused to the tetracycline-inducible plant promoter Triple-Op. After transformation into Nicotiana tabacum W38 TET, transgenic cell cultures were established. Addition of chlorotetracycline to the medium led to specific induction of CPL activity. The optimal chlorotetracycline concentration was approximately 2 mg/l medium. Three to 5 h after induction, the ubiC mRNA concentration reached a maximum, while highest specific CPL activity was detected after 8 days. The artificial secondary metabolite 4HB was converted to glucosides, and their accumulation reached maximum levels after 5 weeks of subculture. The induction was reversible.

Substandard and falsified medicines represent a serious threat for public health and patient safety. Especially in low and middle-income countries, the prevalence of substandard and falsified medicines is reportedly high. However, reliable information on the prevalence of poor-quality medicines is scarce. In this study, 12 essential medicines, including antibiotics, antidiabetics, cardiac drugs and antiasthmatic drugs, were collected from six informal vendors and six licensed pharmacies in the southern part of Togo (regions Maritime and Plateaux). A mystery shopper approach was used in both types of outlets. In total, 64 samples were collected from licensed pharmacies and 30 from informal vendors. Both availability of medicines and prices of medicines were higher in licensed pharmacies than in informal vendors. 92 medicine samples were analyzed by visual examination, followed by chemical analysis for the content and for the dissolution of the active pharmaceutical ingredients according to the respective monographs of the United States Pharmacopoeia. 7 samples (8%) did not comply with the pharmacopoeial specifications, and one sample (1%) showed even extreme deviations. None of the samples was obviously falsified. However, one sample of amoxicillin capsules contained only 47% of the declared content of the active pharmaceutical ingredient, indicating that it may represent amoxicillin capsules 250 mg, rather than 500mg as declared on the label. Medicines stated to originate from Asia (i.e. mainly from India and China) showed a significantly higher proportion (24%) of non-compliant samples than those from Africa and Europe (4%, p = 0.007). High failure rates were observed in medicines both from informal vendors (13%) and from licensed pharmacies (5%), but the difference between both groups was not statistically significant (p = 0.152). The observed high prevalence of substandard medicines requires action from regulatory authorities and health care providers. Testing of selected samples for related substances indicated that inappropriate transport and storage conditions may have been an important cause for substandard quality.

BACKGROUND:Substandard and falsified medical products present a serious threat to public health, especially in low- and middle-income countries. Their identification using pharmacopeial analysis is expensive and requires sophisticated equipment and highly trained personnel. Simple, low-cost technologies are required in addition to full pharmacopeial analysis in order to accomplish widespread routine surveillance for poor-quality medicines in low- and middle-income countries. METHODS:Ten faith-based drug supply organizations in seven countries of Africa and Asia were each equipped with a Minilab of the Global Pharma Health Fund (GPHF, Frankfurt, Germany), suitable for the analysis of about 85 different essential medicines by thin-layer chromatography. Each organization was asked to collect approximately 100 medicine samples from private local medicine outlets, especially from the informal sector. The medicine samples were tested locally according to the Minilab protocols. Medicines which failed Minilab testing were subjected to confirmatory analysis in a WHO-prequalified medicine quality control laboratory in Kenya. RESULTS:Out of 869 medicine samples, 21 were confirmed to be substandard or falsified medical products. Twelve did not contain the stated active pharmaceutical ingredient (API), six contained insufficient amounts of the API, and three showed insufficient dissolution of the API. The highest proportion of substandard and falsified medicines was found in Cameroon (7.1%), followed by the Democratic Republic of Congo (2.7%) and Nigeria (1.1%). Antimalarial medicines were most frequently found to be substandard or falsified (9.5% of all antimalarials). Thin-layer chromatography according to the Minilab protocols was found to be specific and reproducible in the identification of medicines which did not contain the stated API. Since only samples which failed Minilab testing were subjected to confirmatory testing using pharmacopeial methods, this study did not assess the sensitivity of the Minilab methodology in the detection of substandard medicines, and may underestimate the prevalence of poor-quality medicines. CONCLUSIONS:Surveillance for poor-quality medicines can be carried out by local organizations in low- and middle-income countries using a simple, low-cost technology. Such surveillance can identify an important subgroup of the circulating substandard and falsified medical products and can help to prevent them from causing harm in patients. A collaboration of the national drug regulatory authorities with faith-based organizations and other NGOs may therefore represent a promising strategy towards the Sustainable Development Goal of "ensuring access to quality medicines".

Streptomyces sp. CNQ-509 produces the rare O-prenylated phenazines marinophenazines A and B. To identify the enzyme catalyzing the O-prenyl transfer in marinophenazine biosynthesis, we sequenced the genome of S. sp. CNQ-509. This led to the identification of two genomic loci harboring putative phenazine biosynthesis genes. The first locus contains orthologues for all seven genes involved in phenazine-1-carboxylic acid biosynthesis in pseudomonads. The second locus contains two known phenazine biosynthesis genes and a putative prenyltransferase gene termed cnqPT1. cnqPT1 codes for a membrane protein with sequence similarity to the prenyltransferase UbiA of ubiquinone biosynthesis. The enzyme CnqPT1 was identified as a 1,6-dihydroxyphenazine geranyltransferase, which catalyzes the C-O bond formation between C-1 of the geranyl moiety and O-6 of the phenazine scaffold. CnqPT1 is the first example of a prenyltransferase catalyzing O-prenyl transfer to a phenazine.

Coumermycin A1 is an aminocoumarin antibiotic produced by Streptomyces rishiriensis. It exhibits potent antibacterial and anticancer activity. The coumermycin A1 molecule contains two terminal 5-methyl-pyrrole-2-carboxylic acid moieties and one central 3-methylpyrrole-2,4-dicarboxylic acid moiety (CPM). While the biosynthesis of the terminal moieties has been elucidated in detail, the pathway leading to the CPM remains poorly understood. In this work, the minimal set of genes required for the generation of the CPM scaffold was identified. It comprises the five genes couR1, couR2a, couR2b, couR3, and couR4 which are grouped together in a contiguous 4.7 kb region within the coumermycin A1 biosynthetic gene cluster. The DNA fragment containing these genes was cloned into an expression plasmid and heterologously expressed in Streptomyces coelicolor M1146. Thereupon, the formation of CPM could be shown by HPLC and by HPLC-MS/MS, in comparison to an authentic CPM standard. This proves that the genes couR1–couR4 are sufficient to direct the biosynthesis of CPM, and that the adjacent genes couR5 and couR6 are not required for this pathway. The enzyme CouR3 was expressed in Escherichia coli and purified to near homogeneity. The protein exhibited an ATPase activity similar to that reported for its close ortholog, the threonine kinase PduX. However, we could not show a threonine kinase activity of CouR3, and; therefore, the substrate of CouR3 in CPM biosynthesis is still unknown and may be different from threonine.

The biosynthesis of nonribosomally formed peptides (NRPs), which include important antibiotics such as vancomycin, requires the activation of amino acids through adenylate formation. The biosynthetic gene clusters of NRPs frequently contain genes for small, so-called MbtH-like proteins. Recently, it was discovered that these MbtH-like proteins are required for some of the adenylation reactions in NRP biosynthesis, but the mechanism of their interaction with the adenylating enzymes has remained unknown. In this study, we determined the structure of SlgN1, a 3-methylaspartate-adenylating enzyme involved in the biosynthesis of the hybrid polyketide/NRP antibiotic streptolydigin. SlgN1 contains an MbtH-like domain at its N terminus, and our analysis defines the parameters required for an interaction between MbtH-like domains and an adenylating enzyme. Highly conserved tryptophan residues of the MbtH-like domain critically contribute to this interaction. Trp-25 and Trp-35 form a cleft on the surface of the MbtH-like domain, which accommodates the alanine side chain of Ala-433 of the adenylating domain. Mutation of Ala-433 to glutamate abolished the activity of SlgN1. Mutation of Ser-23 of the MbtH-like domain to tyrosine resulted in strongly reduced activity. However, the activity of this S23Y mutant could be completely restored by addition of the intact MbtH-like protein CloY from another organism. This suggests that the interface found in the structure of SlgN1 is the genuine interface between MbtH-like proteins and adenylating enzymes.

SummaryAn artificial operon was synthesized, consisting of the genes for chorismate pyruvate-lyase of E. coli and for 4-hydroxybenzoate 3-hydroxylase of Corynebacterium cyclohexanicum. This operon, directing the biosynthesis of 3,4-dihdroxybenzoate, was expressed in the heterologous expression host Streptomyces coelicolor M512, together with a modified biosynthetic gene cluster for the aminocoumarin antibiotic clorobiocin. The resulting strain produced a clorobiocin derivative containing a 3,4-dihdroxybenzoyl moiety. Its structure was confirmed by MS and NMR analysis, and it was found to be a potent inhibitor of the gyrases from Escherichia coli and Staphylococcus aureus. Bioassays against different E. coli mutants suggested that this compound is actively imported by catechol siderophore transporters in the cell envelope. This study provides an example of the structure of a natural product that can be rationally modified by synthetic biology.

The development of reliable analytic methods, capable of separating mixtures of secondary metabolites as well as providing structural information, is essential for the investigation of secondary metabolites, e.g. from Streptomyces. Here we report a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using a triple quadrupole mass analyzer for the structural elucidation of caprazamycins and liposidomycins from culture extracts of the wild-type producer strains. Comparison of the fragmentation patterns in positive as well as in negative ionization mode revealed several characteristic product ions used for identification of six new caprazamycins. Furthermore, a chromatographic method for the purification of nucleosides from cell cultures using a boronic acid gel was adapted for the partial purification of the culture extracts. Copyright © 2011 John Wiley & Sons, Ltd.

Streptomyces cinnamonensis DSM 1042 produces two types of isoprenoid secondary metabolites: the prenylated naphthalene derivative furanonaphthoquinone I (FNQ I), and isoprenylated phenazines which are termed endophenazines. Previously, a 55 kb gene cluster was identified which contained genes for both FNQ I and endophenazine biosynthesis. However, several genes required for the biosynthesis of these metabolites were not present in this cluster. We now re-screened the cosmid library for genes of the mevalonate pathway and identified a separate genomic locus which contains the previously missing genes. This locus (15 kb) comprised orthologues of four phenazine biosynthesis genes known from Pseudomonas strains. Furthermore, the locus contained a putative operon of six genes of the mevalonate pathway, as well as the gene epzP which showed sequence similarity to a recently discovered class of prenyltransferases. Inactivation and complementation experiments proved the involvement of epzP in the prenylation reaction in endophenazine biosynthesis. This newly identified genomic locus is more than 40 kb distant from the previously identified cluster. The protein EpzP was expressed in Escherichia coli in form of a his-tag fusion protein and purified. The enzyme catalysed the prenylation of 5,10-dihydrophenazine-1-carboxylic acid (dihydro-PCA) using dimethylallyl diphosphate (DMAPP) as isoprenoid substrate. K(m) values were determined as 108 µM for dihydro-PCA and 25 µM for DMAPP.

Highly reproducible production values of the aminocoumarin antibiotic novobiocin were achieved by cultivation of a heterologous Streptomyces producer strain in commercially available square deepwell plates consisting of 24 wells of 3 ml culture volume each. Between parallel cultivation batches in the deepwell plates, novobiocin accumulation showed standard deviations of 4–9%, compared to 39% in baffled Erlenmeyer flasks. Mycelia used as inoculum could be frozen in the presence of 20% peptone and stored at − 70 °C, allowing repeated cultivations from the same batch of inoculum over extended periods of time. Originally, novobiocin titers in the deepwell plate (5–12 mg l− 1) were lower than in Erlenmeyer flasks (24 mg l− 1). Optimization of the inoculation procedure as well as addition of a siloxylated ethylene oxide/propylene oxide copolymer, acting as oxygen carrier, to the production medium increased novobiocin production to 54 mg l− 1. The additional overexpression of the pathway-specific positive regulator gene novG increased novobiocin production to 163 mg l− 1. Harvesting the precultures in a defined section of growth phase greatly reduced variability between different batches of inoculum. The use of deepwell plates may considerably reduce the workload and cost of investigations of antibiotic biosynthesis in streptomycetes and other microorganisms due to the high reproducibility and the low requirement for shaker space and culture medium.

The aminocoumarin antibiotic novobiocin is a gyrase inhibitor formed by a Streptomyces strain. The biosynthetic gene cluster of novobiocin spans 23.4 kb and contains 20 coding sequences, among them the two regulatory genes novE and novG. We investigated the location of transcriptional promoters within this cluster by insertion of transcriptional terminator cassettes and RT-PCR analysis of the resulting mutants. The cluster was found to contain eight DNA regions with promoter activity. The regulatory protein NovG binds to a previously identified binding site within the promoter region located upstream of novH, but apparently not to any of the other seven promoters. Quantitative real-time PCR was used to compare the number of transcripts in a strain carrying an intact novobiocin cluster with strains carrying mutated clusters. Both in-frame deletion of the regulatory gene novG and insertion of a terminator cassette into the biosynthetic gene novH led to a strong reduction of the number of transcripts of the genes located between novH and novW. This suggested that these 16 biosynthetic genes form a single operon. Three internal promoters are located within this operon but appear to be of minor importance, if any, under our experimental conditions. Transcription of novG was found to depend on the presence of NovE, suggesting that the two regulatory genes, novE and novG, act in a cascade-like mechanism. The resistance gene gyrBR , encoding an aminocoumarin-resistant gyrase B subunit, may initially be co-transcribed with the genes from novH to novW. However, when the gyrase inhibitor novobiocin accumulates in the cultures, gyrBR is transcribed from its own promoter. Previous work has suggested that this promoter is controlled by the superhelical density of chromosomal DNA.

The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A1 are inhibitors of gyrase and highly effective antibacterial agents. Their biosynthetic gene clusters have been cloned from the respective Streptomyces producer strains, and the function of nearly all genes contained therein has been elucidated by genetic and biochemical methods. Efficient methods have been developed for the genetic manipulation and the heterologous expression of the clusters, and more than 100 new derivatives of these antibiotics have been generated by metabolic engineering, mutasynthesis and chemoenzymatic synthesis, providing a model for the power of genetic and genomic methods for the generation of new bioactive compounds.

Ergot alkaloids are toxins and important pharmaceuticals that are produced biotechnologically on an industrial scale. The first committed step of ergot alkaloid biosynthesis is catalyzed by dimethylallyl tryptophan synthase (DMATS; EC 2.5.1.34). Orthologs of DMATS are found in many fungal genomes. We report here the x-ray structure of DMATS, determined at a resolution of 1.76 Å. A complex of DMATS from Aspergillus fumigatus with its aromatic substrate L-tryptophan and with an analogue of its isoprenoid substrate dimethylallyl diphosphate reveals the structural basis of this enzyme-catalyzed Friedel-Crafts reaction, which shows strict regiospecificity for position 4 of the indole nucleus of tryptophan as well as unusual independence of the presence of Mg 2+ ions. The 3D structure of DMATS belongs to a rare β/α barrel fold, called prenyltransferase barrel, that was recently discovered in a small group of bacterial enzymes with no sequence similarity to DMATS. These bacterial enzymes catalyze the prenylation of aromatic substrates in the biosynthesis of secondary metabolites (i.e., a reaction similar to that of DMATS).

The bacterium Streptomyces anulatus 9663, isolated from the intestine of different arthropods, produces prenylated derivatives of phenazine 1-carboxylic acid. From this organism, we have identified the prenyltransferase gene ppzP. ppzP resides in a gene cluster containing orthologs of all genes known to be involved in phenazine 1-carboxylic acid biosynthesis in Pseudomonas strains as well as genes for the six enzymes required to generate dimethylallyl diphosphate via the mevalonate pathway. This is the first complete gene cluster of a phenazine natural compound from streptomycetes. Heterologous expression of this cluster in Streptomyces coelicolor M512 resulted in the formation of prenylated derivatives of phenazine 1-carboxylic acid. After inactivation of ppzP, only nonprenylated phenazine 1-carboxylic acid was formed. Cloning, overexpression, and purification of PpzP resulted in a 37-kDa soluble protein, which was identified as a 5,10-dihydrophenazine 1-carboxylate dimethylallyltransferase, forming a C–C bond between C-1 of the isoprenoid substrate and C-9 of the aromatic substrate. In contrast to many other prenyltransferases, the reaction of PpzP is independent of the presence of magnesium or other divalent cations. The Km value for dimethylallyl diphosphate was determined as 116 μm. For dihydro-PCA, half-maximal velocity was observed at 35 μm. Kcat was calculated as 0.435 s-1. PpzP shows obvious sequence similarity to a recently discovered family of prenyltransferases with aromatic substrates, the ABBA prenyltransferases. The present finding extends the substrate range of this family, previously limited to phenolic compounds, to include also phenazine derivatives.

Aromatic prenyltransferases catalyze the transfer of prenyl moieties to aromatic acceptor molecules and give rise to an astounding diversity of primary and secondary metabolites in plants, fungi and bacteria. Significant progress has been made in the biochemistry and genetics of this heterogeneous group of enzymes in the past years. After 30 years of extensive research on plant prenylflavonoid biosynthesis, finally the first aromatic prenyltransferases involved in the formation of these compounds have been cloned. In bacteria, investigations of the newly discovered family of ABBA prenyltransferases revealed a novel type of protein fold, the PT barrel. In fungi, a group of closely related indole prenyltransferase was found to carry out aromatic prenylations with different substrate specificity and regiospecificity, and to catalyze both regular and reverse prenylations.

The depsipeptide antibiotic hormaomycin, which is produced by Streptomyces griseoflavus W-384, contains a 5-chloropyrrole moiety. In the producer strain we identified the gene hrmQ that shows sequence similarity to FADH(2)-dependent halogenases. This gene was cloned and heterologously expressed in Streptomyces roseochromogenes var. oscitans DS12.976, which is the producer of the aminocoumarin antibiotic clorobiocin, which contains a 5-methylpyrrole moiety. For the present experiment, we used a mutant of this strain in which the respective pyrrole-5-methyltransferase had been inactivated. Expression of the halogenase hrmQ in this mutant strain led to the formation of two new clorobiocin derivatives that carried a 5-chloropyrrole moiety. These compounds were isolated on a preparative scale, their structures were elucidated by (1)H NMR spectroscopy and mass spectrometry, and their antibacterial activity was determined. The substrate of HrmQ is likely to be a pyrrole-2-carboxyl-S-[acyl carrier protein] thioester. If this assumption is true, this study presents the first experiment in combinatorial biosynthesis that uses a halogenase that acts on an acyl carrier protein-bound substrate.

The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin contains two putative regulatory genes, i.e., novE and novG. Functional proof for the role of NovG as a positive regulator of novobiocin biosynthesis had been provided previously, and we now investigated the role of novE. Heterologous expression experiments with the novobiocin biosynthetic gene cluster showed that the entire putative promoter region of novE is required to achieve optimal novobiocin production. Overexpression of novE, using a replicative vector, resulted in an increase of novobiocin formation. In contrast, inactivation of novE by in frame deletion resulted in a strong reduction of novobiocin biosynthesis. Novobiocin production could be restored by an intact copy of novE, but also by the regulatory gene novG. These observations suggest that novE is a positive regulator of novobiocin biosynthesis. NovE was expressed in E. coli and purified. However, in contrast to parallel experiments with NovG, no DNA-binding properties could be shown for NovE. RT-PCR experiments showed that expression of novG was detectable in the absence of NovE, and also that expression of novE occurred in absence of NovG.

Many secondary metabolites of clinical importance have been isolated from different Streptomyces species. As most of the natural producers remain difficult to handle genetically, heterologous expression of an entire biosynthetic gene cluster in a well characterised host allows improved possibilities for modifications of the desired compound by manipulation of the biosynthetic genes. However, the large size of a functional gene cluster often prevents its direct cloning into a single cosmid clone. Here we describe a successful strategy to assemble the entire coumermycin A1 biosynthetic gene cluster (38.6 kb) into a single cosmid clone by lambda RED recombination technology. Heterologous expression of the reconstituted gene cluster in Streptomyces coelicolor M512 resulted in the heterologous production of coumermycin A1. Inactivation of the methyltransferase gene couO--responsible for the C-methylation at the 8-positions of the aminocoumarin moieties in coumermycin A1--and heterologous expression of the modified cluster resulted in an accumulation of a C-8-unsubstituted coumermycin A1 derivative. Subsequent expression of the halogenase gene clo-hal from the clorobiocin gene cluster in the heterologous producer strain led to the formation of two new hybrid antibiotics, containing either one or two chlorine atoms. The identities of the new compounds were verified by LC-MS, and their antibacterial activities were tested against Bacillus subtilis in an agar diffusion assay.

Aromatic prenyltransferases (PTases) catalyze the transfer of a C5 (dimethylallyl), C10 (geranyl) or C15 (farnesyl) prenyl group derived from the corresponding isoprenyl diphosphate metabolites onto a variety of electron-rich aromatic acceptors. Prenyl groups appear in a wide variety of bioactive natural products of microbial and plant origin, including amino acids, stilbenes, alkaloids, polyketides and phenylpropanoids such as flavonoids, creating natural product hybrids with altered or enhanced bioactivities. Prenylation of flavonoids enhances some of the desirable pharmacological properties of these plant compounds [1], as demonstrated for apigenin and liquiritigenin [2]. Prenylation appears in many cases to provide a higher level of bioactivity compared to the non-prenylated precursor, often by increasing affinity for biological membranes and interactions with cellular targets [3]. With the recent identification of these enzymes there is increased interest in the role of these regiospecific catalysts in expanding the diversity and bioactivities of several important classes of natural products in vivo and in vitro.

Summary This study reports improved mutasynthetic approaches for the production of aminocoumarin antibiotics. Previously, the mutasynthetic production of aminocoumarins with differently substituted benzoyl moieties was limited by the substrate specificity of the amide synthetase CloL. We expressed two amide synthetases with different substrate specificity, CouL and SimL, in appropriately engineered producer strains. After feeding of precursor analogs that were not accepted by CloL, but by SimL or CouL, a range of aminocoumarins, unattainable in our previous experiments, was produced and isolated in preparative amounts. Further, we developed a two-stage mutasynthesis procedure for the production of hybrid antibiotics that showed the substitution pattern of novobiocin in the aminocoumarin moiety and that of clorobiocin in the deoxysugar moiety. The substitution pattern of the benzoyl moiety was determined by external addition of an appropriate precursor. Twenty-five aminocoumarin compounds were prepared by these methods, and their structures were elucidated with mass and 1 H-NMR spectroscopy.

Streptomyces cinnamonensis DSM 1042 produces the polyketide-isoprenoid compound furanonaphthoquinone I (FNQ I) and isoprenylated phenazines, predominantly endophenazine A. However, the recently identified biosynthetic gene cluster for these compounds only contains a single gene for a mevalonate pathway enzyme, that is, a putative mevalonate kinase gene. This is in strong contrast to all Streptomyces strains examined so far, where all six genes encoding the mevalonate pathway enzymes are clustered in a single operon of 6.8 kb and, thus, raised the question about the biosynthetic origin of the isoprenoid moieties of FNQ I and endophenazine A. In this study, we investigated the incorporation of [13C2]acetate and [2-13C]glycerol into FNQ I and endophenazine A. The results unequivocally prove that the isoprenoid building blocks of both compounds are predominantly formed via the mevalonate pathway (approximately 80%) but that the MEP pathway (approximately 20%) contributes to the biosynthesis of these molecules, too. In actinomycetes, this is the first experimentally proven example of the utilization of both biosynthetic routes for the formation of one single secondary metabolite. The incorporation pattern of [2-13C]glycerol was consistent with a "reverse" prenyl transfer, that is, with the formation of a C-C bond from C-3 of GPP to the polyketide nucleus of FNQ I.

In the biosynthetic gene cluster of the aminocoumarin antibiotic clorobiocin, the small ORF cloY encodes a 71 aa protein which shows significant sequence similarity to mbtH from the mycobactin biosynthetic gene cluster of Mycobacterium tuberculosis. mbtH-like genes are frequently found in the biosynthetic gene clusters of peptide antibiotics and siderophores, but their function has remained enigmatic. In a recent publication it has been suggested that these genes may have no function for secondary metabolite biosynthesis. An in-frame deletion of cloY in the clorobiocin cluster has now been carried out. When the modified cluster was expressed in the heterologous host Streptomyces coelicolor M512, clorobiocin was still formed. However, when the two further mbtH-like genes from elsewhere in the host genome were inactivated as well, clorobiocin formation was reduced dramatically. Complementation with cloY or with any of three other mbtH-like genes restored clorobiocin formation. This is the first report proving the requirement of an mbtH-like gene for secondary metabolite formation, and the first proof that different mbtH-like genes can functionally replace each other. Feeding of an mbtH-defective triple mutant strain with an intact 3-amino-4,7-dihydroxy-coumarin moiety restored antibiotic production, showing that cloY is specifically required for the formation of this moiety of the clorobiocin molecule.

Terpenoids are arguably the largest and most diverse family of natural products, featuring prominently in e.g. signalling, self-defence, UV-protection and electron transfer. Prenyltransferases are essential players in terpenoid and hybrid isoprenoid biosynthesis that install isoprene units on target molecules and thereby often modulate their bioactivity. In our search for new prenyltransferase biocatalysts we focused on the marine-derived Streptomyces sp. CNQ-509, a particularly rich source of meroterpenoid chemistry. Sequencing and analysis of the genome of Streptomyces sp. CNQ-509 revealed seven putative phenol/phenazine-specific ABBA prenyltransferases, and one putative indole-specific ABBA prenyltransferase. To elucidate the substrate specificity of the ABBA prenyltransferases and to learn about their role in secondary metabolism, CnqP1 –CnqP8 were produced in Escherichia coli and incubated with various aromatic and isoprenoid substrates. Five of the eight prenyltransferases displayed enzymatic activity. The efficient conversion of dihydroxynaphthalene derivatives by CnqP3 (encoded by AA958_24325) and the co-location of AA958_24325 with genes characteristic for the biosynthesis of THN (tetrahydroxynaphthalene)-derived natural products indicates that the enzyme is involved in the formation of debromomarinone or other naphthoquinone-derived meroterpenoids. Moreover, CnqP3 showed high flexibility towards a range of aromatic and isoprenoid substrates and thus represents an interesting new tool for biocatalytic applications.

Clorobiocin is an aminocoumarin antibiotic containing a 5-methylpyrrolyl-2-carboxyl moiety, attached by an ester bond to a deoxysugar. This pyrrolyl moiety is important for the binding of the antibiotic to its biological target, the B subunit of gyrase. Inactivation experiments had shown that two putative acyl carrier proteins, CloN5 and CloN1, and two putative acyl transferases, CloN2 and CloN7, are involved in the transfer of the pyrrolyl-2-carboxyl moiety to the deoxysugar. In this study, pyrrolyl-2-carboxyl-N-acetylcysteamine thioester was synthesized and fed to cloN1 ( - ), cloN2 ( - ) and cloN7 ( - ) mutants, and secondary metabolite formation was analyzed by HPLC and HPLC-MS. Transfer of the pyrrolyl-2-carboxyl moiety was observed in the cloN1 ( - ) and cloN2 ( - ) mutants, but not in the cloN7 ( - ) mutant, suggesting that CloN7 is responsible for this reaction. The product of this transfer, novclobiocin 109, was not further methylated to the 5-methylpyrrolyl-2-carboxyl compound, i.e. clorobiocin, suggesting that methylation does not take place after the acyl transfer. Additional investigations for the presence of 5-methylpyrrolyl-2-carboxylic acid in the mutants, and inactivation experiments with the methyltransferase gene cloN6, suggested that methylation by CloN6 and acyl transfer by CloN7 take place in a concerted fashion, requiring the presence of both proteins for efficient product formation. A mechanism for the methylation/acyl transfer process in the late steps of clorobiocin biosynthesis, involving CloN1, CloN2, CloN5, CloN6 and CloN7 is suggested.

The aminocoumarin antibiotic clorobiocin contains an unusual branched deoxysugar with a 5,5-gem-dimethyl structure. Inactivation of the putative C-methyltransferase gene cloU was carried out, which led to the loss of the axial methyl group at C-5 of this deoxysugar moiety. This result establishes the function of cloU, and at the same time it proves that the biosynthesis of the deoxysugar moiety of clorobiocin proceeds via a 3,5-epimerization of the dTDP-4-keto-6-deoxyglucose intermediate. The inactivation was carried out on a cosmid which contained the entire clorobiocin biosynthetic gene cluster. Expression of the modified cluster in a heterologous host led to the formation of desmethyl-clorobiocin and a structural isomer thereof. Both compounds were isolated on a preparative scale, their structures were elucidated by 1H-NMR and mass spectroscopy and their antibacterial activity was assayed.

Novobiocin and clorobiocin are gyrase inhibitors produced by Streptomyces strains. Structurally, the two compounds differ only by substitution at two positions: CH 3 versus Cl at position 8′ of the aminocoumarin ring and carbamoyl versus 5-methyl-pyrrol-2-carbonyl (MePC) at the 3"-OH of noviose. Using genetic engineering, we generated a series of analogs carrying H, CH 3 , or Cl at 8′ and H, carbamoyl, or MePC at 3"-OH. Comparison of the gyrase inhibitory activities of all nine structural permutations confirmed that acylation of 3"-OH is essential for activity, with MePC being more effective than carbamoyl. Substitution at 8′ further enhanced activity, but the effect of CH 3 or Cl depended on the nature of the acyl group at 3": in the presence of carbamoyl at 3", CH 3 resulted in higher activity; in the presence of MePC at 3", Cl resulted in higher activity. This suggests that the structures of both natural compounds are highly evolved for optimal interaction with gyrase. In a second series of experiments, clorobiocin derivatives with and without the methyl group at 4"-OH of noviose, and with different positions of the MePC group of noviose, were tested. Again clorobiocin was superior to all of its analogs. The activities of all compounds were also tested against topoisomerase IV (topo IV). Clorobiocin stood out as a remarkably effective topo IV inhibitor. The relative activities of the different compounds toward topo IV showed a pattern similar to that of the relative gyrase-inhibitory activities. This is the first report of a systematic evaluation of a series of aminocoumarins against both gyrase and topo IV. The results give further insight into the structure-activity relationships of aminocoumarin antibiotics.

Clorobiocin is an aminocoumarin antibiotic containing a pyrrole-2-carboxyl moiety, attached through an ester bond to a deoxysugar. The pyrrole moiety is important for the binding of the antibiotic to its biological target, gyrase. The complete biosynthetic gene cluster for clorobiocin has been cloned and sequenced from the natural producer, Streptomyces roseochromogenes DS 12.976. In this study, the genes cloN1 and cloN7 were deleted separately from a cosmid containing the complete clorobiocin cluster. The modified cosmids were introduced into the genome of the heterologous host Streptomyces coelicolor M512 by using the integration functions of the PhiC31 phage. While a heterologous producer strain harbouring the intact clorobiocin biosynthetic gene cluster accumulated clorobiocin, the cloN1- and cloN7-defective integration mutants accumulated a clorobiocin derivative that lacked the pyrrole-2-carboxyl moiety, while also producing free pyrrole-2-carboxylic acid. The structures of these metabolites were confirmed by NMR and MS analysis. These results showed that CloN1 and CloN7, together with the previously investigated CloN2, are involved in the transfer of the pyrrole-2-carboxyl moiety to the deoxysugar of clorobiocin. A possible mechanism for the role of these three proteins in the acyl-transfer process is suggested.

A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. -Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage C31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids’ inserts showed which genes are essential for antibiotic production. The development of new antimicrobial agents is crucial to reduce the future clinical impact of resistant pathogens (35). Natural products play a dominant role in antimicrobial drug discovery (19), and streptomycetes produce two-thirds of the clinically useful antibiotics of natural origin (12). Structural modification of these natural products is often necessary in drug development, e.g., for improvements in efficacy and pharmacokinetics (32). The manipulation of genes which encode enzymes of the biosynthetic pathways represents a promising approach for introducing structural changes (33). The functional analysis of biosynthetic genes is, however, a prerequisite for such approaches. Moreover, a principal limitation is often presented by the lack of suitable protocols for the genetic manipulation of natural producers (11).

NovW, novU, and novS gene products represent dTDP-4-keto-6-deoxy-D-glucose 3,5 epimarase, C-methyltransferase and dTDP-glucose-4-ketoreductase involved in noviose biosynthetic pathway, respectively. We have expressed three genes to elucidate the functions of NovW, NovU, and NovS in Escherichia coli. NovW and NovU catalyze the formation of dTDP-4-keto-6-deoxy-5-C-methyl-L-lyxo-hexose from dTDP-4-keto-6-deoxy-D-glucose. NovS reduces the product formed from the reaction of NovW with dTDP-4-keto-6-deoxy-D-glucose in the presence of NADH to result in dTDP-l-rhamnose. Furthermore, a pathway for the biosynthesis of noviose is proposed.

In the present study, we produced a hybrid antibiotic, carrying a chlorine atom instead of a methyl group at position 8 of the aminocoumarin moiety of novobiocin. This compound was not accessible by conventional gene inactivation/gene expression experiments due to difficulties in the genetic manipulation of the novobiocin producer Streptomyces spheroides. However, the desired compound was obtained after modification of the novobiocin biosynthetic gene cluster by using λ-Red-mediated recombination in Escherichia coli, followed by integration of the resulting modified cosmid into the φC31 attachment site of Streptomyces coelicolor and coexpression of the halogenase Clo-hal of clorobiocin biosynthesis. The halogenase BhaA, responsible for chlorination of tyrosyl moieties of the glycopeptide antibiotic balhimycin, was unable to functionally replace the halogenase Clo-hal, suggesting that the two enzymes have different substrate specificities.

Coumermycin A(1) is a member of the aminocoumarin family of antibiotics. Unlike its structural relatives, novobiocin and clorobiocin, coumermycin A(1) is a dimer built on a 3-methyl-2,4-dicarboxypyrrole scaffold and bears two decorated noviose sugar components which are the putative target binding motifs for DNA gyrase. Starting with this scaffold, we have utilized the ligase CouL for mono- and bisamide formation with aminocoumarins to provide substrates for the glycosyltransferase CouM. CouM was subsequently shown to catalyze mono- and bisnoviosylation of the resulting CouL products. CouP was shown to possess 4'-O-methyltransferase activity on products from tandem CouL, CouM assays. A fourth enzyme, NovN, the 3'-O-carbamoyltransferase from the novobiocin operon, was then able to carbamoylate either or both arms of the CouP product. The tandem action of CouL, CouM, CouP, and NovN thus generates a biscarbamoyl analogue of the pseudodimer coumermycin A(1). Starting from alternative dicarboxy scaffolds, these four enzymes can be utilized in tandem to create additional variants of dimeric aminocoumarin antibiotics.

Five new aminocoumarin antibiotics were produced by a combined mutational and chemoenzymatic approach. For this purpose, the 3″-carbamoyltransferase NovN from the novobiocin producer Streptomyces spheroides was overexpressed in the heterologous host S. lividans as an N-terminal His 6 fusion protein and purified by nickel affinity chromatography. Five different 3″-unsubstituted aminocoumarin derivatives were isolated from mutants of the clorobiocin producer S. roseochromogenes , carrying single or multiple gene defects. All five compounds were readily accepted as substrates by NovN, and the 3″-carbamoylated products were isolated on a preparative scale. Their structures were elucidated by 1 H-NMR and mass spectroscopy, and their inhibitory activity on gyrase in vitro as well as their antibacterial activity was determined. The results give further insight into the structure-activity relationships of aminocoumarin antibiotics.

The Chinese medicinal plant Artemisia annua L. (Annual Wormwood) contains the antimalarial compound artemisinin. The locally grown herb may offer an additional tool for the control of malaria, especially in poor countries where modern antimalarial drugs are often unavailable. In an open, randomized, controlled pilot trial, we investigated the efficacy and safety of traditional tea preparations of Artemisia annua in the treatment of uncomplicated malaria. Treatment resulted in a quick resolution of parasitaemia and of clinical symptoms. After 7 d of medication, cure rates were on average 74% for the Artemisia preparations compared with 91% for quinine. However, recrudescence rates were high in the Artemisia groups. Therefore, monotherapy with Artemisia annua L. cannot be recommended as alternative to modern antimalarials, but may deserve further investigation.

The aminocoumarin antibiotics clorobiocin, novobiocin, and coumermycin A 1 are inhibitors of bacterial gyrase. Their chemical structures contain amide bonds, formed between an aminocoumarin ring and an aromatic acyl component, which is 3-dimethylallyl-4-hydroxybenzoate in the case of novobiocin and clorobiocin. These amide bonds are formed under catalysis of the gene products of cloL , novL , and couL , respectively. We first examined the substrate specificity of the purified amide synthetases CloL, NovL, and CouL for the various analogs of the prenylated benzoate moiety. We then generated new aminocoumarin antibiotics by feeding synthetic analogs of the 3-dimethylallyl-4-hydroxybenzoate moiety to a mutant strain defective in the biosynthesis of the prenylated benzoate moiety. This resulted in the formation of 32 new aminocoumarin compounds. The structures of these compounds were elucidated using FAB-MS and 1 H-NMR spectroscopy.

Three new antibiotics, vanillobiocin, isovanillobiocin and declovanillobiocin, were isolated from the culture broth of a cloQ-defective mutant of the clorobiocin producer Streptomyces roseochromogenes, which is blocked in the biosynthesis of the prenylated 4-hydroxybenzoic acid moiety of clorobiocin. Spectroscopic analysis showed that the isolated compounds were similar to clorobiocin, but contained vanillic acid as the acyl component instead of the prenylated 4-hydroxybenzoic acid present in clorobiocin. Isovanillobiocin differs from vanillobiocin by the position of the pyrrole unit attached to the sugar moiety of the antibiotic. Declovanillobiocin lacks the chlorine atom at the aminocoumarin ring. All three compounds had lower antibiotic activity against Bacillus subtilis than clorobiocin.