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The visual cliff assay (VCA) has been used in multiple animal models including humans to assess stereopsis, the pinnacle percept of binocular function that is markedly disrupted in amblyopia. Amblyopia is a neurodevelopmental disorder of the visual system caused by refractive error, strabismus, or obscuration of the visual axis during infancy and early childhood. While the current standard of care can improve visual acuity in the amblyopic eye, gains are infrequently associated with improvements in stereoscopic depth perception and typically only achieved when treatment is initiated in early childhood. The mouse model of amblyopia induced by monocular deprivation (MD) is a powerful tool to study the mechanistic pathophysiology of this disorder and test novel therapeutics. However, to date, only one study has used the VCA to assess the effects of MD in mice in a limited capacity. Advantages of the VCA include no need for operant conditioning or training, completion in minutes, and minimal equipment. We comprehensively characterize and validate fundamental aspects of the VCA including test-retest reliability and contributions of binocularity using multiple monocular and binocular manipulations with clinically relevant paradigms. After long-term (3 weeks) MD, mice exhibit reduced predilection for the safe side, decreased latency to cross to the cliff side, and more crosses to the cliff side compared to sham littermate controls. Our data demonstrate that the VCA can detect binocular deficits in amblyopia but with important limitations that guide how the VCA should be applied to read out binocular function in both mechanistic and therapeutic studies in mice.

Brasilicardin A (1) consists of an unusual anti/syn/anti‐perhydrophenanthrene skeleton with a carbohydrate side chain and an amino acid moiety. It exhibits potent immunosuppressive activity, yet its mode of action differs from standard drugs that are currently in use. Further pre‐clinical evaluation of this promising, biologically active natural product is hampered by restricted access to the ready material, as its synthesis requires both a low‐yielding fermentation process using a pathogenic organism and an elaborate, multi‐step total synthesis. Our semi‐synthetic approach included a) the heterologous expression of the brasilicardin A gene cluster in different non‐pathogenic bacterial strains producing brasilicardin A aglycone (5) in excellent yield and b) the chemical transformation of the aglycone 5 into the trifluoroacetic acid salt of brasilicardin A (1 a) via a short and straightforward five‐steps synthetic route. Additionally, we report the first preclinical data for brasilicardin A., The development of a heterologous producer strain that enables the sustainable production of the stereochemically complex natural products brasilicardin C and E at excellent rates is presented. The semi‐synthetic approach allows an efficient gram‐scale conversion of brasilicardin E into the potent immunosuppressant brasilicardin A for which the first preclinical data are reported.

The bacteria of the genus Streptomyces are among the most important producers of biologically active secondary metabolites. Moreover, recent genomic sequence data have shown their enormous genetic potential for new natural products, although many new biosynthetic gene clusters (BGCs) are silent. Therefore, efficient and stable genome modification techniques are needed to activate their production or to manipulate their biosynthesis towards increased production or improved properties. We have recently developed an efficient markerless genome modification system for streptomycetes based on positive blue/white selection of double crossovers using the bpsA gene from indigoidine biosynthesis, which has been successfully applied for markerless deletions of genes and BGCs. In the present study, we optimized this system for markerless insertion of large BGCs. In a pilot test experiment, we successfully inserted a part of the landomycin BGC (lanFABCDL) under the control of the ermEp* promoter in place of the actinorhodin BGC (act) of Streptomyces lividans TK24 and RedStrep 1.3. The resulting strains correctly produced UWM6 and rabelomycin in twice the yield compared to S. lividans strains with the same construct inserted using the PhiBT1 phage-based integration vector system. Moreover, the system was more stable. Subsequently, using the same strategy, we effectively inserted the entire BGC for mithramycin (MTM) in place of the calcium-dependent antibiotic BGC (cda) of S. lividans RedStrep 1.3 without antibiotic-resistant markers. The resulting strain produced similar levels of MTM when compared to the previously described S. lividans RedStrep 1.3 strain with the VWB phage-based integration plasmid pMTMF. The system was also more stable. KEY POINTS: • Optimized genome editing system for markerless insertion of BGCs into Streptomyces genomes • Efficient heterologous production of MTM in the stable engineered S. lividans strain.

We previously developed an efficient deletion system for streptomycetes based on the positive selection of double-crossover events using bpsA, a gene for producing the blue pigment indigoidine. Using this system, we removed interfering secondary metabolite clusters from Streptomyces lividans TK24, resulting in RedStrep strains with dramatically increased heterologous production of mithramycin A (up to 3-g/l culture). This system, however, required a time-consuming step to remove the resistance marker genes. In order to simplify markerless deletions, we prepared a new system based on the plasmid pAMR18A. This plasmid contains a large polylinker with many unique restriction sites flanked by apramycin and kanamycin resistance genes and the bpsA gene for selecting a double-crossover event. The utility of this new markerless deletion system was demonstrated by its deletion of a 21-kb actinorhodin gene cluster from Streptomyces lividans TK24 with 30% efficiency. We used this system to efficiently remove the matA and matB genes in selected RedStrep strains, resulting in biotechnologically improved strains with a highly dispersed growth phenotype involving non-pelleting small and open mycelia. No further increase in mithramycin A production was observed in these new RedStrep strains, however. We also used this system for the markerless insertion of a heterologous mCherry gene, an improved variant of the monomeric red fluorescent protein, under the control of the strong secretory signal sequence of the subtilisin inhibitor protein, into the chromosome of S. lividans TK24. The resulting recombinant strains efficiently secreted mCherry into the growth medium in a yield of 30 mg/l.

This document explores the challenges and perspectives behind the initiative for enabling Mexican universities and research centers to access the Turnitin software, a strategy for promoting greater ethics in academic and scientific communications. It presents information obtained through documentary research, from the experience of using the software during the last academic semester, from interviews with the coordinator of the National Consortium of Scientific and Technological Resources (CONRICyT), and with the Turnitin company representative for Mexico. Given the emerging nature of this project and the complexity of the issue, the results presented consist of a non-comprehensive list of benefits and challenges that emerged from using the software, as well as recommendations to harness its use and consolidate a culture for the ethical use of information in academic and scientific communications.

Purpose This study aims to develop both an activity and an instrument to support college students’ learning of the proper ways to paraphrase. Design/methodology/approach This study used a participatory action research approach, consisting of four phases. A survey was used to collect information, as well as a rubric to evaluate students’ competencies in paraphrasing, before and after an educational intervention activity. The tools were designed by the authors. Findings The findings suggest that students only have a partial understanding of the elements that are needed to write an adequate paraphrase, despite the declared importance in academic dialogue by the majority of them. The findings also suggest that a short-range intervention can help students develop their skills in paraphrasing. Research limitations/implications This research was conducted mostly with a small group of first-year college students and a professor they have known for a year. Therefore, a level of trust had been established. This trust relationship is necessary to achieve a participatory action in an unscheduled activity. Only some of the major results are presented. Originality/value Little actual research has been conducted on college students’ perception on paraphrasing and its importance, as well as on their abilities to paraphrase, especially in the Latin American context. This study demonstrates the usefulness of a short-range intervention that can be easily applied in any course or information literacy program.

Brasilicardin A (BraA) is a promising immunosuppressive compound produced naturally by the pathogenic bacterium Nocardia terpenica IFM 0406. Heterologous host expression of brasilicardin gene cluster showed to be efficient to bypass the safety issues, low production levels and lack of genetic tools related with the use of native producer. Further improvement of production yields requires better understanding of gene expression regulation within the BraA biosynthetic gene cluster (Bra‐BGC); however, the only so far known regulator of this gene cluster is Bra12. In this study, we discovered the protein LysRNt, a novel member of the LysR‐type transcriptional regulator family, as a regulator of the Bra‐BGC. Using in vitro approaches, we identified the gene promoters which are controlled by LysRNt within the Bra‐BGC. Corresponding genes encode enzymes involved in BraA biosynthesis as well as the key Bra‐BGC regulator Bra12. Importantly, we provide in vivo evidence that LysRNt negatively affects production of brasilicardin congeners in the heterologous host Amycolatopsis japonicum. Finally, we demonstrate that some of the pathway related metabolites, and their chemical analogs, can interact with LysRNt which in turn affects its DNA‐binding activity.

La presente investigación expone un conjunto de estándares compuestos de distintas dimensiones e indicadores de rendimiento para evaluar la competencia en la comunicación científica en profesores universitarios mexicanos. El estudio se integró por distintas fases complementarias y subsecuentes: (i) investigación documental sobre elementos normativos y teóricos que sustentan la identificación inicial de indicadores de rendimiento de evaluación comunicación científica; (ii) validación de información por 32 investigadores expertos en ciencias; (iii) validación de información por 62 expertos en alfabetización informacional (ALFIN); y (iv) derivación de un conjunto de estándares, mismos que fueron probados en 28 profesores universitarios del área de las ciencias químicas con potencialidad científica. Los resultados de la investigación definieron un conjunto de estándares integrados por ocho dimensiones y 34 indicadores de rendimiento, para posteriormente probar su funcionalidad diagnosticando niveles individuales y colectivos de competencia en comunicación científica, favoreciendo la identificación de fortalezas y debilidades, con lo cual se posibilita el diseño de propuestas de mejora a través de procesos planeados de cambio y beneficiando el desarrollo de habilidades hacia la producción y comunicación científica. Abstract: The present research exposes a set of standards composed of different dimensions and performance indicators to evaluate the competence in scientific communication in Mexican university professors. The study consisted of different complementary and subsequent phases: (i) documentary research on normative and theoretical elements that support the initial identification of performance indicators for scientific communication evaluation; (ii) validation of information by 32 expert researchers in science; (iii) validation of information by 62 experts in information literacy (IL); and (iv) derivation of a set of standards, which were tested by 28 university professors in the area of chemical sciences with scientific potential. The results of the research defined a set of standards made up of eight dimensions and 34 performance indicators, to later test their functionality by diagnosing individual and collective levels of competence in scientific communication, favoring the identification of strengths and weaknesses, thereby enabling the design of improvement proposals through planned processes of change and benefiting the development of skills towards scientific production and communication.

Mithramycin A is an antitumor compound used for treatment of several types of cancer including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia and Paget's disease. Selective modifications of this molecule by combinatorial biosynthesis and biocatalysis opened the possibility to produce mithramycin analogues with improved properties that are currently under preclinical development. The mithramycin A biosynthetic gene cluster from Streptomyces argillaceus ATCC12956 was cloned by transformation assisted recombination in Saccharomyces cerevisiae and heterologous expression in Streptomyces lividans TK24 was evaluated. Mithramycin A was efficiently produced by S. lividans TK24 under standard fermentation conditions. To improve the yield of heterologously produced mithramycin A, a collection of derivative strains of S. lividans TK24 were constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters using a protocol based on the positive selection of double crossover events with blue pigment indigoidine-producing gene. Mithramycin A production was evaluated in these S. lividans strains and substantially improved mithramycin A production was observed depending on the deleted gene clusters. A collection of S. lividans strains suitable for heterologous expression of actinomycetes secondary metabolites were generated and efficient production of mithramycin A with yields close to 3 g/L, under the tested fermentation conditions was achieved using these optimized collection of strains.

This paper presents an unusual topology of a medium-power seven-level inverter that utilizes a single dc source and a three-phase transformer to generate high-performance seven-level voltage waveforms without complex cascade H-bridges with isolated dc supplies or neutral-point-clamped circuits with capacitor balancing techniques. In contrast to typical topologies of multilevel inverters, the control strategy of the presented circuit facilitates its implementation on a simple 16-bit microcontroller, such that the proposed inverter is operated under a space vector mode. The principle of operation of the seven-level inverter is analyzed, using idealized waveforms for easy and straight implementation together with a description of its practical development, showing experimental results obtained with a 1-kW prototype.

Phoracantha recurva,_Newman, 1840, is a beetle native to Australia. Adults of this species have a bright body, with dark brown and yellow to cream areas on the elytra, antennae as long or longer than the body, pronotum with discs or protuberances on the back, and lateral spines; elytra with at least one spine or process in the apical margin. P. recurva is an important pest, because it causes severe damage to trees belonging to the Myrtaceae family, such as Eucalyptus and Angophora. The National Commission for the Knowledge and Use of Biodiversity in Mexico (CONABIO) has published the list of high-risk exotic insects and arachnids for Mexico. Of the 20 species of insects associated with eucalyptus trees in Mexico, the genus Phoracantha is not registered, probably because reports of this species are only incidental. Here, we report the occurrence of P. recurva, in Mexico based upon captured and observed specimens.

This work corresponds to a practical and transversal integration process of Information Literacy in university curricula, specifically with undergraduate students from the Philosophy program of the Autonomous University of Chihuahua (Mexico), by developing alternatives to evolve traditional classroomteaching practices toward integrating Learning Communities and using Action Research as means ofinfluencing a continuous improvement upon learning processes. This chapter discusses basic concepts from this study and provides the results, which were a product of the data collected from ethnographicprocesses. This practical experience has demonstrated the feasibility of combining this study’scomponents for the achievement of active learning, but also for identifying specific elements that inhibita full implementation.

Ministry of Economy, This work was supported by grants to CM from the Spanish Ministry of Economy and Competitiveness, MINECO (Grants BIO2011-25398, BIO2014-56752-R and PIM2010EEI-00752) and “Apoyo a grupos de excelencia,” Principado de Asturias-FEDER (FC-15-GRUPIN14-014). EntreChem SL acknowledges funding to the ERA-IB program and MINECO (PIM2010EEI-00752). SY and DZ were recipient of predoctoral fellowships from MINECO. We thank Fundación Bancaria Cajastur for financial support to CO, and we also thank Dr. Fernando Reyes from Fundación Medina, and Dr. Javier González-Sabín and Dr. Nicolás Ríos-Lombardía from Entrechem S.L. for technical support in structural elucidation compounds.

This work, including the efforts of Carmen Méndez, was funded by Ministerio de Economía y Competitividad (This work, including the efforts of Carmen Mendez, was funded by Ministerio de Economía y Competitividad (MINECO) (PCIN-2014-066). This work, including the efforts of Francisco Morris, was funded by Ministerio de Economía y Competitividad (MINECO) (PCIN-2014-097). This work, including the efforts of Harald Gross and Pierre Koch, was funded by Bundesministerium für Bildung und Forschung (BMBF) (FKZ 031A568A). This work, including the efforts of Wolfgang Wohlleben, was funded by Bundesministerium für Bildung und Forschung (BMBF) (FKZ 031A568B). This work, including the efforts of Jolanta Zakrzewska-Czerwinska, was funded by MNiSW | Narodowe Centrum Badani Rozwoju (NCBR) (ERA-NET-IB/NeBrasCa/10/2015).). This work, including the efforts of Francisco Morris, was funded by Ministerio de Economía y Competitividad (MINECO) (PCIN-2014-097). This work, including the efforts of Harald Gross and Pierre Koch, was funded by Bundesministerium für Bildung und Forschung (BMBF) (FKZ 031A568A). This work, including the efforts of Wolfgang Wohlleben, was funded by Bundesministerium für Bildung und Forschung (BMBF) (FKZ 031A568B). This work, including the efforts of Jolanta Zakrzewska-Czerwinska, was funded by MNiSW | Narodowe Centrum Badan´ i Rozwoju (NCBR) (ERA-NET-IB/NeBrasCa/10/2015).

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by mutations in the X chromosomal gene Methyl-CpG-binding Protein 2 (MECP2) (1). RTT treatment so far is symptomatic. Mecp2 disruption in mice phenocopies major features of the syndrome (2) that can be reversed upon re-expression of Mecp2 (3. It has recently been reported that transplantation of wild type (WT) bone marrow (BMT) into lethally irradiated Mecp2tm1.1Jae/y mice prevented neurologic decline and early death by restoring microglial phagocytic activity against apoptotic targets (4). Based on this report, clinical trials of BMT for patients with RTT have been initiated (5). We aimed to replicate and extend the BMT experiments in three different RTT mouse models but found that despite robust microglial engraftment, BMT from WT donors did not rescue early death or ameliorate neurologic deficits. Furthermore, early and specific genetic expression of Mecp2 in microglia did not rescue Mecp2-deficient mice. In conclusion our experiments do not support BMT as therapy for RTT.

Autoantibodies against nucleolar antigens are common in systemic sclerosis (SSc). They include autoantibodies against fibrillarin (Fb), which are serological markers for SSc. Fb is associated with the evolutionally-conserved box C/D of small nucleolar RNAs (snoRNAs). We compared indirect immunofluorescence (IIF), Western blot (WB), and immunoprecipitation (IPP) of total small RNAs assays to determine which of these techniques is most specific for the detection of snoRNPs. We also examined the frequency and specificity of autoantibodies from SSc patients to snoRNAs, snRNAs, and scRNAs, and concluded that 1) IIF can not determine autoantibody specificity against Fb, 2) 36% of SSc sera were false-negative by WB, and 3) by IPP, anti-Fb autoantibodies from SSc patients can bind U3, U8, U13, U15, and U22 snoRNAs.

A previously unidentified set of genes encoding a modular polyketide synthase (PKS) has been sequenced in Saccharopolyspora erythraea, producer of the antibiotic erythromycin. This new PKS gene cluster (pke) contains four adjacent large open reading frames (ORFs) encoding eight extension modules, flanked by a number of other ORFs which can be plausibly assigned roles in polyketide biosynthesis. Disruption of the pke PKS genes gave S. erythraea mutant JC2::pSBKS6, whose growth characteristics and pattern of secondary metabolite production did not apparently differ from the parent strain under any of the growth conditions tested. However, the pke PKS loading module and individual pke acyltransferase domains were shown to be active when used in engineered hybrid PKSs, making it highly likely that under appropriate conditions these biosynthetic genes are indeed expressed and active, and synthesize a novel polyketide product.

Sequence comparisons of multiple acyltransferase (AT) domains from modular polyketide synthases (PKSs) have highlighted a correlation between a short sequence motif and the nature of the extender unit selected. When this motif was specifically altered in the bimodular model PKS DEBS1-TE of Saccharopolyspora erythraea, the products included triketide lactones in which acetate extension units had been incorporated instead of propionate units at the predicted positions. We also describe a cassette system for convenient construction of hybrid modular PKSs based on the tylosin PKS in Streptomyces fradiae and demonstrate its use in domain and module swaps.

The soluble, diffusible red-brown pigment produced by a Saccharopolyspora erythraea "red variant" has been shown to contain glycosylated and polymerized derivatives of 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). Flaviolin is a spontaneous oxidation product of 1,3,6,8-tetrahydroxynaphthalene (THN), which is biosynthesized in bacteria by a chalcone synthase-like (CS-like) type III polyketide synthase (PKS). A fragment of the gene responsible for THN biosynthesis in S. erythraea E_8-7 was amplified by polymerase chain reaction (PCR) using degenerate primers based on conserved regions of known plant CS and bacterial CS-like genes. From the isolated fragment, a suicide vector was prepared, which was subsequently used to disrupt the red-brown pigment-producing (rpp) locus in S. erythraea, generating a mutant that displayed an albino phenotype. Chromosomal DNA from the albino mutant was subsequently used in a vector-recapture protocol to isolate a plasmid that contained an insert spanning the entire rpp locus. Sequencing of the insert revealed that the disrupted open reading frame (ORF) encodes a CS-like protein displaying 69% sequence identity to the rppA gene of Streptomyces griseus. The S. griseus rppA gene encodes RppA, the first characterized bacterial CS-like protein, which is sufficient in vitro for the synthesis of THN from malonyl-CoA. The rppA disruption mutant and rppA sequence provided a means by which to address the mechanism of diffusible pigment biosynthesis, as well as to investigate any link between this and the modulation of erythromycin A titre, which has been observed for S. erythraea variants.

Chain initiation on many modular polyketide synthases is mediated by acyl transfer from the CoA ester of a dicarboxylic acid, followed by decarboxylation in situ by KSQ, a ketosynthase-like decarboxylase domain. Consistent with this, the acyltransferase (AT) domains of all KSQ-containing loading modules are shown here to contain a key arginine residue at their active site. Site-specific replacement of this arginine residue in the oleandomycin (ole) loading AT domain effectively abolished AT activity, consistent with its importance for catalysis. Substitution of the ole PKS loading module, or of the tylosin PKS loading module, for the erythromycin (ery) loading module gave polyketide products almost wholly either acetate derived or propionate derived, respectively, instead of the mixture found normally. An authentic extension module AT domain, rap AT2 from the rapamycin PKS, functioned appropriately when engineered in the place of the ole loading AT domain, and gave rise to substantial amounts of C13-methylerythromycins, as predicted. The role of direct acylation of the ketosynthase domain of ex-tension module 1 in chain initiation was confirmed by demonstrating that a mutant of the triketide synthase DEBS1-TE, in which the 4'-phosphopante-theine attachment site for starter acyl groups was specifically removed, produced triketide lactone pro-ducts in detectable amounts.

Multiple ketoreductase activities play a crucial role in establishing the stereochemistry of the products of modular polyketide synthases (PKSs), but there has been little systematic scrutiny of catalysis by individual ketoreductases. To allow this, a diketide synthase, consisting of the loading module, first extension module, and the chain-terminating thioesterase of the erythromycin-producing PKS of Saccharopolyspora erythraea, has been expressed and purified. The DNA encoding the ketoreductase-1 domain in this construct is flanked by unique restriction sites so that another ketoreductase domain can be readily substituted. The purified recombinant diketide synthase catalyzes, at a very low rate (k(cat) equals 2.5 x 10(-3) s(-1)), the specific production of the diketide (2S,3R)-2-methyl-3-hydroxypentanoic acid. The activity of the ketoreductase domain in this model synthase was analyzed using as a model substrate (+/-)-2-methyl-3-oxopentanoic acid N-acetylcysteaminyl (NAC) ester for which k(cat)/K(m) was 21.7 M(-1) s(-1). The NAC thioester of (2S,3R)-2-methyl-3-hydroxypentanoic acid was the major product and was strongly preferred over other stereoisomers as a substrate in the reverse reaction. The bicyclic ketone (9RS)-trans-1-decalone, a known substrate for ketoreductase in fatty acid synthase, was found also to be an effective substrate for the ketoreductase of the diketide synthase. Only the (9R)-trans-1-decalone was reduced, selectively and reversibly, to the (1S,9R)-trans-decalol. The stereochemical course of reduction and oxidation is exactly as found previously for the ketoreductase of animal fatty acid synthase, an additional indication of the close similarity of these enzymes.

The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5' of eryA that lies between the known genes ermE (encoding the erythromycin resistance methyltransferase) and eryBIII (encoding a putative S-adenosylmethionine-dependent methyltransferase), and that contains the gene eryBI (orf2), has now been sequenced. The inferred product of the eryBI gene shows striking sequence similarity to authentic beta-glucosidases. Specific mutants were created in eryBI, and the resulting strains were found to synthesise erythromycin A, showing that this gene, despite its position in the biosynthetic gene cluster, is not essential for erythromycin biosynthesis. A mutant in eryBIII and a double mutant in eryBI and eryBIII were obtained and the analysis of novel erythromycins produced by these strains confirmed the proposed function of EryBIII as a C-methyltransferase. Also, a chromosomal mutant was constructed for the previously sequenced ORF19 and shown to accumulate erythronolide B, as expected for an eryB mutant and consistent with its proposed role as an epimerase in dTDP-mycarose biosynthesis.

The production of erythromycin A by Saccharopolyspora erythraea requires the synthesis of dTDP-D-desosamine and dTDP-L-mycarose, which serve as substrates for the transfer of the two sugar residues onto the macrolactone ring. The enzymatic activities involved in this process are largely encoded within the ery gene cluster, by two sets of genes flanking the eryA locus that encodes the polyketide synthase. We report here the nucleotide sequence of three such ORFs located immediately downstream of eryA, ORFs 7, 8 and 9. Chromosomal mutants carrying a deletion either in ORF7 or in one of the previously sequenced ORFs 13 and 14 have been constructed and shown to accumulate erythronolide B, as expected for eryB mutants. Similarly, chromosomal mutants carrying a deletion in either ORF8, ORF9, or one of the previously sequenced ORFs 17 and 18 have been constructed and shown to accumulate 3-alpha-mycarosyl erythronolide B, as expected for eryC mutants. The ORF13 (eryBIV), ORF17 (eryCIV) and ORF7 (eryBII) mutants also synthesised small amounts of macrolide shunt metabolites, as shown by mass spectrometry. These results considerably strengthen previous tentative proposals for the pathways for the biosynthesis of dTDP-D-desosamine and dTDP-L-mycarose in Sac. erythraea and reveal that at least some of these enzymes can accommodate alternative substrates.

The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5′ of eryA, and lying between eryA and the gene eryK, which is known to encode the C-12 hydroxylase, has been sequenced and shown to contain seven additional open reading frames (ORFs 13–19). On the basis of sequence similarities, roles are proposed for several of these ORFs in the biosynthesis of the deoxysugar mycarose and the deoxyaminosugar desosamine. A chromosomal mutant carrying a deletion in ORF15 has been constructed and shown to accumulate 3-O-mycarosyl-erythronolide B, as expected for an eryC mutant. Similarly, a chromosomal mutant carrying a deletion in ORF16 has been constructed and shown to accumulate erythronolide B, as expected for an eryB mutant.

Lantibiotics are a group of ribosomally synthesised peptides that contain post-translational modifications consisting of (methyl)lanthionine residues forming bridges that confer them characteristic structures. Most lantibiotics are antibacterials that bind to the cell wall precursor lipid II. They can be rod shaped or globular depending on the distribution of the lanthionine residues. Their biosynthetic pathway is relatively simple when compared to other secondary metabolites. Due to this simplicity, genetic manipulation of the pathways is a very attractive tool to obtain variants that might have improved properties. Mutagenesis programmes have shown that the biosynthetic machinery of lantibiotics has relaxed specificity allowing the production of large collections of variants. Lantibiotic gene clusters are a common feature within bacteria, particularly Gram-positive organisms. Genome mining and in vitro synthesis experiments suggest that there is a great potential for discovery of new lantibiotics. With the increasing need for effective antibiotics against multidrug-resistant pathogens, lantibiotics are an attractive option for a new class of molecules. There are two lantibiotics in late preclinical development for use against systemic Gram-positive infections, one lantibiotic in Phase 1 clinical trials for the treatment of Clostridium difficile infections and another lantibiotic in Phase 2b for the treatment of cystic fibrosis. The potential of lantibiotics for the treatment of bacterial infections should become a reality in the next few years with the current compounds going through the corresponding drug development stages and new compounds joining the collection of useful compounds in the fight against multidrug-resistant bacteria.

Background: The 6-deoxyerythronolide B synthase (DEBS) of Saccharopolyspora erythraea , which synthesizes the aglycone core of the antibiotic erythromycin A, contains some 30 active sites distributed between three multienzyme polypeptides (designated DEBS1–3). This complexity has hitherto frustrated mechanistic analysis of such enzymes. We previously produced a mutant strain of S. erythraea in which the chain-terminating cyclase domain (TE) is fused to the carboxyl-terminus of DEBS1, the multienzyme that catalyzes the first two rounds of polyketide chain extension in S. erythraea . This mutant strain produces triketide lactone in vivo . We set out to purify the chimaeric enzyme and to determine its activity in vitro . Results: The purified DEBS1-TE multienzyme catalyzes synthesis of triketide lactones in vitro . The synthase specifically uses the (2S)-isomer of methylmalonyl-CoA, as previously proposed, but has a more relaxed specificity for the starter unit than in vivo . Conclusions: We have obtained a purified polyketide synthase system, derived from DEBS, which retains catalytic activity. This approach opens the way for mechanistic and structural analyses of active multienzymes derived from any modular polyketide synthase.

The lantibiotic actagardine A is nineteen amino acids in length and comprises three intertwined C-terminal methyllanthionine-bridged rings and an N-terminal lanthionine-bridged ring. Produced by the actinomycete Actinoplanes garbadinensis ATCC 31049, actagardine A demonstrates antibacterial activity against important Gram-positive pathogens. This activity combined with its ribosomal synthesis makes it an attractive target for the generation of lantibiotic variants with improved biological activity. A variant generation system designed to allow the specific substitution of amino acids at targeted sites throughout the actagardine A peptide has been used to generate a comprehensive library by site-directed mutagenesis. With the exception of residues involved in bridge formation, each amino acid in the actagardine A peptide as well as the alanine (ala(0)) at position -1 relative to the mature peptide, has been systematically substituted with all remaining 19 amino acids. A total of 228 mutants have been engineered with 44 produced in good yield. The mutant V15F in particular demonstrates improved activity against a range of notable Gram-positive pathogens including Clostridium difficile, when evaluated alongside actagardine A. The scope of variants generated provides an insight into the flexibility of the actagardine A processing machinery and will undoubtedly assist in future mutational studies.