Biomedical subjects

R McDaniel

Publications and source records attributed to R McDaniel.

At least 19 recordsLinked to original sources

PubMed2003

Approaches to stabilization of inter-domain recombination in polyketide synthase gene expression plasmids.

Regions of extremely high sequence identity are recurrent in modular polyketide synthase (PKS) genes. Such sequences are potentially detrimental to the stability of PKS expression plasmids used in the combinatorial biosynthesis of polyketide metabolites. We present two different solutions for circumventing intra-plasmid recombination within the megalomicin PKS genes in Streptomyces coelicolor. In one example, a synthetic gene was used in which the codon usage was reengineered without affecting the primary amino acid sequence. The other approach utilized a heterologous subunit complementation strategy to replace one of the problematic regions. Both methods resulted in PKS complexes capable of 6-deoxyerythronolide B analogue biosynthesis in S. coelicolor CH999, permitting reproducible scale-up to at least 5-l stirred-tank fermentation and a comparison of diketide precursor incorporation efficiencies between the erythromycin and megalomicin PKSs.

Base Sequence↗

PubMed2001

Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations.

Cassette replacement of acyltransferase (AT) domains in 6-deoxyerythronolide B synthase (DEBS) with heterologous AT domains with different substrate specificities usually yields the predicted polyketide analogues. As reported here, however, several AT replacements in module 4 of DEBS failed to produce detectable polyketide under standard conditions, suggesting that module 4 is sensitive to perturbation of the protein structure when the AT is replaced. Alignments between different modular polyketide synthase AT domains and the Escherichia coli fatty acid synthase transacylase crystal structure were used to select motifs within the AT domain of module 4 to re-engineer its substrate selectivity and minimize potential alterations to protein folding. Three distinct primary regions of AT4 believed to confer specificity for methylmalonyl-CoA were mutated into the sequence seen in malonyl-CoA-specific domains. Each individual mutation as well as the three in combination resulted in functional DEBSs that produced mixtures of the natural polyketide, 6-deoxyerythronolide B, and the desired novel analogue, 6-desmethyl-6-deoxyerythronolide B. Production of the latter compound indicates that the identified sequence motifs do contribute to AT specificity and that DEBS can process a polyketide chain incorporating a malonate unit at module 4. This is the first example in which the extender unit specificity of a PKS module has been altered by site-specific mutation and provides a useful alternate method for engineering AT specificity in the combinatorial biosynthesis of polyketides.

Acyltransferases↗

PubMed2001

Process development and metabolic engineering for the overproduction of natural and unnatural polyketides.

Polyketide natural products are a rich source of bioactive substances that have found considerable use in human health and agriculture. Their complex structures require that they be produced via fermentation processes. This review describes the strategies and challenges used to develop practical fermentation strains and processes for polyketide production. Classical strain improvement procedures, process development methods, and metabolic engineering approaches are described. The elucidation of molecular mechanisms that underlie polyketide biosynthesis has played an important role in each of these areas over the past few years.

Biological Factors↗

PubMed2001

Construction of desosamine containing polyketide libraries using a glycosyltransferase with broad substrate specificity.

BACKGROUND: Combinatorial biosynthesis techniques using polyketide synthases (PKSs) in heterologous host organisms have enabled the production of macrolide aglycone libraries in which many positions of the macrolactone ring have been manipulated. However, the deoxysugar moieties of macrolides, absent in previous libraries, play a critical role in contributing to the antimicrobial properties exhibited by compounds such as erythromycin. Since the glycosidic components of polyketides dramatically alter their molecular binding properties, it would be useful to develop general expression hosts and vectors for synthesis and attachment of deoxysugars to expand the nature and size of such polyketide libraries. RESULTS: A set of nine deoxysugar biosynthetic and auxiliary genes from the picromycin/methymycin (pik) cluster was integrated in the chromosome of Streptomyces lividans to create a host which synthesizes TDP-D-desosamine. The pik desosaminyl transferase was also included so that when the strain was transformed with a previously constructed library of expression plasmids encoding genetically modified PKSs that produce different macrolactones, the resulting strains produced desosaminylated derivatives. Although conversion of the macrolactones was generally low, bioassays revealed that, unlike their aglycone precursors, these novel macrolides possessed antibiotic activity. CONCLUSIONS: Based on the structural differences among the compounds that were glycosylated it appears that the desosaminyl transferase from the pik gene cluster is quite tolerant of changes in the macrolactone substrate. Since others have demonstrated tolerance towards modifications in the sugar substituent, one can imagine employing this approach to alter both polyketide and deoxysugar pathways to produce 'unnatural' natural product libraries.

Amino Sugars↗

PubMed2001

Combinatorial biosynthesis of antimicrobials and other natural products.

Combinatorial biosynthesis utilizes the enzymes from antibiotic (and other natural product) biosynthetic pathways to create novel chemical structures. The manipulation of modular polyketide synthases (PKSs) has been the major focus of this effort and has led to the production of, for example, several erythromycin analogs. Many new tools for manipulating and studying these multifunctional enzymes have been developed. These include multiple hosts and expression systems, enzymology tools for in vitro study, and ways to engineer pre-PKS and post-PKS pathways. The result is more rational and faster methods of engineering new compounds for the development of chemotherapeutic agents from natural products. The most significant recent advances in combinatorial biosynthesis are outlined.

Anti-Bacterial Agents↗

PubMed2001

Combinatorial biosynthesis in microorganisms as a route to new antimicrobial, antitumor and neuroregenerative drugs.

Combinatorial biosynthesis utilizes the genes of biosynthetic pathways that produce microbial products to create novel chemical structures. The engineering of mondular polyketide synthase (PKS) genes has been the major focus of this effort and has led to the production of analogs of macrolide antibiotics like the erythromycins and their derived ketolides, and of the immunosuppressive macrolide FK-520 (Fujisawa Pharmaceutical Co Ltd). Approaches to making analogs of the promising antitumor compounds known as epothilones are also being explored. Lead compounds for further study have resulted and routes to analogs of other pharmacologically important compounds have been established. To facilitate this work, many new tools for manipulating and studying the multifunctional PKSs have been developed including the development of Escherichia coli as a PKS expression last. These developments have resulted in faster ways of engineering PKS to produce new compounds for the development of chemotherapeutic agents from natural products.

Animals↗

PubMed2000

Formation of functional heterologous complexes using subunits from the picromycin, erythromycin and oleandomycin polyketide synthases.

BACKGROUND: Recently developed tools for the genetic manipulation of modular polyketide synthases (PKSs) have advanced the development of combinatorial biosynthesis technologies for drug discovery. Although many of the current techniques involve engineering individual domains or modules of the PKS, few experiments have addressed the ability to combine entire protein subunits from different modular PKSs to create hybrid polyketide pathways. We investigated this possibility by in vivo assembly of heterologous PKS complexes using natural and altered subunits from related macrolide PKSs. RESULTS: The pikAI and pikAII genes encoding subunits 1 and 2 (modules 1-4) of the picromycin PKS (PikPKS) and the eryAIII gene encoding subunit 3 (modules 5-6) of the 6-deoxyerythronolide B synthase (DEBS) were cloned in two compatible Streptomyces expression vectors. A strain of Streptomyces lividans co-transformed with the two vectors produced the hybrid macrolactone 3-hydroxynarbonolide. Co-expression of the same pik genes with the gene for subunit 3 of the oleandomycin PKS (OlePKS) was also successful. A series of hybrid polyketide pathways was then constructed by combining PikPKS subunits 1 and 2 with modified DEBS3 subunits containing engineered domains in modules 5 or 6. We also report the effect of junction location in a set of DEBS-PikPKS fusions. CONCLUSIONS: We show that natural as well as engineered protein subunits from heterologous modular PKSs can be functionally assembled to create hybrid polyketide pathways. This work represents a new strategy that complements earlier domain engineering approaches for combinatorial biosynthesis in which complete modules or PKS protein subunits, in addition to individual enzymatic domains, are used as building blocks for PKS engineering.

Anti-Bacterial Agents↗

PubMed2000

Does geographic range affect the attractant-aggregation-attachment pheromone of the tropical bont tick, amblyomma variegatum?

The tropical bont tick, Amblyomma variegatum, transmits heartwater in sub-Saharan Africa and in the Caribbean. This species has a broad geographic distribution, ranging from Madagascar and other islands in the Indian Ocean through most of sub-Saharan Africa, to several islands in the eastern Caribbean Sea. Blood fed male A. variegatum secrete an attraction-aggregation-attachment (AAA) pheromone which, combined with CO2, excites host finding and formation of feeding clusters of these ticks. However, it is not known whether the composition of the pheromone varies throughout A. variegatum's geographic range. Extracts of fed male ticks were examined for phenols and volatile organic acids by gas chromatography and mass spectrometry to determine whether differences occur in the pheromone components of populations of this species across the geographic range (Guadeloupe, Zimbabwe, Zambia and Rwanda). No significant difference in the chemical composition of the pheromone in relation to geographic range was found. No significant differences in rates of attachment in response to native versus foreign extracts were found in on-host attachment tests comparing ticks from two countries. Guadeloupe (Caribbean) and Zimbabwe (African). This finding was confirmed in more detailed studies with ticks from Guadeloupe and four African countries (Kenya, Rwanda, Zambia and Zimbabwe). On-host attachment assays from these countries did not detect consistent differences in response to extracts from different locations. In an olfactometer bioassay, females were not consistently more attracted to extracts from their native locality than from any of the foreign localities. We conclude that despite the widespread distribution of A. variegatum over both hemispheres, no significant differences in pheromone composition or biological responses to male tick pheromone secretions occur.

Animals↗

PubMed2000

Biosynthesis of the anti-parasitic agent megalomicin: transformation of erythromycin to megalomicin in Saccharopolyspora erythraea.

Megalomicin is a therapeutically diverse compound which possesses antiparasitic, antiviral and antibacterial properties. It is produced by Micromonospora megalomicea and differs from the well-known macrolide antibiotic erythromycin by the addition of a unique deoxyamino sugar, megosamine, to the C-6 hydroxyl. We have cloned and sequenced a 48 kb segment of the megalomicin (meg) biosynthetic gene cluster which contains the modular polyketide synthase (PKS) and the complete pathway for megosamine biosynthesis. The similarities and distinctions between the related megalomicin and erythromycin gene clusters are discussed. Heterologous expression of the megalomicin PKS in Streptomyces lividans led to production of 6-deoxyerythronolide B, the same macrolactone intermediate for erythromycin. A 12 kb fragment harbouring the putative megosamine pathway was expressed in Saccharopolyspora erythraea, resulting in the conversion of erythromycin to megalomicin. Considering the extensive knowledge surrounding the genetic engineering of the erythromycin PKS and the familiarity with genetic manipulation and fermentation of S. erythraea, the ability to produce megalomicin in this strain should allow the engineering of novel megalomicin analogues with potentially improved therapeutic activities.

Anti-Bacterial Agents↗

PubMed2000

Cloning, characterization and heterologous expression of a polyketide synthase and P-450 oxidase involved in the biosynthesis of the antibiotic oleandomycin.

The gene cluster encoding the deoxyoleandolide polyketide synthase (OlePKS) was isolated from the oleandomycin producing strain Streptomnyces antibioticus. Sequencing of the first two genes encoding OlePKS, together with the previously identified third gene revealed an overall genetic and protein architecture similar to that of the erythromycin gene cluster encoding the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. When the entire OlePKS (10,487 amino acids) was expressed in the heterologous host Streptomyces lividans, it produced 8,8a-deoxyoleandolide, an aglycone precursor of oleandomycin. The role of the P-450 monooxygenase, OleP, in oleandomycin biosynthesis was also examined in vivo by co-expression with DEBS in S. lividans. The production of 8,8a-dihydroxy-6-deoxyerythronolide B and other derivatives indicates that OleP is involved in the epoxidation pathway of oleandomycin biosynthesis. Since there are currently no genetic systems available for manipulation of the natural oleandomycin producing strain, the heterologous expression system reported here provides a useful tool for studying this important macrolide antibiotic.

Anti-Bacterial Agents↗

PubMed1999

Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel "unnatural" natural products.

The structures of complex polyketide natural products, such as erythromycin, are programmed by multifunctional polyketide synthases (PKSs) that contain modular arrangements of functional domains. The colinearity between the activities of modular PKS domains and structure of the polyketide product portends the generation of novel organic compounds-"unnatural" natural products-by genetic manipulation. We have engineered the erythromycin polyketide synthase genes to effect combinatorial alterations of catalytic activities in the biosynthetic pathway, generating a library of >50 macrolides that would be impractical to produce by chemical methods. The library includes examples of analogs with one, two, and three altered carbon centers of the polyketide products. The manipulation of multiple biosynthetic steps in a PKS is an important milestone toward the goal of producing large libraries of unnatural natural products for biological and pharmaceutical applications.

Catalytic Domain↗

PubMed1999

Novel macrolides through genetic engineering.

Erythromycin, a complex polyketide antibiotic belonging to the macrolide class, is produced as a natural product by the bacterium Saccharopolyspora erythraea. The genes encoding the enzymes responsible for the synthesis of the antibiotic have been cloned and sequenced, revealing that the polyketide backbone of the molecule in produced by a polyketide synthase (PKS) composed of multifunctional proteins that contain discrete functional domains for each step of synthesis. Genetic manipulation of the PKS-encoding genes can result in predictable changes in the structure of the polyketide component of erythromycin, many of which are not easily achievable through standard chemical derivatization or synthesis. Many of the changes can be combined to lead to the further generation of navel structures. Whereas genetic engineering of the erythromycin structure has been practiced for a number of years, the re cent and continuing discoveries of modular PKSs for the synthesis of many other important complex polyketides has raised the possibility of generating novel structures in these molecules by genetic manipulation, as well.

Genetic Engineering↗

PubMed1999

Elucidating the mechanism of chain termination switching in the picromycin/methymycin polyketide synthase.

BACKGROUND: A single modular polyketide synthase (PKS) gene cluster is responsible for production of both the 14-membered macrolide antibiotic picromycin and the 12-membered macrolide antibiotic methymycin in Streptomyces venezuelae. Building on the success of the heterologous expression system engineered using the erythromycin PKS, we have constructed an analogous system for the picromycin/methymycin PKS. Through heterologous expression and construction of a hybrid PKS, we have examined the contributions that the PKS, its internal thioesterase domain (pikTE) and the Pik TEII thioesterase domain make in termination and cyclization of the two polyketide intermediates. RESULTS: The picromycin/methymycin PKS genes were functionally expressed in the heterologous host Streptomyces lividans, resulting in production of both narbonolide and 10-deoxymethynolide (the precursors of picromycin and methymycin, respectively). Co-expression with the Pik TEII thioesterase led to increased production levels, but did not change the ratio of the two compounds produced, leaving the function of this protein largely unknown. Fusion of the PKS thioesterase domain (pikTE) to 6-deoxyerythronolide B synthase (DEBS) resulted in formation of only 14-membered macrolactones. CONCLUSIONS: These experiments demonstrate that the PKS alone is capable of catalyzing the synthesis of both 14- and 12-membered macrolactones and favor a model by which different macrolactone rings result from a combination of the arrangement between the module 5 and module 6 subunits in the picromycin PKS complex and the selectivity of the pikTE domain.

Amino Acid Sequence↗

PubMed1998

Macrolide biosynthesis: a single cytochrome P450, PicK, is responsible for the hydroxylations that generate methymycin, neomethymycin, and picromycin in Streptomyces venezuelae.

The final step in the biosynthesis of methymycin, neomethymycin, and picromycin is an hydroxylation, shown to be carried out by the cytochrome P-450 monooxygenase, PicK. Direct comparison of the relative Kcat/K(m) values for the two substrates, YC-17 and narbomycin, showed a threefold rate preference of picK for narbomycin.

Anti-Bacterial Agents↗

PubMed1998

Characterization of the macrolide P-450 hydroxylase from Streptomyces venezuelae which converts narbomycin to picromycin.

The post-polyketide synthase (PKS) biosynthetic tailoring of macrolide antibiotics usually involves one or more oxidation reactions catalyzed by cytochrome P450 monooxygenases. As the specificities of members from this class of enzymes vary significantly among PKS gene clusters, the identification and study of new macrolide P450s are important to the growing field of combinatorial biosynthesis. We have isolated the cytochrome P450 gene picK from Streptomyces venezuelae which is responsible for the C-12 hydroxylation of narbomycin to picromycin. The gene was located by searching regions proximal to modular PKS genes with a probe for macrolide P450 monooxygenases. The overproduction of PicK with a C-terminal six-His affinity tag (PicK/6-His) in Escherichia coli aided the purification of the enzyme for kinetic analysis. PicK/6-His was shown to catalyze the in vitro C-12 hydroxylation of narbomycin with a kcat of 1.4 s-1, which is similar to the value reported for the related C-12 hydroxylation of erythromycin D by the EryK hydroxylase. The unique specificity of this enzyme should be useful for the modification of novel macrolide substrates similar to narbomycin, in particular, ketolides, a promising class of semisynthetic macrolides with activity against erythromycin-resistant pathogens.

Amino Acid Sequence↗

PubMed1998

Understanding change in primary care practice using complexity theory.

BACKGROUND: Understanding the organization of primary care practices is essential for implementing changes related to delivery of preventive or other health care services. A theoretical model derived from complexity theory provides a framework for understanding practice change. METHODS: Data were reviewed from brief participant observation fieldnotes collected in the 84 practices of the Direct Observation of Primary Care (DOPC) study and in 27 practices from three similar studies investigating preventive services delivery. These data were synthesized with information from an extensive search of the social science, nursing, and health services literature concerning practice organization, and of the literature on complexity theory from the fields of mathematics, physics, biology, management, medicine, and family systems, to create a complexity model of primary care practice. RESULTS: Primary care practices are understood as complex adaptive systems consisting of agents, such as patients, office staff, and physicians, who enact internal models of income generation, patient care, and organizational operations. These internal models interact dynamically to create each unique practice. The particular shape of each practice is determined by its primary goals. The model suggests three strategies for promoting change in practice and practitioner behavior: joining, transforming, and learning. CONCLUSIONS: This model has important implications for understanding change in primary care practice. Practices are much more complex than present strategies for change assume. The complexity model identified why some strategies work in particular practices and others do not.

Family Practice↗

PubMed1998

Establishing mechanisms to conduct multi-institutional research--fatigue in patients with cancer: an exercise intervention.

PURPOSES/OBJECTIVES: To describe the process of establishing a multi-institutional interdisciplinary team of oncology researchers and conducting a pilot study of an exercise intervention for fatigue. DATA SOURCES: Project meeting minutes and records, research team members' logs, subjects' research records, the research study proposal, and team members' individual and collective shared experiences. DATA SYNTHESIS: Site investigators established research teams at five academic medical centers. Fifty subjects were enrolled in the study and tested during their cancer treatment. Study methods, including instrumentation, were evaluated carefully and revised. CONCLUSIONS: The multi-institutional network of researchers is an effective and efficient model for testing an intervention to manage fatigue during cancer treatment. IMPLICATIONS FOR NURSING PRACTICE: Exercise is a feasible and potentially beneficial intervention to combat distressing cancer treatment-related fatigue. A pilot study is essential to determine the best methods for conducting a clinical trial and to develop the teams of researchers necessary for such a project.

Exercise Therapy↗

PubMed1997

Urologic manpower issues for the 21st century: assessing the impact of changing population demographics.

OBJECTIVES: To evaluate the impact of changing population demographics on urologic staffing over the coming decades. METHODS: A model was constructed using data obtained from the U.S. Bureau of the Census for population projections; clinical studies to assess the percentages of men with symptomatic benign prostatic hyperplasia (BPH) and those undergoing prostatectomy; the American Medical Association regarding numbers and annual percent change of practicing urologists; and the American Urological Association regarding numbers of physicians completing residency training programs. Sensitivity analyses were performed varying both the rate of surgical intervention for symptomatic BPH and the annual increase in the number of practicing urologists. RESULTS: Regardless of variations in the surgical rate to as low as 4%, the average number of transurethral resections of the prostate gland/surgical interventions for BPH per urologist will increase by the year 2020 when compared with the known basepoint value obtained for 1990. Additionally, even with an annual net increase of 200 urologists per year, by 2020, the rapidly expanding population over 65 years of age will nearly offset even such a large increase in the number of practicing urologists. CONCLUSIONS: The greatest factor concerning future urologic staffing issues will be the changing population demographics. The need for urologic services will continue to rise. An oversupply of urologists can be avoided as long as the net increase does not exceed an average of 200 urologists annually.

Age Factors↗