Bringing Protein Engineering and Natural Product Biosynthesis Together

In this issue of Chemistry & Biology, Zhang and colleagues developed a yeast cell surface display strategy to effectively evolve the substrate specificity of DhbE, one of the adenylation domains of the bacillibactin synthetase complex. The method yields DbhE variants that have dramatically altered substrate specifities toward unnatural aryl substrates

Design strategy for creating catalytically active metal binding proteins

Metalloenzymes catalyze a wide variety of reactions in nature by taking advantage of the versatility and reactivity of transition metals. Despite the diversity of reactions catalyzed by natural proteins, there is still a demand for designer enzymes. In many cases, all that is needed is routine re-engineering of the native enzymes to perform efficiently under the demanded application conditions. In other cases, the reaction or reaction condition desired differs so much from natural conditions that mere redesign of natural proteins is not practical. De novo enzymes, which are generated entirely from first principles rather than modified from natural proteins, are ideal for these situations. These de novo enzymes would allow us to generate enzymes that can survive at much higher temperatures, work in many different solvents and solutions, or perform completely novel functions. Please click Additional Files below to see the full abstract

Next-generation sequencing approach for connecting secondary metabolites to biosynthetic gene clusters in fungi

Genomics has revolutionized the research on fungal secondary metabolite (SM) biosynthesis. To elucidate the molecular and enzymatic mechanisms underlying the biosynthesis of a specific SM compound, the important first step is often to find the genes that responsible for its synthesis. The accessibility to fungal genome sequences allows the bypass of the cumbersome traditional library construction and screening approach. The advance in next-generation sequencing (NGS) technologies have further improved the speed and reduced the cost of microbial genome sequencing in the past few years, which has accelerated the research in this field. Here, we will present an example work flow for identifying the gene cluster encoding the biosynthesis of SMs of interest using an NGS approach. We will also review the different strategies that can be employed to pinpoint the targeted gene clusters rapidly by giving several examples stemming from our work.Ralph A. Cacho is supported by the NIH Chemistry-Biology Interface Training Grant (NRSA GM-08496) and UCLA Graduate Division. Yi Tang is supported by the NIH grant 1DP1GM106413. Yit-Heng Chooi is currently supported by an Australian Research Council (ARC) Discovery Early Career Researcher Award (DECRA)

Linking Globalization, Economic Growth and Poverty: Impacts of Agribusiness Strategies on Sub-Saharan Africa

Os intérpretes dos manuscritos de Leonardo da Vinci partilham dos mesmos sentimentos de espanto e de fascínio quando examinam sua contribuição para a ciência moderna. Podemos, contudo, perceber uma constante tentativa em prol de uma revisão histórica acerca do papel desempenhado por Leonardo. Observando a história dessas revisões, é possível detectar aspectos significativos das perspectivas históricas e historiográficas dos envolvidos nessa discussão. É o que pretendemos fazer neste trabalho, focando a controvérsia entre Duhem, por um lado, e Sarton, Koyré e Rossi, por outro. Ao fazer isso, buscamos discutir alguns traços que marcam a distinção entre uma historiografia mais antiga e a nova historiografia da ciência, tal como exposta por Thomas Kuhn.392538

Development and Validation of a Risk Score for Chronic Kidney Disease in HIV Infection Using Prospective Cohort Data from the D:A:D Study

Ristola M. on työryhmien DAD Study Grp ; Royal Free Hosp Clin Cohort ; INSIGHT Study Grp ; SMART Study Grp ; ESPRIT Study Grp jäsen.Background Chronic kidney disease (CKD) is a major health issue for HIV-positive individuals, associated with increased morbidity and mortality. Development and implementation of a risk score model for CKD would allow comparison of the risks and benefits of adding potentially nephrotoxic antiretrovirals to a treatment regimen and would identify those at greatest risk of CKD. The aims of this study were to develop a simple, externally validated, and widely applicable long-term risk score model for CKD in HIV-positive individuals that can guide decision making in clinical practice. Methods and Findings A total of 17,954 HIV-positive individuals from the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study with >= 3 estimated glomerular filtration rate (eGFR) values after 1 January 2004 were included. Baseline was defined as the first eGFR > 60 ml/min/1.73 m2 after 1 January 2004; individuals with exposure to tenofovir, atazanavir, atazanavir/ritonavir, lopinavir/ritonavir, other boosted protease inhibitors before baseline were excluded. CKD was defined as confirmed (>3 mo apart) eGFR In the D:A:D study, 641 individuals developed CKD during 103,185 person-years of follow-up (PYFU; incidence 6.2/1,000 PYFU, 95% CI 5.7-6.7; median follow-up 6.1 y, range 0.3-9.1 y). Older age, intravenous drug use, hepatitis C coinfection, lower baseline eGFR, female gender, lower CD4 count nadir, hypertension, diabetes, and cardiovascular disease (CVD) predicted CKD. The adjusted incidence rate ratios of these nine categorical variables were scaled and summed to create the risk score. The median risk score at baseline was -2 (interquartile range -4 to 2). There was a 1: 393 chance of developing CKD in the next 5 y in the low risk group (risk score = 5, 505 events), respectively. Number needed to harm (NNTH) at 5 y when starting unboosted atazanavir or lopinavir/ritonavir among those with a low risk score was 1,702 (95% CI 1,166-3,367); NNTH was 202 (95% CI 159-278) and 21 (95% CI 19-23), respectively, for those with a medium and high risk score. NNTH was 739 (95% CI 506-1462), 88 (95% CI 69-121), and 9 (95% CI 8-10) for those with a low, medium, and high risk score, respectively, starting tenofovir, atazanavir/ritonavir, or another boosted protease inhibitor. The Royal Free Hospital Clinic Cohort included 2,548 individuals, of whom 94 individuals developed CKD (3.7%) during 18,376 PYFU (median follow-up 7.4 y, range 0.3-12.7 y). Of 2,013 individuals included from the SMART/ESPRIT control arms, 32 individuals developed CKD (1.6%) during 8,452 PYFU (median follow-up 4.1 y, range 0.6-8.1 y). External validation showed that the risk score predicted well in these cohorts. Limitations of this study included limited data on race and no information on proteinuria. Conclusions Both traditional and HIV-related risk factors were predictive of CKD. These factors were used to develop a risk score for CKD in HIV infection, externally validated, that has direct clinical relevance for patients and clinicians to weigh the benefits of certain antiretrovirals against the risk of CKD and to identify those at greatest risk of CKD.Peer reviewe

Insights in the Biosynthesis of Griseofulvin and Echinocandin B, two Antifungal Compounds from Ascomycetes

Natural products, since the discovery of the first antibiotic penicillin in 1928, have been the source of and inspiration for drugs. Due the emergence of drug-resistant pathogens and discovery of drug targets in cancer biology, the need for the discovery of new bioactive natural compounds and synthesis of analogs thereof remains present. Fortuitously, the development of next-generation sequencing and tools for the heterologous expression of the biosynthetic genes for natural products has accelerated the discovery of new natural products as well as the elucidation of the respective biosynthetic pathways. This dissertation describes the use of these two emerging technologies in revealing insights in the biosynthesis of two antifungal natural compounds synthesized by filamentous fungi: griseofulvin, an antifungal drug used in treatment against dermatophytes and is in the World Health Organization List of Essential Medicine and echinocandin B, the parent compound of the frontline anti-Candidiasis drug anidulafungin.This dissertation will first present the work in the investigation the biosynthesis of echinocandin B. Invasive candidiasis caused by opportunistic pathogenic strains of genus Candida, accounts for 17% of ICU-related infections and through understanding of the biosynthesis of echinocandin B will aid in the mutasynthesis of analogs with better efficacy than the parent compound. In this work, we characterized the key steps in the biosynthesis of the natural product. Deletion of the biosynthetic genes also allowed for the mutasynthesis of echinocandin B analogs with more potent antifungal activity and showed the potential for re-engineering of the pathway to produce “unnatural” natural compounds.Moreover, this work also describes the full elucidation and reconstitution of the enzymatic reactions leading to the biosynthesis of griseofulvin using a combination of gene knockout and biochemical characterization of the griseofulvin biosynthetic enzymes. Among the important biosynthetic enzymes characterized in this study is the non-reducing polyketide synthase GsfA which was implicated in the biosynthesis of norlichexanthone and the cytochrome P450 GsfF which catalyzes the formation of the spirobicyclic grisan ring in griseofulvin. The culmination of this study is the demonstration of the total in vitro biosynthesis of the compound using purified griseofulvin biosynthetic enzymes

Insights in the Biosynthesis of Griseofulvin and Echinocandin B, two Antifungal Compounds from Ascomycetes

Natural products, since the discovery of the first antibiotic penicillin in 1928, have been the source of and inspiration for drugs. Due the emergence of drug-resistant pathogens and discovery of drug targets in cancer biology, the need for the discovery of new bioactive natural compounds and synthesis of analogs thereof remains present. Fortuitously, the development of next-generation sequencing and tools for the heterologous expression of the biosynthetic genes for natural products has accelerated the discovery of new natural products as well as the elucidation of the respective biosynthetic pathways. This dissertation describes the use of these two emerging technologies in revealing insights in the biosynthesis of two antifungal natural compounds synthesized by filamentous fungi: griseofulvin, an antifungal drug used in treatment against dermatophytes and is in the World Health Organization List of Essential Medicine and echinocandin B, the parent compound of the frontline anti-Candidiasis drug anidulafungin.This dissertation will first present the work in the investigation the biosynthesis of echinocandin B. Invasive candidiasis caused by opportunistic pathogenic strains of genus Candida, accounts for 17% of ICU-related infections and through understanding of the biosynthesis of echinocandin B will aid in the mutasynthesis of analogs with better efficacy than the parent compound. In this work, we characterized the key steps in the biosynthesis of the natural product. Deletion of the biosynthetic genes also allowed for the mutasynthesis of echinocandin B analogs with more potent antifungal activity and showed the potential for re-engineering of the pathway to produce “unnatural” natural compounds.Moreover, this work also describes the full elucidation and reconstitution of the enzymatic reactions leading to the biosynthesis of griseofulvin using a combination of gene knockout and biochemical characterization of the griseofulvin biosynthetic enzymes. Among the important biosynthetic enzymes characterized in this study is the non-reducing polyketide synthase GsfA which was implicated in the biosynthesis of norlichexanthone and the cytochrome P450 GsfF which catalyzes the formation of the spirobicyclic grisan ring in griseofulvin. The culmination of this study is the demonstration of the total in vitro biosynthesis of the compound using purified griseofulvin biosynthetic enzymes

Linking Globalization, Economic Growth and Poverty: Impacts of Agribusiness Strategies on Sub-Saharan Africa

This paper analyzes the increased role of the domestic and multinational private sectors in economic development within SSA. The globalization process demands that private sector strategies must now be assessed by their contributions to emerging economies, as well as by company goals