Luiziana Ferreira da Silva Lab of Bioproducts Department of Microbiology Institute of Biomedical Sciences University of São Paulo - Brazil Microbial Genetics/Biotechnology – Microbial anticancer compounds Nitrogen Biological Fixation in sugarcane Bacterial Metabolism/Bacterial products – biopolymers, biosurfactants Bacterial Metabolism/Biotechnology – bacterial biopolymers Microbial ecology, Plant-microorganism interactions/Biotechnology Harmfull effects Human Animals Agriculture Environment Benefits Medicine Pharmaceutical industry Food Industry Agriculture Environment Roles of microbiomes Studies in humans Skin protection Obesity-associated gut microbiome Brain development & behavior Faecal bacteriotherapy in the treatment of recurrent C. dificile diarrhea. Roles to plant and animals A number of new surveys are helping scientists understand the many ecosystems our bodies offer to microbes and their interactions. A relevant part of our society is still unaware about the benefits and the contribution to our lives that were achieved because of the action of microorganisms Country extension Various climates Different environments Different crops cultivated Variable and heterogeneous natural microbiota New species discovered New genes Generation of bioproducts Platforms for alternative products or alternative routes Nitrogen fixing bacteria Antitumoral agents Endophytes Biopolymers 1986 isolated from Brazilian Savannah (cerrado) a soil showing high acidity, low levels of N and organic matter – Barbosa et al., 1986, Barbosa & Carvalhal, 1988 Physiologic characteristics related to the environment were studied in this bacterium: • Nitrogen fixing ability under adverse conditions: low pH and under high concentrations of toxic compounds • Role of exopolysaccharide in protecting the nitrogenase from oxygen deleterious effects • Stimulation of other bacteria in N-free medium • Liberation of aminoacids in N-free culture medium • Cyanophicin like intracelular reserves Dr.Heloiza R Barbosa E. coli pure & associate cultures B. derxii pure & associate cultures Stimulating both hetero and autothrophs, usually present in soil Biotechnological applications Purified granules can be chemically converted to a derivative with reduced arginine content or to completely biodegradable poly(aspartic acid) , which can be used as a substitute for nonbiodegradable polyacrylates with many technical and medical applications Nitrogenated reserve granules Cyanophycin-like? Under study Gonçalves de Lima et al. 1969 Retamycin Production of antitumoral agents Polyketydes (PKS) Anthracyclines are active against • breast cancer, • lymphomas, • acute leukemias, • neuroblastomas, • bone and soft-tissue sarcomas Dr.Gabriel Padilla Studies on the mechanism of action Chemical structures of cosmomycins produced by Streptomyces olindensis Garrido LM1, Lombó F, Baig I, Nur-E-Alam M, Furlan RL, Borda CC, Braña A, Méndez C, Salas JA, Rohr J, Padilla G. Genetic organization of the sequenced DNA region and constructs used for the generation of the different mutants. The black triangle indicates the apramycin resistance cassette. Restriction sites indicated with an asterisk are not unique sites. SaSau3AI; NcNcoI; BaBamHI; NrNruI Chemical structures of compounds isolated from Streptomyces olindensis ΔcosK (a) and S. olindensis ΔcosG (b) Proposed pathway for glycosylation events in cosmomycin D biosynthesis Dr.Welington L. Araujo Characterization of mutants a-1 b-1 c-1 f a-2 b-2 c-2 g a-3 d e h Random mutagenesis to identify genes related to biosynthetic pathway PKSi 1 KS AT PKSi 2 KS AT PKSi 3 KS AT PKSi 4 KS AT PKSi 5 KS D H D H M M DH K R (ER) D H M D H P P KR 2515 a.a. P P 2585 a.a. P P KR 2576 a.a. P P KR 1777 a.a. AT DH ER P P KR 2262 a.a. PKSi 7 KS P P AT P P TE 2145 a.a. PKSi 8 KS AT DH M ER P P KR K S A T K R D H E R T E Ketosynthase domain Acyl transferase domain Ketoreductase domain Dehidrathese domain Enoil reductase domain Thioesterase domain 2488 a.a. PKSi 9 KS P P AT M R 2576 a.a. PKSi 10 KS P P AT TE M Methyltransferase domain P P Acyl Carrier protrein domain H Hydrolase domain P Acyl CoA reductase domain R Ketosynthase domain 2069 a.a. PKSi 11 KS P P AT TE 2037 a.a. PKSi 12 KS P P AT P P M H 2637 a.a. K PKSIII S P 406 a.a. PKS-NRPS (PKSi6) KS AT D H M KR P P C A P P R 3981 a.a. 13 hypothetical PKS clusters identified in the E. nigrum P16 genome. Aminoacid numbers and domain positions are in scale Epicolactone New metabolite from Epicoccum nigrum Associated to microbial control Two PKS regulators were identified Identification of the PKS gene cluster Dr. J. Gregório C Gomez Dr. Luiziana F. Silva PHA are accumulated as intracelular granules by bacteria C4 & C5 Short-chain length monomers PHA scl Naturally PHA-accumulating bacteria produce either PHAscl or PHAmcl in large amounts C6 & C12 Medium-chain length monomers PHA mcl Monomer composition is responsible for PHA properties and applications 730 Features influencing PHA monomer composition Isolation & evaluation of soil bacteria on the ability to produce PHB from Sucrose, Glucose and Fructose Table 2. Production of P(3HB-co-3HV) from glucose plus propionic acid. PHA Residual CDWc 3HB mol% 3HV mol% Y3HV/Prpb 0,0 71,4 96,1 3,9 0,13 0,95 101,8 14,6 55,0 45,0 0,07 P.cepacia DSM 50181 3,35 1,9 38,4 97,3 2,7 0,04 IPT-040 3,77 1,7 32,3 97,1 2,9 0,05 IPT-044 3,92 1,7 51,1 97,1 2,9 0,07 IPT-045 3,73 0,0 49,4 96,2 3,8 0,08 IPT-048 2,97 0,0 44,3 96,2 3,8 0,06 IPT-055 4,27 72,2 1,5 100,0 0,0 0,00 IPT-056 3,60 31,3 30,9 98,5 1,5 0,02 IPT-076 5,06 1,9 56,8 97,1 2,9 0,10 IPT-083 4,89 5,2 56,8 96.9 3,1 0,10 IPT-086a 2,06 75, 39,0 89,9 10,1 0,09 IPT-098 5,90 0,0 17,7 94,7 5,3 0,07 IPT-101 2,98 41,8 32,3 95,4 4,6 0,05 CDWc (g/L) Carbohydrates (%) A. eutrophus DSM 54 3,92 A. latus DSM 1123 Strains % Gomez et al., 1996 P3HB-co-3HV from sucrose and propionic acid Phenotype/ strategy Strain 3HB 3HV Y3HV/prp* g/g mol% mol% B. sacchari wild type 93.8 6.2 0.10 189 prp UV mutant 43.6 56.4 0.90 189 Sucrose:propionate feeding rates 92.0 18.0 1.27 Improvement to incorporate 3HV units *Y3HV/prp = 3HV yield from propionic acid Maximum theoretical yield = 1.35 g/g 3HB C4-monomer PHA 3HV – C5 4kbp Inactivation on the propionate A EcoRI 2.9 kbp EcoRI 2 kbp 9 kbp EcoRI EcoRI SalI SalI EcoRI catabolic pathway 2MCC = 2 methyl citrate cycle B ORF1 ORF2 ORF3 ORF4 ORF6 ORF5 ORF7 ORF8 52bp ORF9 3HV content is dependent on propionic acid concentration 2MCC is more operative at low prp concentrations. A second prp catabolic pathway do exist? acnM/prpC deletion Control on 3HV content Sucrose:propionic ratio in the feeding media Sucrose Glucose Xylose Glycerol Fatty acids Plant oils Soybean molasses Others... Pseudomonas sp. B. sacchari E. coli Platforms for... PHA Rhamonolipids 1,3-Propanediol Others... Improving product yields biotechnological processes: in •Knockout or gene overexpression is no guarantee of success •A better knowledge of the metabolism is needed to engineer metabolism and improve biotechnological processes. Vallino & Stephanopoulos, 1992 Metabolic engineering advanced analytical tools to identify appropriate targets for genetic modifications mathematical models to perform in silico design of optimized cell factories. Nielsen & Jewett, 2008 FEMS Yeast Res. Fluxes analysis is representative of the phenotype of biotechnological interest .C (negligible) dC/dt = 0 (steady state) Metabolic pathway analysis PHAmcl Fluxes distribution for PHA production by Pseudomonas sp. from glucose. Optimal fluxes distribution for PHA production by Pseudomonas sp. from glucose. PHA production by wild type B. sacchari Fluxes under the theoretical maximum global PHA yield 0,49 0,49 0,48 0,01 0,01 1,24 1.16 1,65 1,73 Glucose Xylose Glycerol Fatty acids Plant oils Soybean molasses Pseudomonas sp. B. sacchari E. coli Platforms for... PHA Rhamonolipids 1,3-Propanediol Others... Brazilian biodiversity as new platforms J.Gregório C. Gomez Luiziana F. Silva Marilda Keico Taciro Karel Olavarria Gamez Rogério S. Gomes Johana K. Bocanegra-Rodriguez Thatiane T. Mendonça Gabriela C. Lozano Linda P. Guaman Bautista Liege A. Kawai Lucas Garbini Cespedes Diana Carolina Tusso Pizon Bernardo Ferreira Camilo Kelli Lopes Rodrigues Cesar W. Guzman Moreno Rafael Nahat Carlos Thandara Garcia Ravelli Juliano Cherix Edmar Ramos de Oliveira Filho Ruben Sanchez (UENF) Aline Carolina C. Lemos Alexandre A. Alves Aelson L. Santos Amanda B. Flora Karen L. Almeida Odalys Rodriguez Gamez Arelis Abalos Rodriguez Jhoanne Hansen Galo A. C. Le Roux - EPUSP Carlos A. M. Riascos Paulo Alexandrino André Fujita IME USP Juliana Cardinali Rezende Andreas K. Gombert (UNICAMP) Walter M. van Gulik Aljoscha Wahl Reza Maleki Seifar J. J. (Sef) Heijnen Kirsten Steinbusch – Waste2Chemical Niels van Stralen – Waste2Chemical NWO MES-Cuba TUDelft