System analysis, Metabolic Engineering and economic analysis on the use of hemicellulosic hydrolysates in bioprocesses

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Edith Atkins
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a soil metagenomic library of Atlantic Forest for genes involved on

2010/51989-4 From 01/01/2011 to 31/12/2012 FAPESP Process

1 System analysis, Metabolic Engineering and economic analysis on the use of hemicellulosic hydrolysates in bioprocesses Bioprospection in a soil metagenomic library of Atlantic Forest for genes involved on the biosynthesis of biodegradable polymers FAPESP Process 2010/ From 01/01/2011 to 31/12/2012 FAPESP Process 2010/ From 01/06/2010 to 30/05/2013 Profa. Dra. Luiziana Ferreira da Silva lukneif@usp.br 1

2 Polyhydroxyalkanoates (PHA )are biopolymers accumulated as intracelular granules by bacteria C4 & C5 Short-chain length monomers PHA scl C6 & C12 Medium-chain length monomers PHA mcl 2 2

3 Biodegradable and biocompatible PHA are thermoplastics Aplications Bucci, semanas Madison & Huisman,

4 Nonato 2012, Int Symp Biopol & other bioproducts 4

5 PHA production integrated to a sugar and etanol mill. Biorefinery Glycerol 5

Estate of the Art : Scenario in Brazil Second-generation ethanol from ligno & hemicellulose Chemical or enzymatic hydrolysis Sugar mixtures available for fermentation: Glucose, xylose,

6 Estate of the Art : Scenario in Brazil Second-generation ethanol from ligno & hemicellulose Chemical or enzymatic hydrolysis Sugar mixtures available for fermentation: Glucose, xylose, arabinose Yeasts are unable to ferment xylose efficiently Xylose & arabinose available in large amounts Perspective 10 7 ton/year of C5 available Use of xylose to generate other products 6

7 Topics addressed in projects FAPESP in the Lab of Bioproducts Biopolymer generation from xylose Production set up on a biorefinery Use of sugar mixtures (glucose, xylose & arabinose). Inhibitors present on hemicellulosic hydrolysates Catabolite repression & productivity Bioprospection of genes to the biosynthesis of polymers with different composition Controlling monomer composition on biopolymers & polymer propeties 7

8 Metodology Proc. 10/ Proc. 10/ Cloning of xylose catabolic genes, catabolite repression & other genes (xylab, xylfgh, crp*, tala) Contruction and evaluation of recombinants to reduce catabolite repression (shaken flasks & 5L bioreactor) Evolutive Engineering to obtain strains with higher m max on xylose Metabolic flux analysis with 13 C xylose (Metatool) PCR screening for PHA synthase genes from a metagenomic library Analysis of PHA synthases to correlate sequences to monomer specificity and to select the best targets for cloning Expression in hosts with different metabolic backgrounds Analysis of monomer composition(cg/ CGMS) Economic Evaluation of the process (Metatool e Super pro design ) 8 8

9 Items proposed vs objectives achieved To increase yields and productivity of bioprocesses using the hemicellulosic fraction of biomass. E coli KO11 and Burkholderia species Catabolite repression partially abolished on recombinant strains constructed Expression of genes (crp*, tala, etc) = increased polymer accumulation, yield * productivity (15%) 40% increase on Burkholderia m max when growing on xylose Economic analysis indicated new targets to make feasible polymer production: control of polymer accumulation, a target productivity value and high cell densities cultivation) Bioprospection of genes involved in the biosynthesis of new biopolymers and gene expression in different host searching for new biomaterials Approximately 300 positive clones were selected, belonging to 4 PHA synthase enzyme classes., currently under second screening, sequencing and cloning 9

10 Abolishing catabolite repression of natural PHB + isolates: Time of xylose consumption was reduced Xylose started to be consumed still in presence of glucose Deletion on PTS genes B. sacchari Growth experiments in triple sugar mixtures with wild type B. sacchari wild type (solid line) and B. sacchari PTS - Glucose + (dotted line): ( ) biomass, (-) glucose, (+) arabinose and ( ) xylose. 10

11 Abolishing catabolite repression of natural PHB + isolates PHB% P PHB were not improved PHB production experiment in triple sugar mixture with ammonium for wild type B. sacchari IPT101 (solid line) and B. sacchari LFM828 PTS - Glucose + (dotted line): (-) glucose, (+) arabinose, ( ) xylose, ( ) biomass, and ( ) PHB content of the cell dry weight (%PHB). 11

12 Abolishing catabolite repression of natural PHB + isolates Deletion on ccpa genes B. megaterium PHB production by B. megaterium (solid line) andb. megaterium DccpA MSL7 (dashed line) in mineral media supplemented with glutamate and sugar mixtures: ( ) glucose, (+) arabinose, ( ) xylose, ( ) cell dry weight (CDW), (x) PHB content from cell dry weight (%PHB) and, ( ) CO 2 production. 12

13 Isolate F24 (Burkholderia sp) can use toxic compounds from sugarcane hydrolysate Growth experiment with F24 in mineral media with xylose (10 g l -1 ) and individual compounds: ( ) 2.5 g l -1 of acetic acid, ( ) 1.25 g l -1 of formic acid, ( ) control experiment only with xylose, ( ) 0.5 g l -1 of HMF, and (x) 0.5 g l -1 of furfural. Effect of the inoculum size (g l -1 ) on utilization of hydrolysates (g l -1 ), cell growth (g l -1 ) and PHA biosynthesis (% of PHA of the cell dry weight) in hydrolysate medium after 48 hours

14 Burkholderia sp F24 using sugarcane bagasse hdrolysate to produce PHB P PHB = 0.28 g.l -1 h -1 Effect of hydrolysate concentration in high cell density cultivation with Burkholderia sp. F24: ( ) cell dry weight ( ) percentage of PHA.of cell dry weight. Solid line represents the hydrolysate and the dotted line represents de hydrolysate concentrated 3 times. Lopes et al., 2012

15 Economic assessment of biopolymer production integrated to a sugar and ethanol mill Raicher,

16 Target productivity 1 g l -1 h -1 Parameters to be improved Projections to improve the process to produce P3HB from hemicellulosic hydrolysate. Evaluating the impact of improving specific growth rates of bacteria and P3HB production Parameters Raicher experiments PHA content simulated scenarios How to improve? 1. Genetic improvement 2. Evolutive engineering 3. Evolutive engineering & Biochemical Engineering strategies 4. Metabolic Flux Analysis 16

17 Team Proc. 10/ Proc. 10/

18 Proc. 10/ B. sacchari genome sequencing 7.3 Mb 18

19 Proc. 10/

20 Further development and new parnerships Partnerships Cooperation with Waste2Chemical n (Netherlands) Lab Prof.Dr. Antonio Aprigio da Silva Curvelo IQSC USP hydrolysates! Prof Ruben Sánchez UENF polymer characterization Developments Biopolymer Iberoamerican Network PRIBOP CYTED Workshop on Sustainable production of biopolymers and other bio-based products 2012 ISBP 2014 International Symposium on Biopolymers Dra Rosane Piccoli mathematical modelling Good perspectives on competitivenes & national & international cooperation 20

21 METABOLIC ENGINEERING APPLIED TO THE PRODUCTION OF BIOPOLYMERS Young Investigator FAPESP process 10/ Coordinator Dr. Marilda Keico Taciro From Mar/2011 to Feb/2015 Team Marilda Keico Taciro PhD - Biochemical Engineer ICB USP Jose Gregório C. Gomez PhD Microbiology ICB USP Luiziana F. Silva PhD Microbiology ICB USP Rogerio Gomes PhD Biotechnology Inter-unities Program USP IB IPT Daniela C. P. Lício MSc Biotechnology Inter-unities Program USP IB IPT Liege A. Kawai MSc Biotechnology Microbioloy ICB USP Elisa B. Gragnani Undergraduation Molecular Sciences USP Lucas G. Cespedes Undergraduation Biological Sciences USP Rafael A. T. P. S. Nahat Undergraduation Polytechnic School USP Rafael F. Chenedezi Undergraduation Polytechnic School USP

22 Context Different meetings & forums indicated the need to incorporate new experimental & computational tools to increase a multidisciplinary approach and develop Metabolic Engineering in Brazil 2007 SINAFERM, Curitiba, PR 2008 CBAB Metabolic Engineering course 2009 SINAFERM Natal RN 2010 Opportunity to nucleate a ME group in the Microbiology Department, Institute of Biomedical Sciences, University of São Paulo 22

23 Items proposed & objectives achieved Case Study: P(3HB-co-3HA MCL ) produced by Pseudomonas strains To nucleate a Metabolic Engineering group at Microbiology Dept. ICB USP To generate polymers with different composition To increase bacterial yields and process productivity 23

24 Methodology Validating flux distribution : Flux Analysis & Metabolic pathway analysis ( metabolic net, softwares, elementary mode analysis) Tracer experiments NMR Batch, fed batch & continuous cultivations in bioreactors Cloning and expression of relevant genes 24

25 Items proposed & objectives achieved Development of a software to Metabolic Flux analysis using 13 C substrates Post graduation course on System Analysis of Metabolism, coordinator: Dr. JGC Gomez, 2011 and 2012 ( 15 students each) Computational & Experimental tools were set up Construction of recombinant strains Polymer with different compositions Recombinants tested on shaken flasks Kinetic parameters obtained in fed batch cultures Construction of metabolic model and experiments in silico Polymer characterization methodology was set up 25

26 PHA presenting new composition produced from glucose by recombinant strains of Pseudomonas sp. LFM461.

27 Metabolic fluxes analysis using 13 C Glucose Fluxes distribution in central metabolism based on isotopomers of PHA monomers MS fragments. Glicose 1.00 CO G6P F6P S7P P5P via de Entner Doudoroff G3P E4P P5P G3P P5P Isotopomer Analysis CO2 PYR AcCoA PHA

28 Publications International conferences: Taciro, M.K., Piccoli, R.A., Silva, L.F., Gomez, J.G., Pradella, J.G.C. Kinetic studies of pseudomonas sp growing in a chemostat culture. ISBP International Symposium on Biopolymers 2012 Cairns Austrália. Piccoli, R.A., Gragnani, E., Kawai, L., Silva, L.F., Gomez, J.G., Taciro, M. K. Metabolic modeling under fed batch cultivation of pseudomonas sp - a PHA producer. ISBP International Symposium on Biopolymers 2012 Cairns Austrália. Riascos, C.A.M., Le Roux, G.A.C., Gombert, A.K., Silva, L.F., Gomez, J.G.C. Metabolic flux analysis of PHAs production employing labeled glucose: developing a programming tool. ISBP International Symposium on Biopolymers 2012 Cairns Austrália. Lício, D. C. P., Lopes, K., Taciro, M.K., Silva, L.F., Gomez, J.G.C. Cloning and evaluation of pha production by recombinant strains harboring the class I pha synthase gene from pseudomonas sp. ISBP International Symposium on Biopolymers 2012 Cairns Austrália. Accepted: Cespedes, L.G., Gomez, J.G.C., Silva, L.F., Tavares, R.R., Mendonça, T.T., Taciro, M.K. Production of plastic films by bacteria from carbohydrates. ISSCT- International Society of Sugar Cane Technologists Congress 2013 São Paulo Brasil. 28

29 Further development and new partnerships Partnerships Prof. Dr. Carlos Riascos IQA UNAL - Colombia Prof. Dr. Galo C. Le Roux POLI - USP Prof. Dr. Andreas K. Gombert POLI - USP Profa. Dra. Regina Baldini IQ USP Dra. Rosane M Piccoli IPT SP Prof. Dr. Ruben Sanchez UENF RJ Developments Sustainable Production of Biopolymers and Other Bio-based Products ISBP 2014 Exchange of students Dra Silvia Batista - Inst Investigaciones Clemente Estable Uruguay Dra. Julia Petinari - Univ. Buenos Aires Argentina 29

Production of biodegradable plastics from bacterial cells

Production of biodegradable plastics from bacterial cells Prof a. Dr a. Luiziana Ferreira da Silva Laboratory of Bioproducts Institute of Biomedical Sciences University of São Paulo Brazil University of