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MANIPULATION OF BACTERIA TO OPTIMIZE SYNTHESIS OF BIOPRODUCTS FROM by bvo16289

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									MANIPULATION OF BACTERIA
TO OPTIMIZE SYNTHESIS OF
   BIOPRODUCTS FROM
       GLYCEROL

     M. Julia Pettinari
    Facultad de Ciencias Exactas y
    Naturales
    Universidad de Buenos Aires, Argentina
Yazdani SS, Gonzalez R..Anaerobic fermentation of glycerol: a path to economic viability for
the biofuels industry. Curr Opin Biotechnol. 2007 Jun;18(3):213-9.
Metabolic Engeneering of microbial
            processes
Strategies:

  Substrate manipulation
  Use of inhibitors
  (Genetic) Manipulation of metabolic
  pathways

 Need for deep knowledge of microorganism´s
 metabolism and genetics
 Recombinant Escherichia coli
       Biopolymers - bioplastics
Polyhydroxyalkanoates       (PHAs):

Linear polymers constituted by
hydroxyesther units.

Accumulated as intracellular
granules by many bacteria as a
response to environmental
stress or nutritional imbalance
                                   Alcaligenes eutrophus


 Their thermoplastic and elastomeric properties make
 them suitable to replace petroleum derived plastics
        PHA biosynthesis pathways
PHA synthesis and degradation are cyclic.

   Synthesis: Acyl
CoA precursors
                                               NADPH
obtained through                              consumed
different
pathways are
converted into
PHAs.                  NADH
                      produced
  Depolimerization
produces Acyl-
CoAs and Acetyl-
CoA, that are used
as carbon and
energy source                               3- hydroxybutyric acid
     Catabolism in PHB accumulating recombinant E. coli
                            Glucose
                                              NADP NADPH
        Glycerol
 NAD+                       Glu-6P                               Gluconate-
 NADH                                          G6PDH             6P             NADP
                      Pgi
                                                                                NADPH
          DHAP         Fru-6P
                                     2X                                   Pentoses-
                                       Gli-3P                             P
                            NAD+
                            NADH                           2NADH 2NAD+
                                      Pyruvate                            Ethanol
          NAD+     NADH


 Formate                             Acetyl-CoA
                                                                Pta-            Acetate
             Ldh
                     NADH
                                                                Ack
  Lactate                              Acetoacetyl-CoA
              NAD+                                                       TCA Cycle
                                   NADPH
                                     NADP                                NAD+   NADH

G6PDH:Glucose -6-P
                                            Hydroxybutiryl-
dehydrogenase                               CoA                           Glycolisis (E-M-P)
Pgi:Phosphoglucose isomerase                     PHB                      Entner-Doudoroff
Ldh:lactate dehydrogenase
Pta:phosphotransacetylase
         The E. coli regulatory network


transcrip
tion
factors




 genes




            Agustino Martínez-Antonio and Julio Collado-Vives (2003) Curr. Opin. Microbiol. 6: 482
      Regulation of aerobic and anaerobic
             metabolism in E. coli

 Transcriptional regulation via two global regulators :
 ArcA (aerobic respiration control) and FNR (fumarate
                  nitrate respiration).
  These regulators respond to O2 and/or alternative
           electron acceptor availability.
         Some genes regulated by ArcA
- Krebs cycle genes (e.g.; sucABCD, sdhCDAB, fumA, mdh) and
glyoxalate cycle (e.g.; icdA, aceB)
- Citochrome o oxidase (cyoABCDE, low affinity for O2) and
citochrome d oxidase (cydAB, high affinity for O2)
           Hypothesis

The E. coli arcA mutants are unregulated for
aerobic pathways in low O2 availability conditions,
and the pool of reducing equivalents as NADH or
NADPH is increased.

We hypothesize that the accumulation of PHB will
be favored in an arcA genetic background due to
the generation of an excess reducing power that
could be funneled into an electron sink.
 Effects of PHB on O2 consumption and NADH pool

                                              Wild
                                              type

                                                                SP314 / pQE32




                                                                   SP314 / pJP24




                                                                   SP314: arcA mutant
       Toluidine blue                             Diamide          pQE32: vector plasmid
         sensitivity                             sensitivity       pJP24: pQE32 with pha genes

FEMS Microbiology Letters (2006). 258: 55–60
Applied and Environmental Microbiology (2006). 72: 2614-2620   Diamide: 1,1´-azobis-(N,N-dimetilformamida)
     Preliminary characterization of arcA
      mutants for PHB accumulation in
              microaerobiosis




                                                         * PHB < 5%
SP314: arcA mutant
pQE32: vector plasmid         Semisynthetic medium with 3% glucose
pJP24: pQE32 with pha genes




SP314/pJP24
                                  K1060 / pJP24
                                     (ArcA+)

                                   CT1061 / pJP24
                                      (arcA)




Bioreactor cultures in
semisynthetic medium with
glucose (MYAG) in
microaerobiosis (no air supply,
magnetic stirrer at 75 rpm)
PHB accumulation in CT1061/pJP24 (arcA2)
 growing on glycerol in microaerobiosis
                      PHB
                   content (%
                     CDW)

          CDW                   Bioreactor cultures
                                in semisynthetic
                                medium with glucose
                                (MYAG) in
                                microaerobiosis (no
                                air supply, magnetic
                                stirrer at 75 rpm)



                    PHB
                 production
    Effect of arcA on PHB producing E. coli
                  Conclusions
Increased redox power avaliability, obtained through mutations
in global regulator ArcA, enabled PHB synthesis in low O2
availiability conditions.

Lower aireation needs could improve the production process
reducing energy utilization.
 increased sustainability
 lower energy-related costs.
Ethanol production from glycerol in arcA
               mutants


                                   Ethanol (mM)
 Growth condition   K1060            CT1062          CT1061
                                     (arcA)         (arcA2)
Aerobic                     ND         0.48  0.07    18.13  0.17
Microaerobic         4.58  0.02      44.43  0.54   111.31  4.97
Metabolic characterization of arcA strains
growing on glycerol
                                      60
     Acetate   Formate    Succinate
     Lactate   Pyruvate   Ethanol     50
                                              CT1062 (arcA)
60
                                      40
50                    K1060 (wt)
                                      30
40
                                      20
30
                                      10
20
                                      0
10
                                      120
 0                                    110
                                      100
                                       90
                                       80   CT1061 (arcA2)
                                       70
                                       60
 Cultures grown for 48 hs in MYA-      50
 glycerol 3% in microaerobic           40
                                       30
 conditions                            20
                                       10
                                        0
                                   K1060 (arcA+) = 0,87
      Ethanol/acetate
                                   CT1062 ( arcA) = 2,01
          Molar ratio
                                   CT1061 (arcA2) = 12,51



 Reducing power formation in microaerobic cultures growing on
                           glicerol
 Strain      NADH          NAD+        Total NAD(H/+)     NADH/NAD+
K1060      0.51  0.06   2.83  0.15     3.34  0.21      0.18  0.03
CT1062     1.96  0.09   3.12  0.22     5.08  0.31      0.63  0.07
CT1061     2.54  0.11   2.62  0.08     5.16  0.19      0.97  0.07

Amounts in micromoles per g CDW
 CT1061 (arcA2) grows more thanT1062 (arcA)
 It has less nutritional requirements
 Higher oxygen consumption rates
 More reduced redox state
 Increased production of reduced compounds



  What is the genetic difference
  bewteen CT1061 and CT1062?
           Genomic Organization of the arcA region
                     in E. coli CT1061

                     3’-IR                                      5’-IR
   513 pb                                                                 212 pb
   arcA´                                                                  ´arcA
(NH2 terminal)                          IS10 (1330 pb)              (COOH terminal)


Detection of ArcA in
   Western blot
                      CT1061
            CT1062
   K1060




                                              ArcA2 Characteristics
                                              Bases = 513
                               29 kDa         aminoacids = 174 (3 aminoacids from IS10-L)
                                              Predicted Mr = 19,42 kDa

                               19 kDa         [wild type ArcA = 29 kDa]
    E. coli CT1061 construction
K1060  P1[ECL618 (lac proAB)XIII arcA2 zij::Tn10]

                               Tet selection

             CT1061 (arcA2 zij::Tn10)



   Genomic Organization of the arcA region
                in E. coli
                                                  1 kb
   rob           creB                creD

          creA          creC                   arcA
        Some CreBC regulated genes
            Gene     Function
Activates   ackA/pta Acetate kinase/phosphotransacetylase
            talA     Non - oxidative pentose phophate branch
                     enzyme
represses   malE     Maltose metabolism
                                Avison et al. 2001. J. Biol. Chem. 276:26955-26961.



 Many E coli lab strains derived from HfrH carry a constitutive
 creC mutation, creC510.
 ECL618, the donor strain from which the arcA2 allelle was
 obtained, is a descendant of HfrH

 Do ECL618 and CT1061 carry the mutation?
 Analysis of creC in E.coli strains
      strain            R77P            Growth on
                     replacement         maltose
      K1060               no                yes
     CT1062               no                yes
     CT1061               yes               no
     ECL618               yes               no
      HfrH                yes               no
 The creC constitutive alelle contains the sustitution of
        an arginine residue by a proline (R77P)

CT1061 contains BOTH an arcA mutation
        AND a creC mutation
Phenotypic characterization of 24 hs cultures of the
strains studied (grown on glucose)




                                             creCc
                                           increases
                                           catabolic
                                             rates
      General conclusions
C and redox balance mechanisms can be
manipulated in order to enhance the use
of the substrate for the production of highly
reduced products, even when using a
reduced substrate such as glycerol, that
will be mostly used in an oxidative
catabolism, which does not lead to the
production of fermentation products.
GROUP PUBLICATIONS ON THE SUBJECT
 •Pablo Iván Nikel, Alejandra I. Nikel, B. Méndez and Miguel A. Ángel Galvagno(arc) Ma. Julia
 •J.A. Ruiz, R. M. Cecilia P. de Almeida, A.Pettinari, M.J.Pettinari. S. Méndez (2008). Poly(3- (2008)
•Pablo I. Nikel,Fernández,RamírezMiguel Evelia Melillo, Miguel Galvagno, Beatriz S. mutants: Insights into an
 •Pablo I. Nikel, M. Julia Pettinari, M. Julia Galvagno and Beatriz (2006). dye and Méndez
 unexplained phenotype andfrom glycerol by a recombinant Escherichia(3-hydroxybutyrate) in Escherichia a
 Pettinari.(2006) New recombinant Escherichia coli strain tailoredofpoly production of poly(3- glycerol
 hydroxybutyrate) synthesis its supression by characterization microaerobic mutant using
Escherichia coli arcA mutants: metabolic profilethe acumulation offor thecoli arcAculturesin fed-batch ascoli
 recombinants. JournalagroindustrialMicrobiology and
 microaerobic cultures of Molecular by-products. Applied and 77:1337-1343
 hydroxybutyrate) fromApplied Microbiology and Biotechnology.Environmental
carbon source. FEMS Microbiology Letters 258: 55–60Biotechnology 15:48-54Microbiology. 72: 3949-3954.
 •Pablo I. Nikel(*), M.Juliade Almeida, MiguelRuiz and Beatriz S. MéndezMéndez (2007). Effects of Granule-
 •Alejandra de Alejandra I. Nikel, Jimena A. A. Galvagno and Beatriz S. (2008). (2006). Poly -3- of HfrH:
•Pablo I. Nikel,Almeida, Pablo I. Nikel,M. Julia M. Giordano, and M. Julia Pettinari ArcA redox mutants as a
 •M. Julia Pettinari, Pablo Pettinari(*), Andrea Pettinari, and Beatriz S. Méndez (2008). The Legacy
 Hydroxybutyric AcidPhaP on Glycerol-Dependentare Responsible for the Unusual Phenotype of an Escherichia
 Associated Protein Synthesis Journal of Molecular Microbiology and Production in Poly(3-
 source of in the Two-Component System CreBC Growth and Polymer Biotechnology 15: 41-47
Mutations reduced bioproducts. by Recombinant Escherichia coli arcA mutants in microaerobiosis. Applied
 and Environmental Microbiology. 72: 2614-2620
 Hydroxybutyrate)-Producing Escherichia 190: 3404-3407
coli arcA Mutant. Journal of Bacteriology. coli. Applied and Environmental Microbiology. 73: 7912-7916




                                     ¡¡MUITO OBRIGADO!!
                                                          http://www.qb.fcen.uba.ar/genbac/
GROUP PUBLICATIONS ON THE SUBJECT
 •Pablo Iván Nikel, Alejandra I. Nikel, B. Méndez and Miguel A. Ángel Galvagno(arc) Ma. Julia
 •J.A. Ruiz, R. M. Cecilia P. de Almeida, A.Pettinari, M.J.Pettinari. S. Méndez (2008). Poly(3- (2008)
•Pablo I. Nikel,Fernández,RamírezMiguel Evelia Melillo, Miguel Galvagno, Beatriz S. mutants: Insights into an
 •Pablo I. Nikel, M. Julia Pettinari, M. Julia Galvagno and Beatriz (2006). dye and Méndez
 unexplained phenotype andfrom glycerol by a recombinant Escherichia(3-hydroxybutyrate) in Escherichia a
 Pettinari.(2006) New recombinant Escherichia coli strain tailoredofpoly production of poly(3- glycerol
 hydroxybutyrate) synthesis its supression by characterization microaerobic mutant using
Escherichia coli arcA mutants: metabolic profilethe acumulation offor thecoli arcAculturesin fed-batch ascoli
 recombinants. JournalagroindustrialMicrobiology and
 microaerobic cultures of Molecular by-products. Applied and 77:1337-1343
 hydroxybutyrate) fromApplied Microbiology and Biotechnology.Environmental
carbon source. FEMS Microbiology Letters 258: 55–60Biotechnology 15:48-54Microbiology. 72: 3949-3954.
 •Pablo I. Nikel(*), M.Juliade Almeida, MiguelRuiz and Beatriz S. MéndezMéndez (2007). Effects of Granule-
 •Alejandra de Alejandra I. Nikel, Jimena A. A. Galvagno and Beatriz S. (2008). (2006). Poly -3- of HfrH:
•Pablo I. Nikel,Almeida, Pablo I. Nikel,M. Julia M. Giordano, and M. Julia Pettinari ArcA redox mutants as a
 •M. Julia Pettinari, Pablo Pettinari(*), Andrea Pettinari, and Beatriz S. Méndez (2008). The Legacy
 Hydroxybutyric AcidPhaP on Glycerol-Dependentare Responsible for the Unusual Phenotype of an Escherichia
 Associated Protein Synthesis Journal of Molecular Microbiology and Production in Poly(3-
 source of in the Two-Component System CreBC Growth and Polymer Biotechnology 15: 41-47
Mutations reduced bioproducts. by Recombinant Escherichia coli arcA mutants in microaerobiosis. Applied
 and Environmental Microbiology. 72: 2614-2620
 Hydroxybutyrate)-Producing Escherichia 190: 3404-3407
coli arcA Mutant. Journal of Bacteriology. coli. Applied and Environmental Microbiology. 73: 7912-7916




                                     ¡¡MUITO OBRIGADO!!
                                                          http://www.qb.fcen.uba.ar/genbac/
GROUP PUBLICATIONS ON THE SUBJECT
 •Pablo Iván Nikel, Alejandra I. Nikel, B. Méndez and Miguel A. Ángel Galvagno(arc) Ma. Julia
 •J.A. Ruiz, R. M. Cecilia P. de Almeida, A.Pettinari, M.J.Pettinari. S. Méndez (2008). Poly(3- (2008)
•Pablo I. Nikel,Fernández,RamírezMiguel Evelia Melillo, Miguel Galvagno, Beatriz S. mutants: Insights into an
 •Pablo I. Nikel, M. Julia Pettinari, M. Julia Galvagno and Beatriz (2006). dye and Méndez
 unexplained phenotype andfrom glycerol by a recombinant Escherichia(3-hydroxybutyrate) in Escherichia a
 Pettinari.(2006) New recombinant Escherichia coli strain tailoredofpoly production of poly(3- glycerol
 hydroxybutyrate) synthesis its supression by characterization microaerobic mutant using
Escherichia coli arcA mutants: metabolic profilethe acumulation offor thecoli arcAculturesin fed-batch ascoli
 recombinants. JournalagroindustrialMicrobiology and
 microaerobic cultures of Molecular by-products. Applied and 77:1337-1343
 hydroxybutyrate) fromApplied Microbiology and Biotechnology.Environmental
carbon source. FEMS Microbiology Letters 258: 55–60Biotechnology 15:48-54Microbiology. 72: 3949-3954.
 •Pablo I. Nikel(*), M.Juliade Almeida, MiguelRuiz and Beatriz S. MéndezMéndez (2007). Effects of Granule-
 •Alejandra de Alejandra I. Nikel, Jimena A. A. Galvagno and Beatriz S. (2008). (2006). Poly -3- of HfrH:
•Pablo I. Nikel,Almeida, Pablo I. Nikel,M. Julia M. Giordano, and M. Julia Pettinari ArcA redox mutants as a
 •M. Julia Pettinari, Pablo Pettinari(*), Andrea Pettinari, and Beatriz S. Méndez (2008). The Legacy
 Hydroxybutyric AcidPhaP on Glycerol-Dependentare Responsible for the Unusual Phenotype of an Escherichia
 Associated Protein Synthesis Journal of Molecular Microbiology and Production in Poly(3-
 source of in the Two-Component System CreBC Growth and Polymer Biotechnology 15: 41-47
Mutations reduced bioproducts. by Recombinant Escherichia coli arcA mutants in microaerobiosis. Applied
 and Environmental Microbiology. 72: 2614-2620
 Hydroxybutyrate)-Producing Escherichia 190: 3404-3407
coli arcA Mutant. Journal of Bacteriology. coli. Applied and Environmental Microbiology. 73: 7912-7916




                                     ¡¡MUITO OBRIGADO!!
                                                          http://www.qb.fcen.uba.ar/genbac/
GROUP PUBLICATIONS ON THE SUBJECT
 •Pablo Iván Nikel, Alejandra I. Nikel, B. Méndez and Miguel A. Ángel Galvagno(arc) Ma. Julia
 •J.A. Ruiz, R. M. Cecilia P. de Almeida, A.Pettinari, M.J.Pettinari. S. Méndez (2008). Poly(3- (2008)
•Pablo I. Nikel,Fernández,RamírezMiguel Evelia Melillo, Miguel Galvagno, Beatriz S. mutants: Insights into an
 •Pablo I. Nikel, M. Julia Pettinari, M. Julia Galvagno and Beatriz (2006). dye and Méndez
 unexplained phenotype andfrom glycerol by a recombinant Escherichia(3-hydroxybutyrate) in Escherichia a
 Pettinari.(2006) New recombinant Escherichia coli strain tailoredofpoly production of poly(3- glycerol
 hydroxybutyrate) synthesis its supression by characterization microaerobic mutant using
Escherichia coli arcA mutants: metabolic profilethe acumulation offor thecoli arcAculturesin fed-batch ascoli
 recombinants. JournalagroindustrialMicrobiology and
 microaerobic cultures of Molecular by-products. Applied and 77:1337-1343
 hydroxybutyrate) fromApplied Microbiology and Biotechnology.Environmental
carbon source. FEMS Microbiology Letters 258: 55–60Biotechnology 15:48-54Microbiology. 72: 3949-3954.
 •Pablo I. Nikel(*), M.Juliade Almeida, MiguelRuiz and Beatriz S. MéndezMéndez (2007). Effects of Granule-
 •Alejandra de Alejandra I. Nikel, Jimena A. A. Galvagno and Beatriz S. (2008). (2006). Poly -3- of HfrH:
•Pablo I. Nikel,Almeida, Pablo I. Nikel,M. Julia M. Giordano, and M. Julia Pettinari ArcA redox mutants as a
 •M. Julia Pettinari, Pablo Pettinari(*), Andrea Pettinari, and Beatriz S. Méndez (2008). The Legacy
 Hydroxybutyric AcidPhaP on Glycerol-Dependentare Responsible for the Unusual Phenotype of an Escherichia
 Associated Protein Synthesis Journal of Molecular Microbiology and Production in Poly(3-
 source of in the Two-Component System CreBC Growth and Polymer Biotechnology 15: 41-47
Mutations reduced bioproducts. by Recombinant Escherichia coli arcA mutants in microaerobiosis. Applied
 and Environmental Microbiology. 72: 2614-2620
 Hydroxybutyrate)-Producing Escherichia 190: 3404-3407
coli arcA Mutant. Journal of Bacteriology. coli. Applied and Environmental Microbiology. 73: 7912-7916




                                     ¡¡MUITO OBRIGADO!!
                                                          http://www.qb.fcen.uba.ar/genbac/
GROUP PUBLICATIONS ON THE SUBJECT
 •Pablo Iván Nikel, Alejandra I. Nikel, B. Méndez and Miguel A. Ángel Galvagno(arc) Ma. Julia
 •J.A. Ruiz, R. M. Cecilia P. de Almeida, A.Pettinari, M.J.Pettinari. S. Méndez (2008). Poly(3- (2008)
•Pablo I. Nikel,Fernández,RamírezMiguel Evelia Melillo, Miguel Galvagno, Beatriz S. mutants: Insights into an
 •Pablo I. Nikel, M. Julia Pettinari, M. Julia Galvagno and Beatriz (2006). dye and Méndez
 unexplained phenotype andfrom glycerol by a recombinant Escherichia(3-hydroxybutyrate) in Escherichia a
 Pettinari.(2006) New recombinant Escherichia coli strain tailoredofpoly production of poly(3- glycerol
 hydroxybutyrate) synthesis its supression by characterization microaerobic mutant using
Escherichia coli arcA mutants: metabolic profilethe acumulation offor thecoli arcAculturesin fed-batch ascoli
 recombinants. JournalagroindustrialMicrobiology and
 microaerobic cultures of Molecular by-products. Applied and 77:1337-1343
 hydroxybutyrate) fromApplied Microbiology and Biotechnology.Environmental
carbon source. FEMS Microbiology Letters 258: 55–60Biotechnology 15:48-54Microbiology. 72: 3949-3954.
 •Pablo I. Nikel(*), M.Juliade Almeida, MiguelRuiz and Beatriz S. MéndezMéndez (2007). Effects of Granule-
 •Alejandra de Alejandra I. Nikel, Jimena A. A. Galvagno and Beatriz S. (2008). (2006). Poly -3- of HfrH:
•Pablo I. Nikel,Almeida, Pablo I. Nikel,M. Julia M. Giordano, and M. Julia Pettinari ArcA redox mutants as a
 •M. Julia Pettinari, Pablo Pettinari(*), Andrea Pettinari, and Beatriz S. Méndez (2008). The Legacy
 Hydroxybutyric AcidPhaP on Glycerol-Dependentare Responsible for the Unusual Phenotype of an Escherichia
 Associated Protein Synthesis Journal of Molecular Microbiology and Production in Poly(3-
 source of in the Two-Component System CreBC Growth and Polymer Biotechnology 15: 41-47
Mutations reduced bioproducts. by Recombinant Escherichia coli arcA mutants in microaerobiosis. Applied
 and Environmental Microbiology. 72: 2614-2620
 Hydroxybutyrate)-Producing Escherichia 190: 3404-3407
coli arcA Mutant. Journal of Bacteriology. coli. Applied and Environmental Microbiology. 73: 7912-7916




                                     ¡¡MUITO OBRIGADO!!
                                                          http://www.qb.fcen.uba.ar/genbac/
Oxidation                                                   Glucose to pyruvate (x mol glu)
                                                            EMP: 2NADH + 2ATP
                                                            ED: 1 NADPH + 1 NADH + 1ATP
                          Glucose
     Glycerol                                NADP           Glycerol to pyruvate (x mol gly)
                                             NADPH          EMP: 2NADH + 1ATP
      NAD+                          2X
      NADH                                                  6 carbons: 4 NADH + 2 ATP

                                    Gli-3P
                          NAD+                               NADH     TCA Cycle
                          NADH                       NAD+
                                    Pyruvate
      NAD+         NADH

                                                      2NADH 2NAD+
Formate
                                    Acetyl-CoA
                      NADH                                             Ethanol
Lactate     NAD+          NADPH
                             NADP                                   Acetate



 Reduction                           PHB                                 Glycolisis (E-M-P)
                                                                         Entner-Doudoroff

								
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