MANIPULATION OF BACTERIA MANIPULATION OF BACTERIA TO OPTIMIZE SYNTHESIS OF TO OPTIMIZE SYNTHESIS OF

BIOPRODUCTS FROM BIOPRODUCTS FROM GLYCEROLGLYCEROL

M. Julia PettinariM. Julia Pettinari

Facultad de Ciencias Exactas y NaturalesUniversidad 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 Metabolic Engeneering of microbial processesprocesses

Strategies:Strategies:

Substrate manipulationSubstrate manipulation

Use of inhibitorsUse of inhibitors

(Genetic) Manipulation of metabolic (Genetic) Manipulation of metabolic pathwayspathways

Need for deep knowledge of microorganism´s metabolism and geneticsRecombinant Escherichia coli

PolyhydroxyalkanoatesPolyhydroxyalkanoates (PHAs): (PHAs):

Linear polymers constituted by Linear polymers constituted by hydroxyesther units.hydroxyesther units.

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

Their thermoplastic and elastomeric properties make Their thermoplastic and elastomeric properties make them suitable to replace petroleum derived plasticsthem suitable to replace petroleum derived plastics

Alcaligenes eutrophus

Biopolymers - bioplastics

PHA synthesis and degradation are cyclic.PHA synthesis and degradation are cyclic.

PHA biosynthesis pathways

Depolimerization Depolimerization produces Acyl-produces Acyl-CoAs and Acetyl-CoAs and Acetyl-CoA, that are used CoA, that are used as carbon and as carbon and energy sourceenergy source

NADPH consumed

NADHproduced

3- hydroxybutyric acid

Synthesis: Acyl Synthesis: Acyl CoA precursors CoA precursors obtained through obtained through different different pathways are pathways are converted into converted into PHAs. PHAs.

Glucose

TCA Cycle

LdhPta-Ack

G6PDH

NADPH

NADP

NADP NADPH

2NADH 2NAD+

NAD+Lactate

NAD+

NADH

Glu-6P

Fru-6P

Gli-3P

Gluconate-6P

Pentoses-P

Pyruvate

Acetyl-CoA

Ethanol

Acetate

Acetoacetyl-CoA

Hydroxybutiryl-CoA

PHB

Pgi

Glycolisis (E-M-P)Entner-Doudoroff

NAD+ NADH

NADP

NADPH

NAD+ NADH

Formate

NADH

G6PDH:Glucose -6-P dehydrogenase

Pgi:Phosphoglucose isomerase

Ldh:lactate dehydrogenase

Pta:phosphotransacetylase

Glycerol

DHAP

NAD+

NADH

2X

Catabolism in PHB accumulating recombinant E. coli

Agustino Martínez-Antonio and Julio Collado-Vives (2003) Curr. Opin. Microbiol. 6: 482

genes

transcription factors

The The E. coliE. coli regulatory network regulatory network

Regulation of aerobic and anaerobic Regulation of aerobic and anaerobic metabolism in metabolism in EE. . colicoli

Transcriptional regulation via two global regulators : ArcA (aerobic respiration control) and FNR (fumarate

nitrate respiration).

- 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)

These regulators respond to O2 and/or alternative electron acceptor availability.

Some genes regulated by ArcA

HypothesisHypothesis

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.

SP314: arcA mutantpQE32: vector plasmidpJP24: pQE32 with pha genes

Effects of PHB on O2 consumption and NADH pool

Diamidesensitivity

SP314 / pQE32

SP314 / pJP24

Wild type

FEMS Microbiology Letters (2006). 258: 55–60Applied and Environmental Microbiology (2006). 72: 2614-2620

Diamide: 1,1´-azobis-(N,N-dimetilformamida)

Toluidine blue sensitivity

Preliminary characterization of Preliminary characterization of arcAarcA mutants for PHB accumulation in mutants for PHB accumulation in

microaerobiosismicroaerobiosis

* PHB < 5%

SP314/pJP24

SP314: arcA mutantpQE32: vector plasmidpJP24: pQE32 with pha genes

Semisynthetic medium with 3% glucose

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 PHB accumulation in CT1061/pJP24 CT1061/pJP24 ((arcA2arcA2)) growing on glycerol in microaerobiosisgrowing on glycerol in microaerobiosis

PHB content (%

CDW)

PHB production

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

Effect of arcA on PHB producing E. coli Effect of arcA on PHB producing E. coli ConclusionsConclusions

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 Ethanol production from glycerol in arcA arcA mutantsmutants

Growth conditionGrowth conditionEthanol (mEthanol (mMM))

K1060K1060 CT1062CT1062((arcAarcA))

CT1061CT1061((arcA2arcA2))

AerobicAerobic NDND 0.48 0.48 0.07 0.07 18.13 18.13 0.17 0.17

MicroaerobicMicroaerobic 4.58 4.58 0.02 0.02 44.43 44.43 0.54 0.54 111.31 111.31 4.97 4.97

Metabolic characterization of arcA strains Metabolic characterization of arcA strains growing on glycerolgrowing on glycerol

0

10

20

30

40

50

60

Acetate Formate Succinate

Lactate Pyruvate Ethanol

K1060 (wt)

CT1062 (arcA)

CT1061 (arcA2)

Cultures grown for 48 hs in MYA-glycerol 3% in microaerobic conditions

StrainStrain NADHNADH NADNAD++ Total NAD(H/Total NAD(H/++)) NADH/NADNADH/NAD++

K1060K1060 0.51 0.51 0.06 0.06 2.83 2.83 0.15 0.15 3.34 3.34 0.21 0.21 0.18 0.18 0.03 0.03

CT1062CT1062 1.96 1.96 0.09 0.09 3.12 3.12 0.22 0.22 5.08 5.08 0.31 0.31 0.63 0.63 0.07 0.07

CT1061CT1061 2.54 2.54 0.11 0.11 2.62 2.62 0.08 0.08 5.16 5.16 0.19 0.19 0.97 0.97 0.07 0.07

Reducing power formation in microaerobic cultures growing on glicerol

Amounts in micromoles per g CDW

K1060 (arcA+) = 0,87

CT1062 ( arcA) = 2,01

CT1061 (arcA2) = 12,51

Ethanol/acetate

Molar ratio

What is the genetic difference bewteen CT1061 and CT1062?

CT1061 (arcA2) grows more thanT1062 (arcA) It has less nutritional requirements Higher oxygen consumption rates More reduced redox state Increased production of reduced compounds

Detection of ArcA in Detection of ArcA in Western blotWestern blot

29 kDa

19 kDa

K10

60

CT

1062

CT

1061

Genomic Organization of the arcA regionin E. coli CT1061

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

[wild type ArcA = 29 kDa]

arcA´ (NH2 terminal)

´arcA (COOH terminal)

3’-IR 5’-IR

513 pb

IS10 (1330 pb)

212 pb

EE. . colicoli CT1061 construction CT1061 construction

K1060 P1[ECL618 (lac proAB)XIII arcA2 zij::Tn10]

Tet selection

CT1061 (arcA2 zij::Tn10)

1 kbrob

creA

creB

creC

creD

arcA

Genomic Organization of the arcA regionin E. coli

Some CreBC regulated genesSome CreBC regulated genesGeneackA/ptatalA

malE

FunctionAcetate kinase/phosphotransacetylaseNon - oxidative pentose phophate branch enzymeMaltose metabolism

Avison et al. 2001. J. Biol. Chem. 276:26955-26961.

Activates

represses

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 Analysis of creC creC inin E.coli E.coli strainsstrains

The creC constitutive alelle contains the sustitution of an arginine residue by a proline (R77P)

strainstrain R77PR77P

replacementreplacement

Growth on Growth on maltosemaltose

K1060K1060 nono yesyes

CT1062CT1062 nono yesyes

CT1061CT1061 yesyes nono

ECL618ECL618 yesyes nono

HfrHHfrH yesyes nono

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 conclusionsGeneral conclusions

C and redox balance mechanisms can be C and redox balance mechanisms can be manipulated in order to enhance the use manipulated in order to enhance the use of the substrate for the production of highly of the substrate for the production of highly reduced products, even when using a reduced products, even when using a reduced substrate such as glycerol, that reduced substrate such as glycerol, that will be mostly used in an oxidative will be mostly used in an oxidative catabolism, which does not lead to the catabolism, which does not lead to the production of fermentation products.production of fermentation products.

http://www.qb.fcen.uba.ar/genbac/

¡¡MUITO OBRIGADO!!

GROUP PUBLICATIONS ON THE SUBJECT

•J.A. Ruiz, R. Fernández, P. I. Nikel, B. Méndez and M.J.Pettinari. (2006). dye (arc) mutants: Insights into an unexplained phenotype and its supression by  the acumulation of poly (3-hydroxybutyrate) in Escherichia coli recombinants. FEMS Microbiology Letters 258: 55–60 •Pablo I. Nikel(*), M.Julia Pettinari(*), Miguel A. Galvagno and Beatriz S. Méndez (2006). Poly -3-Hydroxybutyric Acid Synthesis  by Recombinant Escherichia coli arcA mutants in microaerobiosis. Applied and Environmental Microbiology. 72: 2614-2620

•Pablo Iván Nikel, Alejandra de Almeida, Evelia Melillo, Miguel Ángel Galvagno and Ma. Julia Pettinari.(2006) New recombinant Escherichia coli strain tailored for the production of poly(3-hydroxybutyrate) from agroindustrial by-products. Applied and Environmental Microbiology. 72: 3949-3954. •Alejandra de Almeida, Pablo I. Nikel, Andrea M. Giordano, and M. Julia Pettinari (2007). Effects of Granule-Associated Protein PhaP on Glycerol-Dependent Growth and Polymer Production in Poly(3-Hydroxybutyrate)-Producing Escherichia coli. Applied and Environmental Microbiology. 73: 7912-7916

•Pablo I. Nikel, M. Julia Pettinari, Miguel A. Galvagno and Beatriz S. Méndez (2008). Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant Escherichia coli arcA mutant in fed-batch microaerobic cultures Applied Microbiology and Biotechnology. 77:1337-1343•M. Julia Pettinari, Pablo I. Nikel, Jimena A. Ruiz and Beatriz S. Méndez (2008). ArcA redox mutants as a source of reduced bioproducts. Journal of Molecular Microbiology and Biotechnology 15: 41-47

•Pablo I. Nikel, M. Cecilia Ramírez M. Julia Pettinari, Miguel A. Galvagno, Beatriz S. Méndez (2008)Escherichia coli arcA mutants: metabolic profile characterization of microaerobic cultures using glycerol as a carbon source. Journal of Molecular Microbiology and Biotechnology 15:48-54•Pablo I. Nikel, Alejandra de Almeida, M. Julia Pettinari, and Beatriz S. Méndez (2008). The Legacy of HfrH: Mutations in the Two-Component System CreBC are Responsible for the Unusual Phenotype of an Escherichia

coli arcA Mutant. Journal of Bacteriology. 190: 3404-3407

http://www.qb.fcen.uba.ar/genbac/

¡¡MUITO OBRIGADO!!

GROUP PUBLICATIONS ON THE SUBJECT

•J.A. Ruiz, R. Fernández, P. I. Nikel, B. Méndez and M.J.Pettinari. (2006). dye (arc) mutants: Insights into an unexplained phenotype and its supression by  the acumulation of poly (3-hydroxybutyrate) in Escherichia coli recombinants. FEMS Microbiology Letters 258: 55–60 •Pablo I. Nikel(*), M.Julia Pettinari(*), Miguel A. Galvagno and Beatriz S. Méndez (2006). Poly -3-Hydroxybutyric Acid Synthesis  by Recombinant Escherichia coli arcA mutants in microaerobiosis. Applied and Environmental Microbiology. 72: 2614-2620

•Pablo Iván Nikel, Alejandra de Almeida, Evelia Melillo, Miguel Ángel Galvagno and Ma. Julia Pettinari.(2006) New recombinant Escherichia coli strain tailored for the production of poly(3-hydroxybutyrate) from agroindustrial by-products. Applied and Environmental Microbiology. 72: 3949-3954. •Alejandra de Almeida, Pablo I. Nikel, Andrea M. Giordano, and M. Julia Pettinari (2007). Effects of Granule-Associated Protein PhaP on Glycerol-Dependent Growth and Polymer Production in Poly(3-Hydroxybutyrate)-Producing Escherichia coli. Applied and Environmental Microbiology. 73: 7912-7916

•Pablo I. Nikel, M. Julia Pettinari, Miguel A. Galvagno and Beatriz S. Méndez (2008). Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant Escherichia coli arcA mutant in fed-batch microaerobic cultures Applied Microbiology and Biotechnology. 77:1337-1343•M. Julia Pettinari, Pablo I. Nikel, Jimena A. Ruiz and Beatriz S. Méndez (2008). ArcA redox mutants as a source of reduced bioproducts. Journal of Molecular Microbiology and Biotechnology 15: 41-47

•Pablo I. Nikel, M. Cecilia Ramírez M. Julia Pettinari, Miguel A. Galvagno, Beatriz S. Méndez (2008)Escherichia coli arcA mutants: metabolic profile characterization of microaerobic cultures using glycerol as a carbon source. Journal of Molecular Microbiology and Biotechnology 15:48-54•Pablo I. Nikel, Alejandra de Almeida, M. Julia Pettinari, and Beatriz S. Méndez (2008). The Legacy of HfrH: Mutations in the Two-Component System CreBC are Responsible for the Unusual Phenotype of an Escherichia

coli arcA Mutant. Journal of Bacteriology. 190: 3404-3407

http://www.qb.fcen.uba.ar/genbac/

¡¡MUITO OBRIGADO!!

GROUP PUBLICATIONS ON THE SUBJECT

•J.A. Ruiz, R. Fernández, P. I. Nikel, B. Méndez and M.J.Pettinari. (2006). dye (arc) mutants: Insights into an unexplained phenotype and its supression by  the acumulation of poly (3-hydroxybutyrate) in Escherichia coli recombinants. FEMS Microbiology Letters 258: 55–60 •Pablo I. Nikel(*), M.Julia Pettinari(*), Miguel A. Galvagno and Beatriz S. Méndez (2006). Poly -3-Hydroxybutyric Acid Synthesis  by Recombinant Escherichia coli arcA mutants in microaerobiosis. Applied and Environmental Microbiology. 72: 2614-2620

•Pablo Iván Nikel, Alejandra de Almeida, Evelia Melillo, Miguel Ángel Galvagno and Ma. Julia Pettinari.(2006) New recombinant Escherichia coli strain tailored for the production of poly(3-hydroxybutyrate) from agroindustrial by-products. Applied and Environmental Microbiology. 72: 3949-3954. •Alejandra de Almeida, Pablo I. Nikel, Andrea M. Giordano, and M. Julia Pettinari (2007). Effects of Granule-Associated Protein PhaP on Glycerol-Dependent Growth and Polymer Production in Poly(3-Hydroxybutyrate)-Producing Escherichia coli. Applied and Environmental Microbiology. 73: 7912-7916

•Pablo I. Nikel, M. Julia Pettinari, Miguel A. Galvagno and Beatriz S. Méndez (2008). Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant Escherichia coli arcA mutant in fed-batch microaerobic cultures Applied Microbiology and Biotechnology. 77:1337-1343•M. Julia Pettinari, Pablo I. Nikel, Jimena A. Ruiz and Beatriz S. Méndez (2008). ArcA redox mutants as a source of reduced bioproducts. Journal of Molecular Microbiology and Biotechnology 15: 41-47

•Pablo I. Nikel, M. Cecilia Ramírez M. Julia Pettinari, Miguel A. Galvagno, Beatriz S. Méndez (2008)Escherichia coli arcA mutants: metabolic profile characterization of microaerobic cultures using glycerol as a carbon source. Journal of Molecular Microbiology and Biotechnology 15:48-54•Pablo I. Nikel, Alejandra de Almeida, M. Julia Pettinari, and Beatriz S. Méndez (2008). The Legacy of HfrH: Mutations in the Two-Component System CreBC are Responsible for the Unusual Phenotype of an Escherichia

coli arcA Mutant. Journal of Bacteriology. 190: 3404-3407

http://www.qb.fcen.uba.ar/genbac/

¡¡MUITO OBRIGADO!!

GROUP PUBLICATIONS ON THE SUBJECT

•J.A. Ruiz, R. Fernández, P. I. Nikel, B. Méndez and M.J.Pettinari. (2006). dye (arc) mutants: Insights into an unexplained phenotype and its supression by  the acumulation of poly (3-hydroxybutyrate) in Escherichia coli recombinants. FEMS Microbiology Letters 258: 55–60 •Pablo I. Nikel(*), M.Julia Pettinari(*), Miguel A. Galvagno and Beatriz S. Méndez (2006). Poly -3-Hydroxybutyric Acid Synthesis  by Recombinant Escherichia coli arcA mutants in microaerobiosis. Applied and Environmental Microbiology. 72: 2614-2620

•Pablo Iván Nikel, Alejandra de Almeida, Evelia Melillo, Miguel Ángel Galvagno and Ma. Julia Pettinari.(2006) New recombinant Escherichia coli strain tailored for the production of poly(3-hydroxybutyrate) from agroindustrial by-products. Applied and Environmental Microbiology. 72: 3949-3954. •Alejandra de Almeida, Pablo I. Nikel, Andrea M. Giordano, and M. Julia Pettinari (2007). Effects of Granule-Associated Protein PhaP on Glycerol-Dependent Growth and Polymer Production in Poly(3-Hydroxybutyrate)-Producing Escherichia coli. Applied and Environmental Microbiology. 73: 7912-7916

•Pablo I. Nikel, M. Julia Pettinari, Miguel A. Galvagno and Beatriz S. Méndez (2008). Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant Escherichia coli arcA mutant in fed-batch microaerobic cultures Applied Microbiology and Biotechnology. 77:1337-1343•M. Julia Pettinari, Pablo I. Nikel, Jimena A. Ruiz and Beatriz S. Méndez (2008). ArcA redox mutants as a source of reduced bioproducts. Journal of Molecular Microbiology and Biotechnology 15: 41-47

•Pablo I. Nikel, M. Cecilia Ramírez M. Julia Pettinari, Miguel A. Galvagno, Beatriz S. Méndez (2008)Escherichia coli arcA mutants: metabolic profile characterization of microaerobic cultures using glycerol as a carbon source. Journal of Molecular Microbiology and Biotechnology 15:48-54•Pablo I. Nikel, Alejandra de Almeida, M. Julia Pettinari, and Beatriz S. Méndez (2008). The Legacy of HfrH: Mutations in the Two-Component System CreBC are Responsible for the Unusual Phenotype of an Escherichia

coli arcA Mutant. Journal of Bacteriology. 190: 3404-3407

http://www.qb.fcen.uba.ar/genbac/

¡¡MUITO OBRIGADO!!

GROUP PUBLICATIONS ON THE SUBJECT

•J.A. Ruiz, R. Fernández, P. I. Nikel, B. Méndez and M.J.Pettinari. (2006). dye (arc) mutants: Insights into an unexplained phenotype and its supression by  the acumulation of poly (3-hydroxybutyrate) in Escherichia coli recombinants. FEMS Microbiology Letters 258: 55–60 •Pablo I. Nikel(*), M.Julia Pettinari(*), Miguel A. Galvagno and Beatriz S. Méndez (2006). Poly -3-Hydroxybutyric Acid Synthesis  by Recombinant Escherichia coli arcA mutants in microaerobiosis. Applied and Environmental Microbiology. 72: 2614-2620

•Pablo Iván Nikel, Alejandra de Almeida, Evelia Melillo, Miguel Ángel Galvagno and Ma. Julia Pettinari.(2006) New recombinant Escherichia coli strain tailored for the production of poly(3-hydroxybutyrate) from agroindustrial by-products. Applied and Environmental Microbiology. 72: 3949-3954. •Alejandra de Almeida, Pablo I. Nikel, Andrea M. Giordano, and M. Julia Pettinari (2007). Effects of Granule-Associated Protein PhaP on Glycerol-Dependent Growth and Polymer Production in Poly(3-Hydroxybutyrate)-Producing Escherichia coli. Applied and Environmental Microbiology. 73: 7912-7916

•Pablo I. Nikel, M. Julia Pettinari, Miguel A. Galvagno and Beatriz S. Méndez (2008). Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant Escherichia coli arcA mutant in fed-batch microaerobic cultures Applied Microbiology and Biotechnology. 77:1337-1343•M. Julia Pettinari, Pablo I. Nikel, Jimena A. Ruiz and Beatriz S. Méndez (2008). ArcA redox mutants as a source of reduced bioproducts. Journal of Molecular Microbiology and Biotechnology 15: 41-47

•Pablo I. Nikel, M. Cecilia Ramírez M. Julia Pettinari, Miguel A. Galvagno, Beatriz S. Méndez (2008)Escherichia coli arcA mutants: metabolic profile characterization of microaerobic cultures using glycerol as a carbon source. Journal of Molecular Microbiology and Biotechnology 15:48-54•Pablo I. Nikel, Alejandra de Almeida, M. Julia Pettinari, and Beatriz S. Méndez (2008). The Legacy of HfrH: Mutations in the Two-Component System CreBC are Responsible for the Unusual Phenotype of an Escherichia

coli arcA Mutant. Journal of Bacteriology. 190: 3404-3407

GlucoseNADP

NADPH

2NADH 2NAD+

NAD+Lactate

NAD+

NADH

Gli-3P

Pyruvate

Acetyl-CoAEthanol

AcetateNADPH

NADP

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

NAD+ NADH

Formate

NADH

Glycerol

NAD+

NADH2X

Glucose to pyruvate (x mol glu)EMP: 2NADH + 2ATPED: 1 NADPH + 1 NADH + 1ATP

Glycerol to pyruvate (x mol gly)EMP: 2NADH + 1ATP

6 carbons: 4 NADH + 2 ATP

TCA CycleNAD+

NADH

Oxidation

Reduction