ES2525796T3 - Nuevos microorganismos para la producción de 1,2-propanodiol obtenidos mediante una combinación de evolución y diseño racional - Google Patents

Nuevos microorganismos para la producción de 1,2-propanodiol obtenidos mediante una combinación de evolución y diseño racional Download PDF

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ES2525796T3
ES2525796T3 ES08714246.9T ES08714246T ES2525796T3 ES 2525796 T3 ES2525796 T3 ES 2525796T3 ES 08714246 T ES08714246 T ES 08714246T ES 2525796 T3 ES2525796 T3 ES 2525796T3
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strain
gene
evolution
production
propanediol
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Philippe Soucaille
Francois Voelker
Rainer Figge
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Metabolic Explorer SA
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Metabolic Explorer SA
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B10/00—Directed molecular evolution of macromolecules, e.g. RNA, DNA or proteins

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Abstract

Método para la preparación de una cepa de microorganismo evolucionada para la producción de 1,2-propanodiol a partir de una fuente de carbono, comprendiendo dicho método: - hacer crecer una cepa inicial bajo presión selectiva en un medio de crecimiento apropiado, comprendiendo dicha cepa bacteriana una atenuación de la expresión del gen tpiA y una atenuación de la expresión de al menos uno de los genes involucrados en la conversión de metilglioxal en lactato, con el objetivo de promover la evolución de dicha cepa inicial, - seleccionar y aislar la cepa evolucionada que presenta una tasa de producción de 1,2-propanodiol incrementada, - reconstruir un gen tpiA funcional en la cepa evolucionada.

Description

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E08714246
11-12-2014
El cassette de resistencia a cloramfenicol se eliminó de la cepa E. coli MG1655 lpd*, ∆tpiA, ∆pflAB, ∆adhE, ldhA::km, ∆gloA, ∆aldA, ∆aldB, ∆edd::cm (Ver WO2005073364) de acuerdo con el Protocolo 1.
Protocolo 1: Eliminación de cassettes de resistencia
5 Los cassettes de resistencia a cloramfenicol y/o a kanamicina se eliminaron de acuerdo con la siguiente técnica. El plásmido pCP20 portador de la FLP recombinasa que actúa en los sitios FRT de los cassettes de resistencia a cloramfenicol y/o a kanamicina se introdujo en la cepa mediante electroporación. Después de un cultivo seriado a 41ºC, la pérdida de los cassettes de resistencia a antibiótico se verificó mediante un análisis PCR con los
10 oligonucleótidos detallados en la Tabla 1.
La presencia de modificaciones previamente construidas en la cepa se verificó utilizando los oligonucleótidos detallados en la Tabla 1.
15 La cepa obtenida se nombró E. coli MG1655 lpd*, ∆tpiA, ∆pflAB, ∆adhE, ldhA::km, ∆gloA, ∆aldA, ∆aldB, ∆edd.
Tabla 1: Oligonucleótidos utilizados para la verificación de la inserción de un cassette de resistencia o la pérdida de un cassette de resistencia
Nombre de la región
Nombre de los oligos SEQ ID Homología con la región cromosómica
gen tpiA
cdh N°1 Ver WO2005073364
(deleción)
YIIQ N°2
gen pflAB
pflABF N°3 Ver WO2005073364
pflABR
N°4
gen adhE
ychGf N°5 Ver WO2005073364
adhECr
N°6
gen ldhA
hsIJC N°7 Ver WO2005073364
(inserción cassette)
ldhAC2 N°8
gen gloA
NemACd N°9 Ver WO2005073364
Rnt Cr
N°10
gen aldA
Ydc F C f N°11 Ver WO2005073364
gapCCr
N°12
gen aldB
aldB C f N°13 Ver WO2005073364
YiaYCr
N°14
gen edd
Edad N°15 Ver WO2005073364
Zwf r
N°16
gen ldhA gene
ldhAF N°17 1439724 hasta 1439743
(deleción)
ldhAR N°18 1441029 hasta 1441007
gen arcA
arcAF N°19 4638292 hasta 4638273
arcAR
N°20 4636854 hasta 4636874
gen ndh
ndhF N°21 1164722 hasta 1164742
ndhR
N°22 1167197 hasta 1167177
gen tpiA
YIIQ N°2 4109599 hasta 4109580
(reconstrucción)
tpiA R N°23 4108953 hasta 4108973
promotor gapA
yeaAF N°24 1860259-1860287
(Ptrc16-gapA)
gapAR N°25 1861068-1861040
gen pykA
pykAF N°26 1935338 hasta 1935360
pykAR
N°27 1937425 hasta 1937401
gen pykF
pykFF N°28 1753371 hasta 1753392
11 E08714246
11-12-2014
Nombre de la región
Nombre de los oligos SEQ ID Homología con la región cromosómica
pykFR
N°29 1755518 hasta 1755495
genes ackA-pta
B2295 N°30 2410900 hasta 2410919
YfcCR
N°31 2415164 hasta 2415145
genes poxB
poxBF N°32 908475 hasta 908495
poxBR
N°33 910375 hasta 910352
b) Construcción de una cepa modificada E. coli MG1655 lpd*, ∆tpiA, ∆pflAB, ∆adhE, ∆ldhA::cm, ∆gloA, ∆aldA, ∆aldB, ∆edd
5 Para eliminar el cassette de resistencia a kanamicina y para desactivar el gen ldhA, el cassette de resistencia a cloramfenicol se insertó en el gen ldhA delecionando la mayor parte del gen en cuestión de acuerdo con el Protocolo
2.
Protocolo 2: Introducción de un producto de PCR para recombinación y selección de los recombinantes
10 Los oligonucleótidos elegidos para la sustitución de un gen o de una región intergénica y detallados en la Tabla 2 se utilizaron para amplificar ya sea el cassette de resistencia a cloramfenicol a partir del plásmido pKD3 o el cassette de resistencia a kanamicina a partir del plásmido pKD4 (Datsenko, K.A. & Wanner, B.L. (2000)). El producto de PCR obtenido se introdujo posteriormente mediante electroporación en la cepa receptora portadora del plásmido pKD46
15 en el que el sistema λ Red (y, β, exo) expresado favorece enormemente la recombinación homóloga. Los transformantes resistentes a antibiótico fueron entonces seleccionados y la inserción del cassette de resistencia fue verificada por análisis PCR utilizando los oligonucleótidos apropiados que se detallan en la Tabla 1.
Las demás modificaciones de la cepa fueron verificadas con los oligonucleótidos detallados en la Tabla 1. 20 La cepa resultante se nombró E. coli MG1655 lpd*, ∆ldhA::cm, ∆tpiA, ∆pflAB, ∆adhE. ∆gloA, ∆aldA, ∆aldB, ∆edd.
Tabla 2: Oligonucleótidos utilizados para la sustitución de una región cromosómica por recombinación con un producto de PCR en la cepa E. coli MG1655 25
Nombre de la región
Nombre de los oligos SEQ ID Homología con la región cromosómica
gen ldhA
DldhAF N°34 1440865-1440786
DldhAR
N°35 1439878-1439958
gen arcA
DarcAF N°36 4637868-4637791
DarcAR
N°37 4637167-4637245
gen ndh
DndhF N°38 1165071-1165149
DndhR
N°39 1166607-1166528
gen tpiA
tpiA::kmF N°40 4109264 -4109195
(reconstrucción)
tpiA::kmR N°41 4109109 -4109193
promotor gapA
Ptrc-gapAF N°42 1860478-1860536
(Ptrc 16-gapA)
Ptrc-gapAR N°43 1860762-1860800
gen pykA
DpykAF N°44 1935756-1935836
DpykAR
N°45 1937055-1937135
gen pykF
DpykFF N°46 1753689-1753766
DpykFR
N°47 1755129-1755051
genes ackA-pta
DackAF N°48 2411494-2411573
DptaR
N°49 2414906-2414830
genes poxB
DpoxBF N°50 908557-908635
DpoxBR
N°51 910262-910180
12
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imagen14

Claims (1)

  1. imagen1
ES08714246.9T 2007-03-23 2008-03-21 Nuevos microorganismos para la producción de 1,2-propanodiol obtenidos mediante una combinación de evolución y diseño racional Active ES2525796T3 (es)

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IB2007001680 2007-03-23
WOPCT/IB2007/001680 2007-03-23
PCT/EP2008/053445 WO2008116852A1 (en) 2007-03-23 2008-03-21 New micro-organisms for the production of 1,2-propanediol obtained by a combination of evolution and rational design.

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AR (1) AR066196A1 (es)
BR (1) BRPI0808972B1 (es)
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Publication number Priority date Publication date Assignee Title
US8647642B2 (en) 2008-09-18 2014-02-11 Aviex Technologies, Llc Live bacterial vaccines resistant to carbon dioxide (CO2), acidic PH and/or osmolarity for viral infection prophylaxis or treatment
MX2011004842A (es) * 2008-11-07 2011-05-30 Metabolic Explorer Sa Uso de sacarosa como sustrato para la produccion fermentativa de 1,2-propanodiol.
EP2233562A1 (en) 2009-03-24 2010-09-29 Metabolic Explorer Method for producing high amount of glycolic acid by fermentation
WO2010141920A2 (en) * 2009-06-04 2010-12-09 Genomatica, Inc. Microorganisms for the production of 1,4-butanediol and related methods
US8969053B2 (en) 2009-07-30 2015-03-03 Metabolic Explorer Mutant YqhD enzyme for the production of a biochemical by fermentation
EP2459720B1 (en) 2009-07-30 2016-04-27 Metabolic Explorer Mutant glycerol dehydrogenase (glydh) for the production of a biochemical by fermentation
CN102575235B (zh) 2009-07-30 2015-05-20 代谢探索者公司 用于通过发酵生产生物化学品的突变甲基乙二醛合酶(mgs)
SG10201504733XA (en) 2010-07-12 2015-07-30 Univ Gent Metabolically engineered organisms for the production of added value bio-products
EP2532751A1 (en) * 2011-06-10 2012-12-12 Metabolic Explorer Use of inducible promoters in the fermentative production of 1,2-propanediol
US9353386B2 (en) 2011-06-15 2016-05-31 B.R.A.I.N. Biotechnology Research And Information Network Ag Means and methods for producing propanediol
EP3050970B1 (en) 2015-01-28 2019-09-18 Metabolic Explorer Modified microorganism for optimized production of 1,4-butanediol
CN107690482B (zh) 2015-04-07 2022-11-29 代谢探索者公司 用于2,4-二羟基丁酸的优化生产的经修饰的微生物
ES2811337T3 (es) 2015-04-07 2021-03-11 Metabolic Explorer Sa Microorganismo modificado para la producción optimizada de 2,4-dihidroxibutirato con eflujo de 2,4- dihidroxibutirato aumentado
US10676723B2 (en) 2015-05-11 2020-06-09 David Gordon Bermudes Chimeric protein toxins for expression by therapeutic bacteria
BR112018002048A2 (pt) 2015-08-07 2018-09-18 Evonik Degussa Gmbh ?micro-organismo geneticamente modificado e método para a produção fermentativa de metionina?
WO2017042602A1 (en) 2015-09-10 2017-03-16 Metabolic Explorer New lactaldehyde reductases for the production of 1,2-propanediol
US10801050B2 (en) 2015-10-23 2020-10-13 Metabolic Explorer Microorganism modified for the assimilation of levulinic acid
KR102771842B1 (ko) 2015-11-27 2025-02-21 에보닉 오퍼레이션스 게엠베하 L-메티오닌을 제조하는 방법
EP3481850A1 (en) 2016-07-08 2019-05-15 Evonik Degussa GmbH Method for the fermentative production of methionine or its hydroxy analog form by microorganisms comprising genes coding sugar phosphotransferase system (pts)
US11180535B1 (en) 2016-12-07 2021-11-23 David Gordon Bermudes Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria
US11129906B1 (en) 2016-12-07 2021-09-28 David Gordon Bermudes Chimeric protein toxins for expression by therapeutic bacteria
EP3342873A1 (en) 2016-12-29 2018-07-04 Metabolic Explorer Conversion of methylglyoxal into hydroxyacetone using enzymes and applications thereof
EP3470512A1 (en) 2017-10-10 2019-04-17 Metabolic Explorer Mutant phosphoserine aminotransferase for the conversion of homoserine into 4-hydroxy-2-ketobutyrate
KR102245274B1 (ko) 2019-01-28 2021-04-27 재단법인 지능형 바이오 시스템 설계 및 합성 연구단 최소 유전체를 갖는 신규 미생물 및 이의 제조 방법

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US6087140A (en) 1997-02-19 2000-07-11 Wisconsin Alumni Research Foundation Microbial production of 1,2-propanediol from sugar
WO2004033646A2 (en) * 2002-10-04 2004-04-22 E.I. Du Pont De Nemours And Company Process for the biological production of 1,3-propanediol with high yield
US20060270013A1 (en) * 2003-02-18 2006-11-30 Michel Chateau Method for the production of evolved microorganisms which permit the generation or modification of metabolic pathways
FR2864967B1 (fr) * 2004-01-12 2006-05-19 Metabolic Explorer Sa Microorganisme evolue pour la production de 1,2-propanediol

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JP5570821B2 (ja) 2014-08-13
US20100285547A1 (en) 2010-11-11
KR101528943B1 (ko) 2015-06-15
DK2109681T3 (en) 2014-12-08
KR20100015810A (ko) 2010-02-12
CA2679989C (en) 2015-07-07
IL200705A0 (en) 2010-05-17
EP2109681B1 (en) 2014-10-15
CN101641446A (zh) 2010-02-03
MX2009010229A (es) 2009-10-26
US8298807B2 (en) 2012-10-30
WO2008116852A1 (en) 2008-10-02
AR066196A1 (es) 2009-08-05
CA2679989A1 (en) 2008-10-02
TW200904984A (en) 2009-02-01

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