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

Info

Publication number
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
Authority
ES
Spain
Prior art keywords
strain
gene
evolution
production
propanediol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
ES08714246.9T
Other languages
English (en)
Inventor
Philippe Soucaille
Francois Voelker
Rainer Figge
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Metabolic Explorer SA
Original Assignee
Metabolic Explorer SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Metabolic Explorer SA filed Critical Metabolic Explorer SA
Application granted granted Critical
Publication of ES2525796T3 publication Critical patent/ES2525796T3/es
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; 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
    • 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
    • C12N15/01—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

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

imagen1
imagen2
imagen3
imagen4
imagen5
imagen6
imagen7
imagen8
imagen9
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
imagen10
imagen11
imagen12
imagen13
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)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
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.

Publications (1)

Publication Number Publication Date
ES2525796T3 true ES2525796T3 (es) 2014-12-30

Family

ID=38814484

Family Applications (1)

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

Country Status (14)

Country Link
US (1) US8298807B2 (es)
EP (1) EP2109681B1 (es)
JP (1) JP5570821B2 (es)
KR (1) KR101528943B1 (es)
CN (1) CN101641446A (es)
AR (1) AR066196A1 (es)
BR (1) BRPI0808972B1 (es)
CA (1) CA2679989C (es)
DK (1) DK2109681T3 (es)
ES (1) ES2525796T3 (es)
IL (1) IL200705A0 (es)
MX (1) MX2009010229A (es)
TW (1) TW200904984A (es)
WO (1) WO2008116852A1 (es)

Families Citing this family (22)

* Cited by examiner, † Cited by third party
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
BRPI0823256B8 (pt) * 2008-11-07 2020-05-26 Metabolic Explorer Sa uso de sacarose como substrato para produção 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
MY187676A (en) 2009-06-04 2021-10-08 Genomatica Inc Microorganisms for the production of 1,4-butanediol and related methods
WO2011012702A1 (en) 2009-07-30 2011-02-03 Metabolic Explorer Mutant glycerol dehydrogenase (glydh) for the production of a biochemical by fermentation
BR112012002153A2 (pt) 2009-07-30 2015-09-15 Metabolic Explorer Sa metilglioxal sintase mutante (mgs) para a produção de material bioquímico por fermentação
WO2011012697A2 (en) 2009-07-30 2011-02-03 Metabolic Explorer Mutant yqhd enzyme for the production of a biochemical by fermentation
EP2532751A1 (en) * 2011-06-10 2012-12-12 Metabolic Explorer Use of inducible promoters in the fermentative production of 1,2-propanediol
EP2721167A1 (en) 2011-06-15 2014-04-23 B.R.A.I.N. Biotechnology Research And Information Network AG New 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
US10415062B2 (en) 2015-04-07 2019-09-17 Metabolic Explorer Modified microorganism for the optimized production of 2,4-dihydroxybutyrate
CN107771214B (zh) 2015-04-07 2022-01-18 代谢探索者公司 用于具有增加的2,4-二羟基丁酸外排物的优化的2,4-二羟基丁酸产生的修饰的微生物
PL3331998T3 (pl) 2015-08-07 2020-12-28 Evonik Operations Gmbh Wytwarzanie przez fermentację l-metioniny zależne od tiokarboksylanu białka
EP3347478B1 (en) 2015-09-10 2022-09-21 Metabolic Explorer New lactaldehyde reductases for the production of 1,2-propanediol
EP3365427B1 (en) 2015-10-23 2020-08-19 Metabolic Explorer Microorganism modified for the assimilation of levulinic acid
MX2018006434A (es) 2015-11-27 2018-09-28 Evonik Degussa Gmbh Metodo para producir l-metionina.
KR102430878B1 (ko) 2016-07-08 2022-08-09 메타볼릭 익스플로러 당 포스포트랜스퍼라제 시스템 (pts)을 코딩하는 유전자를 포함하는 미생물에 의한 관심 분자의 발효적 생산을 위한 방법
US11129906B1 (en) 2016-12-07 2021-09-28 David Gordon Bermudes Chimeric protein toxins for expression by therapeutic bacteria
US11180535B1 (en) 2016-12-07 2021-11-23 David Gordon Bermudes Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from 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

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6087140A (en) 1997-02-19 2000-07-11 Wisconsin Alumni Research Foundation Microbial production of 1,2-propanediol from sugar
ES2356526T3 (es) * 2002-10-04 2011-04-08 E.I. Du Pont De Nemours And Company Procedimiento para la producción biológica de 1,3-propanodiol con elevado rendimiento.
JP2006517796A (ja) 2003-02-18 2006-08-03 メタボリック エクスプローラー 代謝経路の生成または改変を可能とする進化した微生物の生産方法
FR2864967B1 (fr) 2004-01-12 2006-05-19 Metabolic Explorer Sa Microorganisme evolue pour la production de 1,2-propanediol

Also Published As

Publication number Publication date
WO2008116852A1 (en) 2008-10-02
TW200904984A (en) 2009-02-01
BRPI0808972B1 (pt) 2016-11-29
CA2679989C (en) 2015-07-07
CN101641446A (zh) 2010-02-03
EP2109681B1 (en) 2014-10-15
DK2109681T3 (en) 2014-12-08
US8298807B2 (en) 2012-10-30
CA2679989A1 (en) 2008-10-02
JP2010521958A (ja) 2010-07-01
IL200705A0 (en) 2010-05-17
US20100285547A1 (en) 2010-11-11
BRPI0808972A2 (pt) 2014-08-26
MX2009010229A (es) 2009-10-26
EP2109681A1 (en) 2009-10-21
JP5570821B2 (ja) 2014-08-13
KR101528943B1 (ko) 2015-06-15
KR20100015810A (ko) 2010-02-12
AR066196A1 (es) 2009-08-05

Similar Documents

Publication Publication Date Title
ES2525796T3 (es) Nuevos microorganismos para la producción de 1,2-propanodiol obtenidos mediante una combinación de evolución y diseño racional
US20220347271A1 (en) Sequence-specific antimicrobials by blocking dna repair
Vandecraen et al. The impact of insertion sequences on bacterial genome plasticity and adaptability
Doublet et al. Antibiotic marker modifications of λ Red and FLP helper plasmids, pKD46 and pCP20, for inactivation of chromosomal genes using PCR products in multidrug-resistant strains
Thomason et al. Recombineering: genetic engineering in bacteria using homologous recombination
JP6621464B2 (ja) 欠失突然変異
Ellermeier et al. Construction of targeted single copy lac fusions using λ Red and FLP-mediated site-specific recombination in bacteria
Murphy Phage recombinases and their applications
Huguet et al. A toxin antitoxin system promotes the maintenance of the IncA/C-mobilizable Salmonella Genomic Island 1
Cooper et al. Determining the specificity of cascade binding, interference, and primed adaptation in vivo in the Escherichia coli type IE CRISPR-Cas system
Riccobono et al. Characterization of IncI1 sequence type 71 epidemic plasmid lineage responsible for the recent dissemination of CTX-M-65 extended-spectrum β-lactamase in the Bolivian Chaco region
US20130164790A1 (en) Self-deleting plasmid
Xin et al. Identification and functional analysis of potential prophage-derived recombinases for genome editing in Lactobacillus casei
EP2532751A1 (en) Use of inducible promoters in the fermentative production of 1,2-propanediol
Kalivoda et al. New vector tools with a hygromycin resistance marker for use with opportunistic pathogens
Wang et al. IcgA is a virulence factor of Rhodococcus equi that modulates intracellular growth
Brantl et al. An antisense RNA-mediated transcriptional attenuation mechanism functions in Escherichia coli
Jacoby et al. Properties of IncP-2 plasmids of Pseudomonas spp
van Aartsen et al. An optimized method for suicide vector-based allelic exchange in Klebsiella pneumoniae
DE102014212675A1 (de) T7-Expressionssystem, Verfahren zu seiner Herstellung und seine Verwendung zur Herstellung von rekombinanten Proteinen
Hossain et al. Genome modifications and cloning using a conjugally transferable recombineering system
Xin et al. Targeted and repetitive chromosomal integration enables high-level heterologous gene expression in Lactobacillus casei
Ji et al. Development of a dual-antimicrobial counterselection method for markerless genetic engineering of bacterial genomes
Tark et al. Dual role of NER in mutagenesis in Pseudomonas putida
US7919322B2 (en) Targeted deletions using retroelement-mediated placement of recombination sites