ES2529254T3 - Proceso de fermentación usando velocidades de incorporación de oxígeno específicas como un control del proceso - Google Patents

Proceso de fermentación usando velocidades de incorporación de oxígeno específicas como un control del proceso Download PDF

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ES2529254T3
ES2529254T3 ES03756237.8T ES03756237T ES2529254T3 ES 2529254 T3 ES2529254 T3 ES 2529254T3 ES 03756237 T ES03756237 T ES 03756237T ES 2529254 T3 ES2529254 T3 ES 2529254T3
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lactic acid
specific oxygen
incorporation rates
oxygen incorporation
fermentation
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Pim Van Hoek
Aristos Aristidou
Brian Rush
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Cargill Inc
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    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid
    • C—CHEMISTRY; METALLURGY
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    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; 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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

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Abstract

Un proceso de fermentación en donde una levadura genéticamente modificada que tiene una alteración de una ruta nativa de la piruvato descarboxilasa (PDC) fermenta un sustrato de fermentación, se mantiene la concentración de oxígeno disuelto en menos del 1% de la cantidad de saturación y se determina la velocidad de incorporación de oxígeno específica durante una fase de producción del proceso de fermentación, y se controla al menos un parámetro operativo en respuesta a la velocidad de incorporación de oxígeno medida.

Description

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E03756237
30-01-2015
preferiblemente de al menos 2,5 g/l/h. Con densidades celulares preferidas de hasta 3-6 g de células/litro de medio de fermentación, las productividades máximas tienden a ser hasta aproximadamente 5,0 g/l/h, y más típicamente hasta aproximadamente 4,0 g/l/h. Es muy preferido llevar la fermentación a cabo de modo que se alcancen estas productividades volúmicas cuando el pH del medio o la temperatura, o ambos parámetros, están dentro de los intervalos descritos en el párrafo precedente.
El ácido láctico producido de acuerdo con la invención es útil para producir lactida, un anhídrido cíclico de dos moléculas de ácido láctico. Dependiendo del estereoisómero del ácido láctico, la lactida puede ser D-lactida (preparada a partir de dos moléculas de ácido D-láctico), L-lactida (preparada a partir de dos moléculas de ácido Lláctico) o D-L-lactida (preparada a partir de una molécula de ácido L-láctico y una molécula de ácido D-láctico). Un método conveniente para producir lactida a partir de ácido láctico es a través de un método de polimerización/despolimerización como el descrito en la Patente de EE.UU. nº 5.142.023, concedida a Gruber et al.
A su vez, la lactida es particularmente útil como un monómero para la producción de polímeros [PLA; del inglés, poly(lactic acid)] y copolímeros de polilactida. En la Patente de EE.UU. nº 5.142.023, concedida a Gruber et al., también se describen procedimientos para preparar estos polímeros. Los productos de PLA preferidos son polímeros estables en estado fundido como los descritos en la Patente de EE.UU. nº 5.338.822, concedida a Gruber et al. El PLA puede ser semicristalino o amorfo.
Los ejemplos siguientes sirven para ilustrar ciertas realizaciones de la invención y no limitan su alcance ni su espíritu. Todas las partes y porcentajes son en peso a menos que se indique otra cosa.
Ejemplo 1
Se prepara una reserva madre para inoculación de una célula de levadura modificada, denominada CD 587, en un matraz sacudido de 250 ml de capacidad que contiene 100 ml de medio de extracto de levadura (10 g/l) – peptona (20 g/l) tamponado con CaCO3 (42 g/l), con 100 g/l de glucosa. A una OD600 = 10, las células son recolectadas por centrifugación y son posteriormente resuspendidas en una disolución de glicerol al 15% (peso/volumen) y almacenadas a -80 °C en partes alícuotas de 1,5 ml.
La célula CD 587 es una célula de K. marxianus que tiene su gen PDC suprimido y un gen D-LDH exógeno de L. helveticus integrado en su genoma, en el sitio del gen PDC suprimido, bajo el control de secuencias promotoras y terminadoras de PDC nativas. La célula CD 587 y su preparación se describen más detalladamente en la Solicitud Provisional de EE.UU. nº 60/384.333, presentada el 30 de mayo de 2002.
La fase de crecimiento se inicia inoculando una reserva madre de 1,5 ml en glicerol en un fermentador de 3 l de capacidad, lo que da lugar a una OD600 inicial de 0,05. La fase de crecimiento se lleva a cabo aeróbicamente burbujeando continuamente aire a un caudal de 1,5 l/min (0,5 vvm), con una velocidad constante del agitador de 800 rpm. Se continúa el crecimiento hasta que el DO se reduce al 5% de la saturación de aire. Esto coincide con una concentración constante de CO2 en el gas desprendido. Se mide la OUR determinando la cantidad de aire suministrado y analizando el oxígeno de los gases desprendidos por medio de espectrometría de masas. Durante la fase de crecimiento, la OUR es aproximadamente 20,8 ± 2,5 milimoles de O2/gdw/h. Bajo estas condiciones, la OUR viene limitada por la capacidad de la célula para metabolizar el oxígeno disponible. La densidad celular final es aproximadamente 4 g/l.
Una vez que el DO ha alcanzado el valor de cero, se mantienen las condiciones de aireación durante una hora antes de comenzar la fase de producción. La fase de producción se inicia cambiando instantáneamente el caudal de aire a 0,1 l/min (0,033 vvm) y disminuyendo la velocidad del agitador a 500 rpm. Este cambio en las condiciones de aireación da lugar a una OUR de 1,5 ± 0,1 milimoles de O2/gdw/h y un DO de cero durante la fase de producción. Se continúa la fermentación durante aproximadamente 60 horas. Se miden la velocidad de consumo de glucosa, la velocidad de producción de lactato y el rendimiento a lactato extrayendo periódicamente muestras para análisis por HPLC/IC/GC-MS. En la Tabla 1 se muestran los resultados en la fase de producción.
Tabla 1
Título máximo de ácido láctico
106 ± 3,1 g/kg
Velocidad de consumo de glucosa
1,2 ± 0,05 g/gdw/h
Velocidad de producción de ácido láctico
1,1 ± 0,04 g/gdw/h
Rendimiento a ácido láctico (fase de producción)
0,92 ± 0,03 g de ácido láctico/g de glucosa
% de carbono recuperado (fase de producción)
99% ± 3,0
Pureza óptica del ácido láctico
> 99,9
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Claims (1)

  1. imagen1
ES03756237.8T 2002-05-30 2003-05-30 Proceso de fermentación usando velocidades de incorporación de oxígeno específicas como un control del proceso Expired - Lifetime ES2529254T3 (es)

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US38433302P 2002-05-30 2002-05-30
US384333P 2002-05-30
PCT/US2003/016825 WO2003102200A2 (en) 2002-05-30 2003-05-30 Fermentation process using specific oxygen uptake rates as a process control

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ES03731489T Expired - Lifetime ES2290466T3 (es) 2002-05-30 2003-05-30 Metodos y materiales para la produccion de acido d-lactico en levadura.

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