ES2656790T3 - Microorganismos para la producción de ácido adípico y otros compuestos - Google Patents

Microorganismos para la producción de ácido adípico y otros compuestos Download PDF

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ES2656790T3
ES2656790T3 ES09763021.4T ES09763021T ES2656790T3 ES 2656790 T3 ES2656790 T3 ES 2656790T3 ES 09763021 T ES09763021 T ES 09763021T ES 2656790 T3 ES2656790 T3 ES 2656790T3
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coa
route
adipyl
aminocaproic acid
adipil
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Anthony P. Burgard
Priti Pharkya
Robin E. Osterhout
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Genomatica Inc
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Abstract

Un organismo microbiano de origen sintético, que comprende un organismo microbiano que tiene una ruta para convertir adipil-CoA en ácido 6-aminocaproico (ruta de ácido 6-aminocaproico) y al menos una ruta para producir adipil-CoA (ruta adipil-CoA), comprendiendo dicha ruta de ácido 6-aminocaproico dos ácidos nucleicos exógenos que codifican cada uno una enzima de la ruta de ácido 6-aminocaproico expresada en una cantidad suficiente para producir ácido 6-aminocaproico, comprendiendo dicha ruta de ácido 6-aminocaproico aldehído deshidrogenasa dependiente de CoA, y transaminasa o 6-aminocaproato deshidrogenasa, en el que dicha al menos una ruta de adipil-CoA comprende al menos una de una ruta para convertir adipato en adipil CoA y una ruta para convertir succinil-CoA más acetil-CoA en adipil-CoA, en el que dicha ruta para convertir adipato en adipil CoA comprende adipil-CoA sintetasa o fosfotransadipilasa/adipato cinasa; y en el que dicha ruta para convertir succinil-CoA más acetil-CoA en adipil-CoA comprende succinil-CoA:acetil-CoA acil transferasa, 3-oxoadipil-CoA transferasa, 3-oxoadipato reductasa, 3-hidroxiadipato deshidratasa, 2-enoato reductasa, y adipil-CoA sintetasa, fosfotransadipilasa/adipato cinasa, adipil-CoA:acetil-CoA transferasa o adipil-CoA hidrolasa.

Description

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han descrito previamente y se conocen bien en la técnica. Las condiciones aerobias y anaerobias ejemplares se describen, por ejemplo, en la Solicitud de Patente de Estados Unidos N.º de serie 11/891.602, presentada el 10 de agosto de 2007. Las fermentaciones pueden realizarse de forma discontinua, en lote alimentado o de forma continua, como se divulga en el presente documento.
5 Si se desea, el pH del medio se puede mantener a un pH deseado, en particular pH neutro, como un pH de aproximadamente 7 mediante la adición de una base, tal como NaOH u otras bases, o ácido, según sea necesario para mantener el medio de cultivo a un pH deseable. La velocidad de crecimiento puede determinarse midiendo la densidad óptica usando un espectrofotómetro (600 nm), y la tasa de captación de glucosa controlando el
10 agotamiento de la fuente de carbono a lo largo del tiempo.
El medio de crecimiento puede ser, por ejemplo, cualquier fuente de carbohidratos que pueda suministrar una fuente de carbono al microorganismo de origen sintético. Dichas fuentes incluyen, por ejemplo, azúcares tales como glucosa, xilosa, arabinosa, galactosa, manosa, fructosa y almidón. Otras fuentes de carbohidratos incluyen, por 15 ejemplo, materias primas renovables y biomasa. Los tipos ejemplares de biomasas que se pueden usar como materias primas en los métodos de la invención incluyen biomasa celulósica, biomasa hemicelulósica y materias primas de lignina o porciones de materias primas. Dichas materias primas de biomasa contienen, por ejemplo, sustratos de carbohidratos útiles como fuentes de carbono tales como glucosa, xilosa, arabinosa, galactosa, manosa, fructosa y almidón. Dadas las enseñanzas y la orientación proporcionadas en el presente documento, los
20 expertos en la técnica entenderán que las materias primas renovables y la biomasa distintas de las ilustradas anteriormente también pueden usarse para cultivar los organismos microbianos de la invención para la producción de adipato, ácido 6-aminocaproico o caprolactama.
Además de materias primas renovables tales como las ilustradas anteriormente, los organismos microbianos de
25 adipato, ácido 6-aminocaproico o caprolactama de la invención también pueden modificarse para su crecimiento en singás como su fuente de carbono. En esta realización específica, una o más proteínas o enzimas se expresan en los organismos productores de adipato, ácido 6-aminocaproico o caprolactama para proporcionar una ruta metabólica para la utilización de singás u otra fuente de carbono gaseoso.
30 El gas de síntesis, también conocido como singás o gas productor, es el producto principal de la gasificación de carbón y de materiales carbonosos tales como materiales de biomasa, incluyendo cultivos agrícolas y residuos. El singás es una mezcla principalmente de H2 y CO y puede obtenerse a partir de la gasificación de cualquier materia prima orgánica, incluyendo, pero sin limitación, carbón, aceite de carbón, gas natural, biomasa y materia orgánica residual. La gasificación generalmente se realiza bajo una alta relación de combustible con respecto a oxígeno.
35 Aunque principalmente contiene H2 y CO, el singás también puede incluir CO2 y otros gases en cantidades inferiores. Por lo tanto, el gas de síntesis proporciona una fuente rentable de carbono gaseoso tal como CO y, adicionalmente, CO2.
La ruta de Wood-Ljungdahl cataliza la conversión de CO y H2 en acetil-CoA y otros productos, tal como acetato. Los
40 organismos capaces de utilizar CO y singás también tienen generalmente la capacidad de utilizar CO2 y mezclas de CO2/H2 a través del mismo conjunto básico de enzimas y transformaciones incluidas por la ruta de Wood-Ljungdahl. La conversión dependiente de H2 de CO2 en acetato por microorganismos se reconoció mucho antes de que se revelara que el CO también podría ser utilizado por los mismos organismos y que estaban implicadas las mismas rutas. Se ha demostrado que muchos acetógenos crecen en presencia de CO2 y producen compuestos tales como
45 el acetato, siempre que haya hidrógeno presente para suministrar los equivalentes reductores necesarios (véase, por ejemplo, Drake, Acetogenesis, págs. 3-60 Chapman y Hall, Nueva York, (1994)). Esto se puede resumir por la siguiente ecuación:
2 CO2 + 4 H2 + n ADP + n Pi → CH3COOH + 2 H2O + n ATP
50 Por lo tanto, los microorganismos de origen sintético que poseen la ruta de Wood-Ljungdahl pueden utilizar mezclas de CO2 y H2, también para la producción de acetil-CoA y otros productos deseados.
La ruta de Wood-Ljungdahl se conocen bien en la técnica y consiste en 12 reacciones que pueden separarse en dos
55 ramificaciones: (1) ramificación de metilo y (2) ramificación de carbonilo. La ramificación de metilo convierte el singás en metil-tetrahidrofolato (metil-THF), mientras que la ramificación de carbonilo convierte el metil-THF en acetil-CoA. Las reacciones en la ramificación de metilo son catalizadas en orden por las siguientes enzimas: ferredoxina oxidorreductasa, formato deshidrogenasa, formiltetrahidrofolato sintetasa, meteniltetrahidrofolato ciclodeshidratasa, metilentetrahidrofolato deshidrogenasa y metilentetrahidrofolato reductasa. Las reacciones en la ramificación de
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Claims (1)

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ES09763021.4T 2008-03-27 2009-03-27 Microorganismos para la producción de ácido adípico y otros compuestos Active ES2656790T3 (es)

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