ES2567305T3 - Sistemas de PUFA policétido sintasa y usos de los mismos - Google Patents

Sistemas de PUFA policétido sintasa y usos de los mismos Download PDF

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ES2567305T3
ES2567305T3 ES10178950.1T ES10178950T ES2567305T3 ES 2567305 T3 ES2567305 T3 ES 2567305T3 ES 10178950 T ES10178950 T ES 10178950T ES 2567305 T3 ES2567305 T3 ES 2567305T3
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acid sequence
nucleic acid
culture
amino acid
polyketide synthase
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James G. Metz
William R. Barclay
James H. Flatt
Jerry M. Kuner
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Abstract

Una molécula aislada de ácido nucleico que comprende una secuencia de ácido nucleico seleccionada entre el grupo que consiste en: a. una secuencia de ácido nucleico que codifica una secuencia de aminoácidos de: SEC ID Nº 20, y fragmentos biológicamente activos de la misma, en la que los fragmentos biológicamente activos muestran una actividad ß-ceto acil-ACP sintasa (KS); b. una secuencia de ácido nucleico que codifica una secuencia de aminoácidos que es al menos un 60 % idéntica a dicha secuencia de aminoácidos de (a), en la que dicha secuencia de aminoácidos muestra una actividad ß-ceto acil-ACP sintasa (KS); y c. una secuencia de ácido nucleico que es completamente complementaria a la secuencia de ácido nucleico de (a) o (b).

Description

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Después del periodo de cultivo, las células se recogen y analizan para el contenido de compuestos bioactivos de interés (por ejemplo, lípidos), pero más particularmente, para compuestos que contengan dos o más enlaces insaturados, y más preferiblemente tres o más dobles enlaces, e incluso más preferiblemente cuatro o más dobles enlaces. Para lípidos, estas cepas que poseen dichos compuestos de más de aproximadamente el 5 %, y más preferiblemente más de aproximadamente el 10 %, y más preferiblemente más de aproximadamente el 15 %, e incluso más preferiblemente más de aproximadamente el 20 % del peso seco del microorganismo se identifican como que contienen de forma predecible un nuevo sistema PKS del tipo descrito anteriormente. Para otros compuestos bioactivos, tales como antibióticos o compuestos que se sintetizan en cantidades más pequeñas, esas cepas que poseen dichos compuestos a más de aproximadamente el 0,5 %, y más preferiblemente más de aproximadamente el 0,1 %, y más preferiblemente más de aproximadamente el 0,25 %, y más preferiblemente más de aproximadamente el 0,5 %, y más preferiblemente más de aproximadamente el 0,75 %, y más preferiblemente más de aproximadamente el 1 %, y más preferiblemente más de aproximadamente el 2,5 %, y más preferiblemente más de aproximadamente el 5 % del peso seco del microorganismo se identifican como que contienen de forma predecible un nuevo sistema PKS del tipo descrito anteriormente.
Como alternativa, o junto con este método, pueden identificarse cepas microbianas potenciales que contienen nuevos sistemas PUFA PKS como se describe en este documento examinando el perfil de ácidos grasos de la cepa (obtenida por cultivo del organismo o a través de fuentes publicadas u otras fuentes fácilmente disponibles). Si el microbio contiene más de aproximadamente el 30 %, y más preferiblemente más de aproximadamente el 40 %, y más preferiblemente más de aproximadamente el 45 %, e incluso y más preferiblemente más de aproximadamente el 50 %, de sus ácidos grados totales como C14:0, C16:0 y/o C16:1, aunque produciendo también al menos un ácido graso de cadena larga con tres o más enlaces insaturados, y más preferiblemente 4 o más dobles enlaces, y más preferiblemente 5 o más dobles enlaces, e incluso más preferiblemente 6 o más dobles enlaces, entonces esta cepa microbiana se identifica como un candidato probable a poseer un nuevo sistema PUFA PKS del tipo descrito en esta invención. La selección de este organismo en las condiciones de bajo oxígeno descritas anteriormente, y la confirmación de la producción de moléculas bioactivas que contienen dos o más enlaces insaturados sugeriría la existencia de un nuevo sistema PUFA PKS en el organismo, que podría confirmase adicionalmente por análisis del genoma de los microbios.
El éxito de este método también puede potenciarse seleccionando cepas eucariotas que se sabe que contienen ácidos grasos C17:0 y o C17:1 (junto con los grandes porcentajes de ácidos grasos C14:0, C16:0 y C16:1 descritos anteriormente) -porque los ácidos grados C17:0 y C17:1 son marcadores potenciales para un sistema de producción de ácidos grasos basado o influenciado por bacterias (procariotas). Otro marcador para identificar cepas que contienen nuevos sistemas PUFA PKS es la producción de perfiles simples de ácidos grasos por el organismo. De acuerdo con la presente invención, un "perfil simple de ácidos grasos" se define como 8 o menos ácidos grasos que se producen por la cepa a niveles mayores del 10 % de los ácidos grasos totales.
El uso de cualquiera de estos métodos o marcadores (individualmente o preferiblemente en combinación) posibilitaría a un experto en la materia identificar fácilmente cepas microbianas con alta predicción de contener un nuevo sistema PUFA PKS del tipo descrito en esta invención.
Combinando muchos de los métodos y marcadores descritos anteriormente, se ha desarrollado un nuevo filtro biorracional (usando cultivos en matraz de agitación) para detectar microorganismos que contienen sistemas PKS productores de PUFA. Este sistema de selección se realiza del siguiente modo:
Una parte de un cultivo de la cepa/microorganismo a ensayar se coloca en matraz de agitación con separadores de 250 ml con 500 ml de medio de cultivo (tratamiento aeróbico), y otra parte de cultivo de la misma cepa se coloca en un matraz de agitación sin separadores de 250 ml con 250 ml de medio de cultivo (tratamiento anóxico/de bajo oxígeno). Pueden emplearse diversos medios de cultivo dependiente del tipo y cepa de microorganismo que se está evaluando. Ambos matraces se colocan en una mesa oscilatoria a 200 rpm. Después de 48-72 horas de tiempo de cultivo, los cultivos se recogen por centrifugación y las células se analizan para el contenido de éster metílico de ácido graso mediante cromatografía de gases para determinar los siguientes datos para cada cultivo: (1) perfil de ácidos grasos; (2) contenido de PUFA; y (3) contenido de grasas (aproximadamente como cantidad total de ácidos grasos/peso seco de la célula).
Estos datos después se analizan haciendo las siguientes cinco preguntas (Sí/No):
Comparación de los datos del matraz de bajo O2/anóxico con los datos del matraz aeróbico:
(1)
¿El DHA (u otro contenido de PUFA) (como % FAME (ésteres metílicos de ácido graso)) permanece aproximadamente igual o preferiblemente aumentado en el cultivo de bajo oxígeno en comparación con el cultivo aeróbico?
(2)
¿Es C14:0 + C16:0 + C16:1 mayor de aproximadamente el 40 % de TFA en el cultivo anóxico?
(3)
¿Existen muy pocos (<1 % como FAME) o ningún precursor (C18:3n-3 + C18:2n-6+C18:3n-6) para la ruta convencional de elongasa/desaturasa dependiente de oxígeno en el cultivo anóxico?
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Claims (1)

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