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Sustitución funcional de la histidina en las proteínas para generar organismos dependientes de aminoácidos no

Fei Gan¹, Renhe Liu¹, Feng Wang¹

¹California Institute for Biomedical Research (Calibr) , La Jolla , California 92037 , United States.

Journal of the American Chemical Society

|March 7, 2018

Resumen

Los investigadores desarrollaron un método para crear microbios que necesitan un aminoácido no canónico específico para crecer, mejorando la seguridad de las vacunas y la contención biológica. Esto implica la sustitución de los aminoácidos esenciales de histidina por análogos funcionales.

Área de la Ciencia:

Biología sintética
Biotecnología
Genética microbiana

Sus antecedentes:

Los organismos que requieren aminoácidos no canónicos son cruciales para las vacunas vivas y la contención biológica.
La manipulación genética de los residuos de aminoácidos esenciales ofrece una vía para lograr la auxotropía.

Objetivo del estudio:

Desarrollar una estrategia sencilla para crear organismos con una estricta dependencia de crecimiento de un aminoácido no canónico.
Demostrar la sustitución funcional de un residuo clave de histidina en una metalloenzima por un análogo no canónico.

Principales métodos:

Sustitución genética de un residuo de histidina (His) en la enzima manosa-6-fosfato isomerasa (ManA) que contiene zinc con 3-metil-l-histidina (MeH).
Ingeniería de una cepa de Escherichia coli con una variante ManA evolucionada (Opt5) que alberga sustituciones de N262S y H264MeH.
Evaluación de la dependencia del crecimiento y la tasa de reversión de la cepa de E. coli modificada.

Principales resultados:

3-metil-l-histidina (MeH) sustituido funcionalmente por un ligando de unión al metal clave (H264) en el ManA.
La variante modificada de ManA (Opt5) mostró una actividad comparable a la de la enzima de tipo salvaje.
La cepa de Escherichia coli modificada exhibió una estricta dependencia de crecimiento del MeH con una tasa de reversión muy baja.

Conclusiones:

Esta estrategia proporciona un método robusto para la generación de organismos auxotróficos.
El enfoque es potencialmente aplicable a otras metaloproteínas o no metaloproteínas con residuos esenciales de histidina.
Este método mejora la contención biológica y la seguridad de los organismos recombinantes y las vacunas vivas.

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