ES2643046T3 - Microorganismos fermentadores de pentosa - Google Patents

Microorganismos fermentadores de pentosa Download PDF

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ES2643046T3
ES2643046T3 ES13706186.7T ES13706186T ES2643046T3 ES 2643046 T3 ES2643046 T3 ES 2643046T3 ES 13706186 T ES13706186 T ES 13706186T ES 2643046 T3 ES2643046 T3 ES 2643046T3
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fermenting microorganisms
pcr
pentose fermenting
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Zdravko Dragovic
Christian GAMAUF
Christoph Reisinger
Ulrich Kettling
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Clariant Produkte Deutschland GmbH
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Abstract

Una célula de levadura que expresa una proteína que comprende una secuencia de aminoácidos que tiene al menos un 80% de identidad, lo más preferiblemente un 90% de identidad, lo más altamente preferible un 95% de identidad con SEQ ID NO. 2 y que tiene actividad de xilosa isomerasa en una célula de levadura.

Description

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Todos dichos genes candidato se trataron como en la entrada ZP_07904696.1 (SEQ ID NO. 2) que es xilosa isomerasa (EsXI) de Eubacterium saburreum DSM 3986 como se describe en los párrafos siguientes. La entrada de secuencia de codificación unida (NZ_AEPW01000073 REGION: 2583..3956: SEQ ID NO.1) se tomó como base para la construcción de cebadores de cloranción.
2. Amplificación del gen de xilosa isomerasa (EsXI) de Eubacterium saburreum DSM 3986
Los métodos para la manipulación de moléculas de ácido nucleico son generalmente conocidos por la persona experta en la materia y se introducen aquí por referencia (1. Molecular cloning: a laboratory manual, Joseph Sambrook, David William Russell; 2. Current Protocols in Molecular Biology. Last Update: January 11, 2012. Page Count: approx, 5300, Print ISSN: 1934-3639:.). Las plantillas del DNA genómico del Eubacterium saburreum DSM 3986 se obtuvo de DSMZ (Deutsche Stammsammlung für Mikroorganismen und Zellkulturen). Los pares de cebadores flaqueantes se diseñaron para que coincidieran con la terminación N-y C-terminal de SEQ ID NO. 1. Para la amplificación de una versión truncada en el extremo N de SEQ ID NO.1 la región de unión del cebador sensorial se desplazó 54 bp posteriores (empezando con A55). La reacción de PCR se establece utilizando Finnzymes Phusiontm High Fidelity Polymerase (sistema tampón HF) siguiendo las recomendaciones del suministrador para las concentraciones de dNTP, cebador y tampón. La amplificación de los productos de PCR se realiza en un termociclador Eppendorf utilizando el programa estándar para Phusion Polymerase (98ºC 30’’ de desnaturalización inicial seguido de 35 ciclos de etapas de 98ºC (20’’) – 60ºC (20’’) – 72ºC (1’20’’) y una fase de elongación final a 72ºC durante 10 minutos. Los productos de PCR de tamaño esperado se purifican mediante electroforesis en gel TAE-agarosa teñido con bromuro de etidio preparativo y se recuperan del gel utilizando el kit SV-PCR y de purificación en gel de Promga Wizard. Para la unión de un 6xHis-Tag C-terminal se utilizan los productos de PCR primarios como plantilla para la reamplificación del fragmento de DNA entero utilizando la imprimación inversa extendida con la correspondiente extensión 5’, bajo idénticas condiciones (fusión PCR de 6xHIS-Tag). Los productos de PCR obtenidos se purifican otra vez por electroforesis en gel de Agarosa y se recuperan utilizando el kit SV-PCR y de purificación en gel de Promga Wizar. Contiene la Version 6xHis-TAG Cterminal del gen EsXI o 6xHis-TAG C-terminal del gen-gen (EsXI truncado) Es-sh XI, respectivamente.
La amplificación de genes de xilosa isomerasa optimizados con codones se hizo a partir de plantillas genéticas optimizadas solicitadas por Geneart Regensburg, Alemania. Los algoritmos de optimización para la optimización de la secuencia se utilizaron según lo proporcionado por la empresa.
3. Clonación del EsXI y Es-shXI ORF en el plásmido de expresión de Saccharomyces cerevisiae
Una preparación de plásmido del plásmido pSCMB454 aislado del cultivo de Escherichia coli se linealizó mediante restricción con endonucleasa XmnI y los fragmentos digeridos separados de las especies no procesadas por electroforesis en gel de Agarosa. El esqueleto del vector linealizado se recuperó del gel siguiendo las instrucciones del kit SV-PCR y de purificación en gel de Promga Wizard. El producto de PCR amplificado se clonó en el esqueleto del vector digerido con XmnI utilizando métodos de clonación estándar. La transformación se llevó a cabo en células de Escherichia coli W Mach1 químicamente competentes de acuerdo con el protocolo del proveedor. Los transformantes se hicieron crecer durante una noche en placas de LB-Ampicilina y se probaron para la corrección mediante la preparación del plásmido MINI y el control de la digestión así como la secuenciación del DNA. Se preparó una mayor cantidad de ADN plasmídico a partir de un clon confirmado utilizando el sistema PureYieldTM Plasmid Midiprep System de Promega. Un ejemplo de la secuencia del casete de expresión resultante que incluye la secuencia promotora GPD y terminadora cyc1 se da en SEQ ID NO. 6.
4. Transformación en Saccharomyces cerevisiae
La cepa ATCC 204667 de Saccharomyces cerevisiae (MATa, ura3-52, mal GAL+, CUP(r)) se utilizó como huésped para todos los experimentos de transformación.
La transformación se llevó a cabo utilizando métodos estándar conocidos por los expertos en la técnica (por ejemplo, véase Gietz, R.D. y R.A. Woods. (2002) Transformation of yeast by the LiAc/ss Carrier DNA/PEG method. Methods in Enzymology 350: 87-96). Una versión intacta del gen ura3 de S. cerevisiae contenido en el vector de expresión se utilizó como marcador de selección y los transformantes se seleccionaron para el crecimiento en medio mínimo sin uracilo. El medio mínimo consistió en 20 g·l-1 de glucosa, 6,7 g·l-1 de base nitrogenada de levadura sin aminoácidos, 40 mg·l-1 de L-tirosina, 70 mg·l-1 de L-fenilalanina, 70 mg·l-1 de L-triptófano, 200 mg·l-1 de L-valina y 50 mg·l-1 de hemisulfato de adenina, clohidrato de L-arginina, clorhidrato de L-histidina monohidrato, L-isoleucina, L-leucina, clorhidrato de L-lisina, L-metionina, L-serina y L-treonina. El pH se ajustó a 5,6 y se añadieron 15 g·l-1 de agar para medio sólido.
5. Crecimiento de xilosa isomerasas que expresan cepas de Saccharomyces en medio de xilosa
A) Colonias individuales de cepas de Saccharomyces transformadas con el vector de expresión para xilosa isomerasa de Eubacterium saburreum (Es XI), y Clostridum phytofermentas (Cp XI) así como el vector de expresión simple pSCMB454 se transfirieron en placas de medio mínimo con glucosa como única fuente de carbono. Las colonias individuales se trasfirieron después en placas de medio mínimo con xilosa como única fuente de carbono (20 g·l-1) y se incubaron a 30ºC. Después de 7 días solo los transformantes con los vectores de expresión de xilosa
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SEQ ID NO. 4: Xilosa isomerasa de Clostridium phytofermentas (Proteína) (Cp XI)
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SEQ ID NO. 5: Xilosa isomerasa de Piromyce sp (Proteína – PI_XI)
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SEQ ID NO. 6: Casete de expresión Ex XI (Negrita y mayusculas: secuencia de codificación del gen EsXI 6x-His-tag C-terminal y un enlazador de fusión; Mayúsculas pequeñas: Promotor GPD; subrayado: restos del sitio XnmI; cursiva: terminador CYCI)
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ES13706186.7T 2012-02-07 2013-02-07 Microorganismos fermentadores de pentosa Active ES2643046T3 (es)

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EP12000783.6A EP2626423A1 (en) 2012-02-07 2012-02-07 Pentose-fermenting microorganism
PCT/EP2013/052407 WO2013117631A1 (en) 2012-02-07 2013-02-07 Pentose fermenting microorganisms

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US9187743B2 (en) 2013-03-11 2015-11-17 E I Du Pont De Nemours And Company Bacterial xylose isomerases active in yeast cells
CA2991707C (en) 2015-07-13 2023-08-01 MARA Renewables Corporation Enhancing microbial metabolism of c5 organic carbon
BR112018002284A2 (pt) 2015-08-05 2018-12-11 Cargill Inc método de fermentação para produzir um bioproduto, levedura crabtree negativa geneticamente engenheirada e método para reduzir a produção de glicerol por uma levedura engenheirada
EP3559210A1 (en) * 2016-12-21 2019-10-30 Vib Vzw Xylose isomerases that confer efficient xylose fermentation capability to yeast
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