ES2525150T3 - Ácidos grasos de cadena ramificada y producción biológica de los mismos - Google Patents
Ácidos grasos de cadena ramificada y producción biológica de los mismos Download PDFInfo
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- ES2525150T3 ES2525150T3 ES10803687.2T ES10803687T ES2525150T3 ES 2525150 T3 ES2525150 T3 ES 2525150T3 ES 10803687 T ES10803687 T ES 10803687T ES 2525150 T3 ES2525150 T3 ES 2525150T3
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
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- C12Y102/04—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with a disulfide as acceptor (1.2.4)
- C12Y102/04004—3-Methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring) (1.2.4.4), i.e. branched-chain-alpha-ketoacid dehydrogenase
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Abstract
Un método para producir ácido graso anteiso, comprendiendo el método cultivar una célula que comprende al menos un polinucleótido expresado en exceso, en donde un polinucleótido expresado en exceso es un polinucleótido en donde un polinucleótido expresado en exceso es un polinucleótido natural para la célula, cuyo producto se genera en mayor cantidad del que se encuentra normalmente en la célula, o un polinucleótido exógeno, comprendiendo dicho polinucleótido exógeno o expresado en exceso una secuencia de ácido nucleico que codifica un polipéptido que cataliza al menos una de las siguientes reacciones: (aa) conversión de piruvato a citramalato; (bb) conversión de citramalato a citraconato; (cc) conversión de citraconato a ß-metil-D-malato; (dd) conversión de ß-metil-D-malato a 2-oxobutanoato; o (ee) conversión de treonina a 2-oxobutanoato bajo condiciones que permitan la expresión del polinucleótido o polinucleótidos y la producción de ácido graso anteiso, en donde la célula produce más ácidos grasos anteiso que una célula por otra parte similar que no comprende el polinucleótido o polinucleótidos.
Description
E10803687
28-11-2014
respecto, en algunos casos, se introducen copias adicionales del polinucleótido en la célula huésped para aumentar la cantidad de enzima disponible para la producción de ácido graso. La sobreexpresión de un polinucleótido nativo también se consigue regulando en exceso la actividad del promotor endógeno, o uniendo de forma operativa el polinucleótido a un promotor más sólido.
5 Las enzimas exógenas y sus correspondientes polinucleótidos son también adecuados para usar en el contexto de la invención, y los rasgos de la ruta de biosíntesis o del producto final se pueden hacer a medida dependiendo de la enzima concreta utilizada. Si se desea, el polinucleótido o polinucleótidos se aíslan o derivan de los organismos productores de ácido graso de cadena ramificada descritos en la presente memoria. Por ejemplo, FabH de E. coli, una 3-cetoacil-ACP sintetasa, utiliza preferiblemente la acetil-CoA como sustrato en lugar de la
10 acil-CoA ramificada, mientras que FabH de B. subtilis impulsa de manera más eficaz la síntesis de ácido graso ramificado. Así, en un aspecto, la célula es una célula de E. coli que comprende un polinucleótido que codifica un FabH de B. subtilis.
Una citramalato sintetasa ilustrativa producida por las células se deriva de CimA de M. jannaschii. AHAS ilustrativas incluyen E. coli IlvIH, E. coli IlvIH (G14D), E. coli IlvGM, y B. subtilis IlvBH. Una BCDH ilustrativa es Bkd de B. 15 subtilis, y una 3-cetoacil-ACP sintetasa ilustrativa es FabH de B. subtilis. Una treonina desaminasa ilustrativa es TdcB de E. coli. Las tioesterasas ilustrativas incluyen, aunque no de forma limitativa, TesA de E. coli, tioesterasa de la glándula uropigial de Mallard, y tioesterasa de glándula mamaria de rata. Una isopropilmalato isomerasa ilustrativa es LeuCD de E. coli, y una isopropilmalato deshidrogenasa ilustrativa es LeuB de E. coli. En un aspecto, la célula comprende una secuencia de ácido nucleico que tiene al menos aproximadamente 90 por ciento de identidad con la
20 secuencia de ácido nucleico definida en la SEC ID N.°: 32, 36, 42, 43, 46, 51, 57, 62, 68, u 83, o codifica una polipéptido que comprende una secuencia de aminoácidos que tiene al menos aproximadamente 90 por ciento de identidad con la secuencia de aminoácido definida en la SEC ID N.°: 33, 39, 40, 41, 47, 48, 52, 53, 58, 65, 66, 67, 84, u 85. Las enzimas ilustrativas que median en la producción de ácidos grasos anteiso y/o iso también se han descrito en la Tabla A.
25 Tabla A
- Actividad
- Nombre del gen Organismo N.° acceso
- Aminoácido de cadena ramificada transaminasa
- ilvE Salmonella enteric NP_457845
- ilvE
- Yersinia pestis YP_002348774
- ilvE
- Shigella flexneri NP_709575
- ilvE
- Pectobacterium carotovorum YP_003018265
- ilvE
- Ralstonia solanacearum YP_003753411
- 2-cetoácido de cadena ramificada deshidrogenasa, componente E1, subunidad alfa
- bkdAA Anoxybacillus flavithermus YP_002315323
- bfmBAA
- Staphylococcus aureus NP_374631
- bkdA1
- Sphingobium japonicum YP_003544745
- bkdA1
- Brevibacillus brevis YP_002771850
- bkdA
- Lactobacillus casei YP_001987607
- 2-cetoácido de cadena ramificada deshidrogenasa, componente E1, subunidad beta
- bkdAB Anoxybacillus flavithermus YP_002315324
- bfmBAB
- Staphylococcus aureus NP_374630
- bkdA2
- Sphingobium japonicum YP_003544746
- bkdA2
- Brevibacillus brevis YP_002771851
- bkdB
- Lactobacillus casei YP_001987606
- 2-cetoácido de cadena ramificada deshidrogenasa, componente E2
- bkdB Anoxybacillus flavithermus YP_002315325
- bfmBB
- Staphylococcus aureus NP_374629
- pdhC
- Sphingobium japonicum YP_003544747
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- bkdB
- Brevibacillus brevis YP_002771852
- bkdC
- Lactobacillus casei YP_001987605
- 2-cetoácido de cadena ramificada deshidrogenasa, componente E3
- lpdV Anoxybacillus flavithermus YP_002315322
- Staphylococcus aureus
- NP_374632
- pdhD
- Sphingobium japonicum YP_003545508
- Lpd
- Brevibacillus brevis YP_002771849
- bkdD
- Lactobacillus casei YP_001987608
- 3-cetoacil-ACP sintetasa III
- fabH Geobacillus kaustophilus YP_146657.1
- Bacillus megaterium
- YP_003561163.1
- fabH
- Staphylococcus aureus ZP_05601460.1
- fabH1
- Streptomyces coelicolor P72392
- Beutenbergia cavernae
- YP_002881824
- citramalato sintetasa
- cimA Methanobrevibacter ruminantium YP_003424156
- cimA
- Leptospira interrogans ABK13754
- Ignicoccus hospitalis
- ABU82163
- Cyanothece 51142
- YP_001801665
- Geobacter sulfurreducens
- NP_952848
- 3-isopropilmalato deshidrogenasa
- leuB Cronobacter turicensis YP_003209069.1
- leuB
- Shigella boydii YP_406624
- leuB
- Actinobacillus pleuropneumoniae YP_001651485
- leuB
- Cronobacter turicensis YP_003209069
- leuB
- Pantoea ananatis YP_003519000
- isopropilmalato isomerasa subunidad grande
- leuC Salmonella enteric NP_459116
- leuC
- Serratia proteamaculans YP_001476977
- leuC
- Photorhabdus asymbiotica YP_003039932
- leuC
- Klebsiella pneumoniae YP_002917788
- leuC
- Haemophilus influenzae NP_439151
- isopropilmalato isomerasa subunidad pequeña
- leuD Shigella dysenteriae YP_401823
- leuD
- Buchnera aphidicola YP_002477704
- leuD
- Actinobacillus pleuropneumoniae YP_001967934
- leuD
- Haemophilus somnus YP_718599
- leuD
- Xanthomonas campestris YP_365316
- treonina desaminasa
- Cha1 Saccharomyces cerevisiae NP_001018030
- ilvA
- Vibrio fischeri YP_002157347
- ilvA
- Shewanella violacea YP_003558613
18
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- ilvA
- Metilococcus capsulatus YP_112886
- ilvA
- Dichelobacter nodosus YP_001209243
- treonina deshidratasa
- tdcB Pantoea ananatis YP_003519179
- tdcB
- Klebsiella pneumoniae YP_001335951
- tdcB
- Shigella boydii YP_409322
- tdcB
- Acinetobacter baumannii YP_001706275
- tdcB
- Psychrobacter arcticum YP_264671
- acetolactato sintetasa (AHAS) subunidad III grande
- ilvI Yersinia enterocolitica YP_001005008
- ilvI
- Salmonella enterica YP_002113134
- ilvI
- Buchnera aphidicola NP_240056
- ilvI
- Xenorhabdus bovienii YP_003469370
- ilvI
- Klebsiella pneumoniae YP_001333772
- acetolactato sintetasa (AHAS) subunidad III pequeña
- ilvH Laribacter hongkongensis YP_002794162
- ilvH
- Burkholderia mallei YP_103450
- ilvH
- Nitrosomonas europaea NP_841373
- ilvH
- Campylobacter jejuni YP_178690
- ilvH
- Desulfomicrobium baculatum YP_003156951
- acetolactato sintetasa (AHAS) subunidad II grande
- ilvG Yersinia pestis YP_002348776
- ilvG
- Ralstonia solanacearum YP_003752653
- ilvG
- Bordetella bronchiseptica NP_887973
- ilvG
- Aeromonas salmonicida YP_001140066
- ilvG
- Stenotrophomonas maltophilia YP_001973605
- acetolactato sintetasa (AHAS) subunidad II pequeña
- ilvM Shigella dysenteriae YP_405398
- ilvM
- Dickeya dadantii YP_002989351
- ilvM
- Xenorhabdus bovienii YP_003470064
- ilvM
- Photorhabdus luminescens NP_931846
- ilvM
- Xanthomonas oryzae YP_199583
- cetol-ácido reductoisomerasa
- ilvC Buchnera aphidicola NP_240398
- ilvC
- Pseudomonas aeruginosa YP_002442658
- ilvC
- Francisella tularensis YP_001122021
- ilvC
- Vibrio fischeri YP_205911
- ilvC
- Actinobacillus pleuropneumoniae YP_001652891
- dihidroxiácido deshidratasa
- ilvD Citrobacter rodentium YP_003367413
- ilvD
- Buchnera aphidicola YP_002468875
- ilvD
- Xanthomonas campestris YP_001901776
19
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asoció con un aumento en los ácidos grasos anteiso y una disminución en los ácidos grasos iso, según muestra la Figura 16.
Los resultados de este Ejemplo muestran que aumentar el flujo de carbono hacia la ruta de la isoleucina de la síntesis de ácido graso ramificado aumenta la cantidad de ácido graso ramificado anteiso producido en la célula 5 huésped.
Ejemplo 12. Análisis de ácidos grasos.
Este ejemplo describe un método para analizar ácidos grasos, tal como los ácidos grasos producidos en células bacterianas mediante cromatografía de gases.
Las muestras para análisis se prepararon de la siguiente forma. Los cultivos bacterianos (aproximadamente 1,5 ml)
10 se congelaron en viales de vidrio de 2,0 ml y se guardaron a -15 °C hasta estar listos para su procesamiento. Las muestras se enfriaron en hielo seco durante 30 minutos y a continuación se liofilizaron durante la noche (~16 horas) hasta sequedad. Se añadió a cada vial una alícuota de 10 µl de patrón interno (trinonadecanoato de glicerol (n.° de catálogo Sigma T4632-1G)), seguido por adición de 400 µl de NaOH 0,5 N (en metanol). El vial se tapó y se vortizó durante 10 segundos. A continuación, las muestras se incubaron a 65 °C durante 30-50 minutos, se retiraron de la
15 incubadora, y se añadieron 500 µl de reactivo de trifluoruro de boro (número de catálogo Aldrich B1252). Las muestras se vortizaron durante 10 segundos. A continuación, las muestras se incubaron a 65 °C durante 10-15 minutos y se enfriaron a temperatura ambiente (aproximadamente 20 minutos). Se añadió hexano (350 µl), y las muestras se vortizaron durante 10 segundos. Si las fases no se separan, se añadieron 50-100 µl de una solución salina saturada (5 g NaCl en 5 ml de agua), y la muestra se volvió a vortizar durante 10 segundos. Al menos 100 µl
20 de la capa superior de hexano se introdujo en un vial de cromatografía de gases (CG), que se cerró y guardó a 4 °C o -20 °C hasta análisis.
La cromatografía de gases se llevó a cabo como se describe en la Tabla B siguiente. Un patrón de éster metílico de ácido bacteriano (número de catálogo Sigma 47080-U) y un patrón de éster metílico de ácido graso (número de catálogo Sigma 47885-U) se utilizaron para identificar picos en las muestras. Se utilizó una muestra de
25 comprobación con tripalmitato de glicerilo l (número de catálogo Sigma T5888-1G) para confirmar la esterificación de las muestras. Se utilizó un patrón de blanco (solamente el patrón interno) para evaluar el ruido de fondo.
Tabla B
- Cromatógrafo de gases
- HP 5890 GC Series II
- Detector
- FID 360 °C 40 ml/min Hidrógeno, 400 ml/min Aire
- Gas portador
- Helio
- Programa cuantitativo
- GC Chemstation A.09.03. (Agilent)
- Columna
- VF-5 ms 15 M x 0,150 mm x 0,15 mm número de catálogo Varian CP9035
- Revestimiento de inyección
- Gooseneck (con envoltura de lana de vidrio)
- Inyector
- HP 7673
- Jeringa de inyección
- 10 ml
- Modo de inyección
- División 25:1
- Volumen de inyección
- 4 ml (velocidad del émbolo = rápida; 5 bombeos de la muestra)
- Lavados con disolvente antes de la inyección
- 2 muestras
- Lavados con disolvente después de la inyección
- 3 tanto con acetona como con hexano
- Temperatura del inyector
- 325 °C
- Tiempo total del programa
- 16 minutos
27
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| US12/972,822 US20110151526A1 (en) | 2009-12-22 | 2010-12-20 | Branched-Chain Fatty Acids And Biological Production Thereof |
| PCT/US2010/061544 WO2011087787A1 (en) | 2009-12-22 | 2010-12-21 | Branched-chain fatty acids and biological production thereof |
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| US20150064782A1 (en) | 2012-04-02 | 2015-03-05 | REG Life Sciences, LLC | Production of fatty acid derivatives |
| WO2014144461A2 (en) * | 2013-03-15 | 2014-09-18 | Consejo Nacional De Investigaciones Cientificas Y Tecnicas (Conicet) | Engineered organisms for production of novel lipids |
| KR101747542B1 (ko) * | 2015-10-23 | 2017-06-15 | 씨제이제일제당 (주) | L-이소루신 생산능을 가지는 코리네박테리움 속 미생물 및 이를 이용하여 l-이소루신을 생산하는 방법 |
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| WO2020070699A1 (en) * | 2018-10-04 | 2020-04-09 | Anna University | Production of l-2-aminobutyrate from citramalate,citraconate or 2-oxobutanoate |
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