Engineering xylose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Kurosawa, Kazuhiko, Sandra J Wewetzer, and Anthony J Sinskey. 2013 Engineering Xylose Metabolism in Triacylglycerol-producing Rhodococcus Opacus for Lignocellulosic Fuel Production. Biotechnology for Biofuels 6(1): 134. As Published http://dx.doi.org/10.1186/1754-6834-6-134 Publisher BioMed Central Ltd. Version Final published version Citable link http://hdl.handle.net/1721.1/81356 Terms of Use Creative Commons Attribution Detailed Terms http://creativecommons.org/licenses/by/2.0 Stachyose D-Malic Acid Oxalic Acid Turanose Pyruvic Acid N-Acetyl-D-Glucosaminitol D-Melezitose L-Malic Acid D-Glucose-6-Phosphate D-Fructose Citric Acid α-Hydroxy Glutaric Acid-γ-Lactone D-Melibiose L-Arginine Glyoxylic Acid D-Raffinose L-Isoleucine α-Methyl-D-Mannoside M-Inositol Citramalic Acid Sec-Butylamine Sucrose Tween 40 L-Lyxose N-Acetyl-D-Glucosamine Glycyl-L-Proline Mucic acid Lactulose L-Tartaric Acid Formic Acid Palatinose Bromo Succinic Acid Acetamide m-Hydroxy Phenyl Acetic Acid Mono Methyl Succinate D-Ribono-1,4-Lactone α-D-Lactose Succinic Acid L-Homoserine L-Rhamnose Lactitol L-Fucose β-Methyl-D-Galactoside Malonic Acid Salicin Sebacic Acid Methyl Pyruvate β-Methyl-D-Xyloside α-D-Glucose L-Alaninamide Mannan Maltotriose 5-Keto-D-Gluconic Acid Hydroxy-L-Proline α-Methyl-D-Galactoside L-Proline 3-Methyl Glucose L-Alanine Glycerol β-Cyclodextrin D-Trehalose L-Threonine Dulcitol D-Arabitol Xylitol D-Glucose-1-Phosphate γ-Amino Butyric Acid D,L-Octopamine Glycogen D-Sorbitol Succinamic Acid β-Methyl-D-Glucuronic Acid L-Alanyl-Glycine Glycine L-Methionine p-Hydroxy Phenyl Acetic Acid D-Arabinose Inulin D-Galactose L-Phenylalanine D-Fructose-6-Phosphate Melibionic Acid D-Ribose I-Erythritol Itaconic Acid Sorbic Acid L-Glucose α-Methyl-D-Glucoside D-Galacturonic Acid M-Tartaric Acid L-Valine L-Sorbose Tricarballylic Acid α-Keto-Butyric Acid D-Lactic Acid Methyl Ester α-Cyclodextrin α-Hydroxy butyric Acid D-Threonine Gentiobiose Maltose Carbon substrateCarbon Thymidine L-Arabitol D-Mannitol δ-Amino Valeric Acid Sedoheptulosan α-Keto-Glutaric Acid γ-Hydroxy Butyric Acid D,L-α-Glycerol Phosphate Tyramine Amygdalin β-Hydroxy Butyric Acid Acetoacetic Acid L-Leucine D-Glucosamine Inosine D-Alanine L-Ornithine N-Acetyl-L-Glutamic Acid L-Pyroglutamic Acid 2,3-Butanediol D-Serine 2-Aminoethanol D-Glucuronic Acid L-Histidine Butyric Acid 1,2-Propanediol D-Tagatose D-Glucosaminic Acid N-Acetyl-β-D-Mannosamine Citraconic Acid L-Asparagine Oxalomalic Acid Gelatin Phenylethyl-amine L-Glutamic Acid N-Acetyl-Neuraminic Acid Caproic Acid D-Xylose Glycolic Acid Maltitol Glucuronamide Chondroitin Sulfate C D-Galactonic Acid-γ-Lactone L-Lysine L-Serine α-Keto Valeric Acid 2,3-Butanone 2-Deoxy-D-Ribose D-Saccharic Acid Glycyl-L-Glutamic Acid D-Aspartic Acid D-Gluconic Acid D-Mannose Adonitol L-Glutamine D-Fucose Pectin Propionic Acid N-Acetyl-D-Galactosamine D-Tartaric Acid L-Aspartic Acid 3-Hydroxy 2-Butanone Dihydroxy Acetone L-Lactic Acid Laminarin 3-O-β-D-Galacto-pyranosyl-D-Arabinose 4-Hydroxy Benzoic Acid Arbutin β-D-Allose Fumaric Acid Dextrin Adenosine Quinic Acid L-Arabinose 2-Deoxy Adenosine D,L-Malic Acid Uridine Tween 20 Acetic Acid γ-Cyclodextrin Capric Acid Putrescine β-Methyl-D-Glucoside D-Cellobiose Tween 80 Glycyl-L-Aspartic Acid 2-Hydroxy Benzoic Acid D,L-Carnitine D-Psicose L-Galactonic Acid-γ-Lactone 0.0 0.4 0.8 1.2 1.6 0.0 0.4 0.8 1.2 1.6 0.0 0.4 0.8 1.2 1.6 Overall well color development (OD595) Wild-type PD630 Transformant Xsp8 Figure S1 Carbon utilization profiles of R. opacus strains. Turbidity data measure bacterial growth, indicative of substrate utilization. Washed cells grown on LB plates for 3 days were suspended in 28 ml IF-0a GN/GP Base Inoculating Fluid (Biolog) and 0.3 ml Redox Dye Mix D (Biolog) and adjusted to an OD660 of 0.01. Aliquots of 50 µl the mixture were dispensed into each well of the BIOLOG PM microplates. Data are based on the absorbance following incubation of the plates for 5 days. The well color development for each sample was calculated as the mean value of the OD595 in the three replicates of each substrate, subtracted of the average OD595 in the background wells. The order of the carbon sources is the rank of the growth on 190 substrates for PD630. Error bars show the standard deviations. Hyg-XI MNYQPTPEDRFTFGLWTVGWQGRDPFGDATRRALDPVETVQRLAELGAHGVTFHDDDLIPFGSSDTERESHIKRFRQALD Ave-XI MNYQPTPEDRFTFGLWTVGWQGRDPFGDATRRALDPVETVQRLAGLGAHGVTFHDDDLIPFGSSDTERESHIKRFRQALD Cha-XI MSYQPTPEDRFTFGLWTVGWQGRDPFGDATRRALDPVETVQRLAELGAHGVTFHDDDLIPFGSSDTEREAHIKRFRQALD Coe-XI MNYQPTPEDRFTFGLWTVGWQGRDPFGDATRQALDPAESVRRLSELGAYGVTFHDDDLIPFGSSDTERESHIKRFRQALD Sp8-O1 MSFQPTPEDRFTFGLWTVGWQGRDPFGDATRPALDPVETVQRLAELGAYGVTFHDDDLIPFGSSDTERESHIKRFRQALD 80 *.:**************************** ****.*:*:**: ***:********************:********** Hyg-XI ATGMTVPMATTNLFTHPVFKDGAFTANDRDVRRYALRKTIRNIDLAAELGAKTYVAWGGREGAESGAAKDVRSALDRMKE Ave-XI ATGMAVPMATTNLFTHPVFKDGAFTANDRDVRRYALRKTIRNIDLAAELGAKTYVAWGGREGAESGAAKDVRVALDRMKE Cha-XI ATGMKVPMATTNLFAHPVFKDGAFTANDRDVRRYALRKTIRNIDLAVELGAQVYVAWGGREGAESGAAKDVRAALDRMKE Coe-XI ATGMKVPMATTNLFTHPVFKDGAFTANDRDVRRYALRKTIRNIDLAVELGASVYVAWGGREGAESGAAKDVRDALDRMKE Sp8-O1 ATGMTVPMATTNLFTHPVFKDGGFTANDRDVRRYALRKTIRNIDLAAELGAKTYVAWGGREGAESGGAKDVRDALDRMKE 160 **** *********:*******.***********************.****..*************.***** ******* Hyg-XI AFDLLGEYVTSQGYDLRFAIEPKPNEPRGDILLPTVGHALAFIERLERPELYGVNPEVGHEQMAGLNFPHGIAQALWAGK Ave-XI AFDLLGEYVTAQGYDLRFAIEPKPNEPRGDILLPTVGHALAFIERLERPELYGVNPEVGHEQMAGLNFPHGIAQALWAGK Cha-XI AFDLLGEYVTSQGYDLRFAIEPKPNEPRGDILLPTVGHALAFIERLERPELYGVNPEVGHEQMAGLNFPHGIAQALWAGK Coe-XI AFDLLGEYVTEQGYDLKFAIEPKPNEPRGDILLPTVGHALAFIERLERPELYGVNPEVGHEQMAGLNFPHGIAQALWAGK Sp8-O1 AFDLLGEYVIAQGYDLRFAIEPKPNEPRGDILLPTVGHALAFIERLERPELYGVNPEVGHEQMAGLNFPHGIAQALWAGK 240 ********* *****:*************************************************************** Hyg-XI LFHIDLNGQSGIKYDQDLRFGAGDLRSAFWLVDLLESAGYEGPRHFDFKPPRTEDLDGVWASAAGCMRNYLILKERAAAF Ave-XI LFHIDLNGQSGIKYDQDLRFGAGDLRAAFWLVDLLESAGYEGPKHFDFKPPRTEDLDGVWASAAGCMRNYLILKERTAAF Cha-XI LFHIDLNGQSGIKYDQDLRFGAGDLRAAFWLVDLLESAGYTGPRHFDFKPPRTEDLDGVWASAAGCMRNYLILKERAAAF Coe-XI LFHIDLNGQSGIKYDQDLRFGAGDLRAAFWLVDLLERAGYAGPRHFDFKPPRTEDFDGVWASAAGCMRNYLILKDRAAAF Sp8-O1 LFHIDLNGQSGIKYDQDLRFGAGDLRAAFWLVDLLETAGYEGPRHFDFKPPRTEDFDGVWASAAGCMRNYLILKDRAAAF 320 **************************:********* *** **:***********:******************:*:*** Hyg-XI RADPEVQEALRASRLDQLAQPTAADGLEDLLADRAAFEDFDVEAAAARGMAFERLDQLAMDHLLGARG Ave-XI RADPEVQEALRAARLDELAQPTAGDGLTALLADRTAFEDFDVEAAAARGMAFEQLDQLAMDHLLGARG Cha-XI RADPEVQEALRAARLDELAQPTAADGLQALLADRSAFESFDVEAAAARGMAFEHLDQLAMDHLLGARG Coe-XI RADPQVQEALAAARLDELARPTAEDGLAALLADRSAYDTFDVDAAAARGMAFEHLDQLAMDHLLGAR- Sp8-O1 RADPEVQEALRAARLDQLAQATAADGLDALLADRSAFEDFDVDAAAARGMAFEHLDQLAMDHLLGARG 388 ****:***** *:***:**:.** *** *****:*:: ***:**********:************* Figure S2 Comparison of ORF-1 in pXsp8 insert with several xylose isomerases at the deduced amino acid sequence level using the CLUSTALW program. Hyg-XI, xylose isomerase of S. hygroscopicus accession no. AEY87893; Ave-XI, xylose isomerase of S. avermitilis accession no. NP_828358; Cha-XI, xylose isomerase of S. chartreusis accession no. ZP_09957234; Coe-XI, xylose isomerase of S. coelicolor accession no. NP_625460; Sp8-O1, ORF-1 of the 3603-bp insert. Numbering of the amino acids starts at the N-terminus of all proteins. The introduction of gaps is indicated by dashes. Asterisks, colons and periods indicate identical, strongly similar and weakly similar conserved residues, respectively, among all of these enzymes. *************:***********.:**:***********::* ************* *** ***:**:****:*:****** Cha-XK MSAAEGPLVVGVDSSTQSTKALVVDVSTGRVVASGQAPHTVSSGSGRESDPRQWWDALCEALRQCGDAAHEAAAVSVGGQQHG Coe-XK MSAAEGPLVVGVDTSTQSTKALVVDAATGRVVASGQAPHTVSSGTGRESDPRQWWDALGEALSQCGEAAREAAAVSVGGQ QHG Ave-XK MSAAEGPLVVGVDTSTQSTKALVVDASTGRVVASGQAPHTVSAGAGRESDPRQWWDALCEALHQCGDAAHEAAAISVGGQ QHG Hyg-XK MSAAEGPLVVGVDTSTQSTKALVVDAATGQVVASGQAPHTVSSGAGRESDPRQWWDALCEALRQCGDAAHEAAAVSIGGQ QHG Sp8-O2 MSAAEGPLVVGVDTSTQSTKALVVDAATGQVVASGQAPHTVTTGGGRESDPRQWWDALCEALRQCGDAAHEAAAISVGGQ QHG 83 ***** :*:***************** ** :****.* *********..***.:*****:******.**. ************ Cha-XK LVTLDDRGEPVRPALLWNDVRSAPQARRLTEELGGAKFWAERTGSVPAASFTVTKWAWLAEHEPEAARATKAVRLPHDYLTER Coe-XK LVTLDARGEPVRPALLWNDVRSAPQARRLIDELGGAKAWAERTGSVPSASFTVTKWAWLTEHEPEAARAVKAVRLPHDYLTER Ave-XK LVTLDAHGDPVRPALLWNDVRSAPQARRLIEELGGPKAWAERTGSVPGPSFTVTKWAWLTEHEPEAVRATAAVRLPHDYLTER Hyg-XK LVTLDERGEPVRPALLWNDVRSAPQAGRLVEELGGPKAWAERTGSVPGASFTVTKWAWLAEHEPEAVRATRAVRLPHDYLTER Sp8-O2 LVTLDERGEPVRPALLWNDVRSAPQARRLVEELGGPKAWAERTGSVPGASFTASKWAWLAEHEPEAARATRAVRLPHDYLTER 166 ***:..*****.********.** ***: *:::.**************:.*****.*.**********.*************: Cha-XK LTGQGTTDRGDASGTGWWASGTERYDEEILAQVGLDPALLPRVVRPGEIAGTVRDSHDLPFSKGTLVAPGTGDNAAAALGLGV Coe-XK LTGEGTTDRGDVSGTGWWASGTEAYDEEILARVALDPALLPRVVRPGEVAGTVRDGHGLPFSKGTLVAAGTGDNAAAALGLGL Ave-XK LTGQGTTDRGDVSGTGWWASATESYDEEVLTHVGLDPALLPRVVRPGEVVGTVRDSHDLPFSKGTLVAPGTGDNAAAALGLGL Hyg-XK LTGQGTTDRGDASGTGWWASGTEAYDEETLAHVGLDPALLPRVVRPGEVAGTVRDSHDLPFSKGTLVAPGTGDNAAAALGLGL Sp8-O2 LTGQAVTDRGDASGTGWWASGTERYDEDTLAHLGLDPALLPRVVRPGEVAGTVRDGHGLPFSKGTLVASGTGDNAAAALGLGL 249 **.**:*******.**** :**:**:******************************:********* .* *****:***** Cha-XK RPGVPVMSLGTSGTVYAVSQRRPADPSGTVAGFADARGDWLPLACTLNCTLAVDRVATLLGLDREAVEPGTAVTLLPYLDGER