Engineering Xylose Metabolism in Triacylglycerol-Producing Rhodococcus Opacus for Lignocellulosic Fuel Production

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Engineering xylose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production

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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

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 substrate Carbon 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 Coe-XK RPGVPVMSLGTSGTAYAVSQRRPADPTGTVAGFADARGDWLPLACTLNCTLAVDRVASLLGLDREAVEPGTDVTLLPFLDGER Ave-XK RPGTPVLSLGTSGTVYAVSTRRPADPTGTVAGFADARGDWLPLACTLNCTLAVDRVAALLGLDREAVEPGTGVTLLPYLDGER Hyg-XK RPGTPVLSLGTSGTVYAVSRRRPTDPTGTVAGFADARGDWLPLACTLNCTLAVDRVAALLGLDREAVESGGSVTLLPYLDGER Sp8-O2 LPGTPVLSLGTSGTVYAVSRHRPADPTGTVAGFADARGDWLPLACTLNCTLAVDRVATLLGLDREAVAPGGSVTLLPFLDGER 332

**:**::**:********* *************:********** *** .:*********** ***************: . Cha-XK TPNLPNASGLLHGLRHDTTAGQLLQAAYDGAVHSLLGALDLVLDEDADPSAPLLLIGGGARGTAWQQTVRRLSGRPVQVPEAK Coe-XK TPNLPHSSGLLHGLRHDTTAGQLLQAAYDGAVHSLLGALDLVLDADADPSAPLLLIGGGARGTAWQQTVRRLSGRPVQIPEAR Ave-XK TPDLPHASGLLHGLRHDTTGGQLLQAAYDGAVHALLGALDLVLDADADRSAPLLLIGGGARGTAWQQTVRRLSGRPVQVPEAR Hyg-XK TPNLPHASGVLHGLRHDTTAGQLLQAAYDGAVHALLGALDLVLDEDADRDTPLLLIGGGARGTAWQQTVRRLSGRPVQVPEAR Sp8-O2 TPDLPNASGLLHGLRHDTTPGQLLQAAYDGAVHSLLGALDLVLDEDADRDSPLLLIGGGARGHAWQQTVRRLSGRPVQ ILRLA 415 Hyg-CB MSDTSPYGFELVRRGYDRAQVDERISKLVSDRDSALARITALEKRIEELHLETQNAQAQITDAEPSYAGLGARVEKILRLA 81 Ave-CB MSDTSPYGFELVRRGYDRAQVDERISKLVSDRDSALARITALEKRIEELHLETQNAQAQVTDAEPSYAGLGARVEKILRLA 81 Cha-CB MSDTSPYGFELVRRGYDRAQVDERISKLVSDRDSALARITALEKRIEELHLETQNAQAQISDAEPSYAGLGARVEKILRLA 81 Coe-CB MSDTSPYGFELVRRGYDRAQVDERISKLVSDRDSALARITALEKRIEELHLETQNAQAQVNDAEPSYAGLGARVEKILRLA 81 .:. :. :*:. .*: : . . . . :. *. .: * .. *: : : *..* :*****

* * :* . * : :. .. *.**. : . : ::..* .: Cha-XK ELVALGAAAQAAGVLTGEDPAAVARRWNTAAGPVLDAVERDEETLARIAGVLSDAAPLLERPTDTH 481 Coe-XK ELVALGAAAQAAGLLTGEDAAAVARRWNTAAGPVLDAVERDEATLNRITGVLSDAAPLLERDAASR 481 Ave-XK ELVALGAAAQAAGVLTGEDPAAVARRWDTAHGPVLDAVERDEATLARISGVLSDAAPLLEREPDRR 481 Hyg-XK ELVALGAAAQAAGLLTGEDPAAVARRWNTAAGPVLEAVERDRTTLDRITGVLSDAAPLLERGTDTE 481 Sp8-O2 EEEAKELREEARRAAEQHRELAESAAQQVRNDAESYATERKAKAEDEGSRIVEKAKGEASQLRADAQKDAQSKREEADALFEE 498 Hyg-CB EEEAKELREEARRAAEQHRDLAESAAQQVRNDAESYAAERKAKAEDEGVRIVEKAKSDAAQLRAEAQKDAQSKREEADALFEE 164 Ave-CB EEEAKDLREEARRASEQHRELAESAAQQVRNDAESFAADRKSKAEDEGVRIVEKAKSDASQLRQEAQKDAQSKREEADALFEE 164 Cha-CB EEEAKDLREEARRAAEQHRELAESAAQQVRNDAESYSAERKAKAEDEGLRIVEKAKSDAAQLRAEAQKDAQSKREEADALFEE 164 Coe-CB EEEAKDLREEARRAAEQHRELAESSAQQVRNDAESYAAERKAKAEDEGVRIVEKAKGDASQLRSEAQKDAQSKRDEADALFEE 164 *****:********:****:****:**********::::**:****** *******.:*:*** :*********:********

Sp8-O2 TRAKAAQAAADFETNLAKRREQSERDLASRQQKAEKRLAEIEHRAEQLRLEAEKLRTDAERRARQTVETAQRQAEDIVADANA 581 Hyg-CB TRAKAAQAAADFETNLAKRREQSERDLASRQQKAEKRLAEIEHRAEQLRLEAEKLRTDAERRARQTVETAQRQAEDIVADANA 247 Ave-CB TRAKAAQAAADFETNLAKRREQSERDLASRQAKAEKRLAEIEHRAEQLRLEAEKLRTDAERRARQTVETAQRQAEDIVADANA 247 Cha-CB TRAKAAQAAADFETNLAKRREQSERDLASRQAKAEKRLAEIEHRAEQLRLEAEKLRTDAERRARQTVETAQRQAEDIVADANA 247 Coe-CB TRAKAAQAAADFETNLAKRREQSERDLASRQAKAEKRLAEIEHRAEQLRLEAEKLRTDAERRARQTVETAQRQSEDIVADANA 247 ******************************* *****************************************:*********

Sp8-O2 KADRIRSESERELAALTNRRDSINAQLTNVREMLATLTGAAVAAAGT-TGEDEPVSRGVPAQQTR 645 Hyg-CB KADRIRSESERELAALTNRRDSINAQLTNVREMLATLTGAAVAAAGT-STDDEPISRGVPAQQSR 311 Ave-CB KADRIRSESERELAALTNRRDSINAQLTNVREMLATLTGAAVAAAGTPSTDDEPISRGVPAQQSR 312 Cha-CB KADRIRSESERELAALTNRRDSINAQLTNVREMLATLTGAAVAAAGT-PAEDEPISRGVPAQQSR 311 Coe-CB KADRIRSESERELAALTNRRDSINAQLTNVREMLASLTGAAVAAAPS--VEDESVSRGVPAQQSR 310 ***********************************:********* : :**.:********:* Figure S3 Comparison of ORF-2 in pXs8 insert with several xylulose kinases and cellulose-binding proteins at the deduced amino acid sequence level using the CLUSTALW program. Cha-XK, xylulose kinase of S. chartreusis accession no. ZP_09957235; Coe-XK, xylulose kinase of S. coelicolor accession no. NP_733525; Ave-XK, xylulose kinase of S. avermitilis accession no. NP_828357; Hyg-XK, xylulose kinase of S. hygroscopicus accession no. AEY87894; Sp8-O2, ORF-2 of the 3603-bp insert; Hyg-CB, cellulose-binding protein of S. hygroscopicus accession no. AEY91743; Ave-CB, cellulose-binding protein of S. avermitilis accession no. NP_824035; Cha-CB, cellulose-binding protein of S. chartreusis accession no. ZP_09956458; Coe-CB, cellulose-binding protein of S. coelicolor accession no. NP_629535. Numbering of the amino acids starts at the N-terminus of all proteins. Sequences of xylulose kinases and cellulose-binding proteins are depicted in black and blue letters, respectively. 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. Figure S4 Construction of plasmid pX0, pX1, pX2, pX3 and pX4. These plasmids contain the NG2 origin of replication from pEP2, the RP4 mob element from pSUP301, a spectinomycin resistance marker derived from the omega interposon and the trc promoter from pTrc99A47. See for methods for details. a b Derivative 6 1 6 1 Derivative 6 1 6 1 PD630- Xsp8- 5 X1 - 2 5 X3 - 2 5 X1 - 2 5 X3 - 2

6 1 4 3 4 3 6 1 4 3 4 3

5 X0 - 2 (xylA) (xylA/xylB) (xylA) (xylA/xylB) 5 X0 - 2

4 3 6 1 6 1 6 1 6 1 4 3

(gene deletion) 5 2 5 2 5 2 5 X4 - 2 X2- X4- (gene deletion) X2-

4 3 4 3 4 3 4 3

(xylB) (xylA/xylB-CBD) (xylB) (xylA/xylB-CBD)

B DNA of X1- D DNA of X3- G DNA of X1- I DNA of X3- c pX1 pX3 d pX1 1 2 3 4 5 6 pX3 1 2 3 4 5 6 Derivative 1 2 3 4 5 6 1 2 3 4 5 6 Derivative PD630- Xsp8-

A DNA of X0- F DNA of X0- pX0 1 2 3 4 5 6 pX0 1 2 3 4 5 6 (xylA) (xylA/xylB) (xylA) (xylA/xylB) 8 kb C DNA of X2- E DNA of X4- 8 kb H DNA of X2- J DNA of X4-

pX2 1 2 3 4 5 6 pX4 1 2 3 4 5 6 pX2 1 2 3 4 5 6 pX4 1 2 3 4 5 6 3 kb 3 kb

1 kb 1 kb (gene deletion) (gene deletion)

(xylB) (xylA/xylB-CBD) (xylB) (xylA/xylB-CBD)

Figure S5 Elucidation of the molecular targets involved in improvement of xylose metabolism in R. opacus PD630. (a,b) Growth of Rhodococcus isolates on LB plates. Six colonies each plate in Figure 6a, b were randomly streaked onto LB plate and incubated for 3 days. The isolates were designated PD630X0-1 to -6, PD630X1-1 to -6, PD630X2-1 to -6, PD630X3-1 to -6 and PD630X4-1 to -6, respectively, from pX0, pX1, pX2, pX3 and pX4 plates in Figure 6a, and Xsp8X0-1 to -6, Xsp8X1-1 to -6, Xsp8X2-1 to -6, Xsp8X3-1 to -6 and Xsp8X4-1 to -6, respectively, from pX0, pX1, pX2, pX3 and pX4 plates in Figure 6b. (c,d) Detection of the plasmid DNA in R. opacus derivatives. Each strain was grown in LB medium supplemented with spectinomycin for 24 h in a flask. The total genomic DNA of each strain was prepared and subjected to 0.7% agarose gel electrophoresis. Arrows in panels A and F, pX0; arrows in panels B and G, pX1; arrows in panels C and H, pX2; arrows in panels D and I, pX3; arrows in panels E and J, pX4. Filled arrowheads indicate size of relevant markers (1 kb ladder).