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Fatty Acid I ('D IIIIII IIIIIIII Ill lllll lllll lllll lllll lllll lllll lllll lllll 111111111111111111 US009487804B2 c12) United States Patent (IO) Patent No.: US 9,487,804 B2 San et al. (45) Date of Patent: Nov. 8, 2016 (54) HYDROXY- AND DICARBOXYLIC-FAT Golyshin P.N., et al., Oleiphilaceae fam. nov., to include Oleiphilus SYNTHSIS BY MICROBES messinensis gen. nov., sp. nov., a novel marinebacterium that obligately utilizes hydrocarbons. Int J Syst Evol Microbiol 52:901- (71) Applicant: William Marsh Rice University, 911 (2002). Houston, TX (US) Heath, R.J. & Rock, C.O. Inhibition of beta-ketoacyl-acyl carrier protein synthase III (FabH) by acyl-acyl carrier protein in (72) Inventors: Ka-Yiu San, Houston, TX (US); Xixian Escherichia coli, J Biol Chem 271, 10996 (1996a). Xie, Houston, TX (US); Leepika Tuli, Heath, R.J. & Rock, C.O., Regulation of fatty acid elongation and Houston, TX (US); Hui Wu, Houston, initiation by acyl-acyl carrier protein in Escherichia coli, J Biol TX (US) Chem 271, 1833 (1996b). Ishizuka M., et al., Overexpression of human acyl-CoA thioesterase (73) Assignee: William Marsh Rice University, upregulates peroxisome biogenesis, Exp Cell Res. Jul. 1, Houston, TX (US) 2004;297( 1): 127-41. Jing F., et al., Phylogenetic and experimental characterization of an ( *) Notice: Subject to any disclaimer, the term ofthis acyl-ACP thioesterase family reveals significant diversity in enzy­ patent is extended or adjusted under 35 matic specificity and activity, BMC Biochemistry 12:44 (2011). U.S.C. 154(b) by 28 days. Julsing M.K., Outer membrane protein AikL boosts biocatalytic oxyfunctionalization of hydrophobic substrates in Escherichia coli, (21) Appl. No.: 14/607,624 Appl Environ Microbiol. 78(16):5724-33 (2012). Lee S., et al., Heterologous co-expression of accA, fabD, and (22) Filed: Jan. 28, 2015 thioesterase genes for improving long-chain fatty acid production in Pseudomonas aeruginosa and Escherichia coli. Appl Biochem (65) Prior Publication Data Biotechnol. May 2012;167(1):24-38. US 2015/0225753 Al Aug. 13, 2015 Lee Sl, Jeon E, Jung Y, Lee J. Liu C. & Shao Z., Alcanivorax dieselolei sp. nov., a novel alkane-degrading bacterium isolated Related U.S. Application Data from sea water and deep-sea sediment. Int J Syst Evol Microbiol (60) Provisional application No. 61/939,567, filed on Feb. 55(3):1181-1186 (2005). 13, 2014. Marchant R., et al., The degradation of n-hexadecane in soil by Thermophilic geobacilli. FEMS Microbiol Ecol 56 (1):44-54 (51) Int. Cl. (2006). C12N 9/16 (2006.01) Meintanis C, et al., Biodegradation of crude oil by thermophilic C12P 7164 (2006.01) bacteria isolated from a volcano island. Biodegradation 17:3-9 C12N 15152 (2006.01) (2006). C12P 7142 (2006.01) Saerens S.M.G., et al., The Saccharomyces cerevisiae EHTl and C12P 7144 (2006.01) EEBl genes encode novel enzymes with medium-chain fatty acid (52) U.S. Cl. ethyl ester synthesis and hydrolysis capacity. J Biol Chem 281(7): CPC ............... C12P 716409 (2013.01); C12N 9/16 4446-4456 (2006). (2013.01); C12N 15152 (2013.01); C12P 7142 Soyombo A., et al., Molecular Cloning and Expression of (2013.01); C12P 7144 (2013.01); C12Y Palmitoyl-protein Thioesterase 2 (PPT2), a Homolog ofLysosomal 301/02014 (2013.01); Y02P 20/52 (2015.11) Palmitoyl-protein Thioesterase with a Distinct Substrate Specificity, (58) Field of Classification Search JBC 272(43): 27456-27463 (1997). CPC ...... C12N 9/0008; C12N 9/16; C12P 7/6409 (Continued) USPC .......................................................... 435/134 See application file for complete search history. (56) References Cited Primary Examiner - Tekchand Saidha (74) Attorney, Agent, or Firm - Boulware & Valoir U.S. PATENT DOCUMENTS 7,223,567 B2 5/2007 Ka-Yiu et al. (57) ABSTRACT 7,901,924 B2 3/2011 San et al. 2013/0052700 Al 2/2013 Poetter et al. Systems, methods and microbes that allow the biological 2014/0093921 Al 4/2014 San et al. 2014/0193867 Al 7/2014 San et al. production of hydroxy fatty acids and dicarboxylic fatty 2014/0212935 Al 7/2014 San et al. acids are provided. Specifically, hydroxy fatty acids and dicarboxylic fatty acids are produced by microbes that have FOREIGN PATENT DOCUMENTS been engineered to overexpress acyl ACP thioesterase plus WO W02011116279 9/2011 an alkane degration pathway, such as AlkBGT or AlkJH WO W02013024114 2/2013 These can be in separate microbes or the same microbe, and WO W02013096665 6/2013 separate microbes can be co-cultured or sequentially cul­ OTHER PUBLICATIONS tured. Continuously fed systems transferring secreted fats from one microbial culture to another can also be used. Davis, M.S.; Cronan, J.E., Inhibition of Escherichia coli acetyl coenzyme A carboxylase by acyl-acyl carrier protein, Jr. J Bacteriol 183, 1499 (2001). 16 Claims, 9 Drawing Sheets US 9,487,804 B2 Page 2 (56) References Cited of a plant medium-chain acyl-acyl carrier protein thioesterase, J. Bacteriol. 176:7320-7327 (1994). OTHER PUBLICATIONS Whyte, L.G. Gene Cloning and Characterization of Multiple Alkane Hydroxylase Systems in Rhodococcus Strains Ql5 and NRRL Throne-Holst, M., Identification of Novel Genes Involved in Long­ B-16531, Appl. Environ. Microbiol.68(12): 5933-5942 (2002). Chain n-Alkane Degradation by Acinetobacter sp. Strain DSM Yakimov M.M., et al., Oleispira antarctica gen. nov., sp. nov., a 17874, Appl Environ Microbiol. 73(10): 3327-3332 (2007). van Beilen J.B., DNA sequence determination and functional char­ novel hydrocarbonoclastic marine bacterium isolated from Antarc­ acterization of the OCT-plasmid-encoded aikJKL genes of tic coastal sea water. Int J Syst Evol Microbiol 53:779-785 (2003). Pseudomonas oleovorans, Mo! Microbiol. 6(21):3121-36 (1992). Yakimov M.M., et al., Thalassolituus oleivorans gen. nov., sp nov., Voelker T.A. & Davies H.M., Alteration of the specificity and a novel marine bacterium that obligately utilizes hydro-carbons. Int regulation of fatty acid synthesis of Escherichia coli by expression J Syst Evol Microbiol 54:141-148 (2004). e• 00 • Glucose ~ ~ ?roxyacyl~ ~ ~ ! =~ PEP 3-Ketoacyl-ACP Enoyl-ACP l z Pyruvate 0 ~ .) ~CIO N l Acyl-ACP 0.... Acetyl-CoA--.Malonyl-CoA--. Malonyl-ACP O'I Thio-;t;-a;;I -t rJJ ('D=­ Fatty acid I ('D..... - I .... 0 Oxaloacetate Citrate AlkB .... l,O AlkG I AlkT I ( l ' AlkJ Succinyl-CoA a-ketoglutarate IDicarboxylic AlkH Hydroxy I fatty acid I d ~ ~_:~-~-- r.,;_ \C ~ 00 FIG. 1: Diagram showing the genetically engineered biosynthetic pathway of hydroxy fatty acid and dicarboxylic fatty acid. The -....l 00 bold arrows in the dotted box represent the newly introduced pathway of fatty acid derivatives. =~ =N e• ~ 7J). • j>~<\iY.lgl,> -~·o(:' ~ B '"\' \ , :x.1;,,1 ~ p> t····i ~ ::~-> ~ !Ufo,lffi pBiffl::\13 (AT. J CX! =~ ,,(>Kb ·~ \ ::l..'oal llfo,filI \. .fl·:-.:dg1:t1 z \., !>htll.! rrnBilTl ,/ 0 ,.,/· \c:JII "--- ~ f,>·tB:C ~CIO N 0.... O'I pT« I y,AlkBGI rJ'1 r ('D =­ ,i_ ni.-;1, ('D '*:B!tw:~i• ..... N 0.... .S3d Xbol l,O \ // .\ .,. ...... A!idJ, AlkG; AlkT FIG. 2: Schematic diagram showing the conshuction of pBDTUM3 and pAlkBGT. Abbreviations: jatB2 gene from Cuphea hookeriana; AlkBGT genes from Pseudomonas putida Pl; pTum3, mutational trc promoter; rrnB TIT2, rrnB terminator; pl5A d r.,;_ origin, origin of replication of plasmid pl5A; CAT, chloromycctin resistance gene; pTrc, trc promoter; lacl: lac operon rcprcssor; \C ~ 00 Amp, arnpicillin resistant gene; pBR322 origin, origin of replication of plasmid pBR322; restriction enzyme sites: Xbal, HindIII, -....l 00= SacI. ~ =N e• 00 • ~ ~ ~ ~ lnd =~ z 0 ~ pA.lkJH ~CIO N 0.... 7.3Kb O'I pBR322 origin rJJ ('D=­ ('D..... ~ 0.... l,O FIG. 3. Schematic diagram of the pAlkJH. Abbreviations: AlkH.J genes from Pseudomonas putida Pl; pTrc, trc promoter; lacl: lac d operon repressor; Amp, ampicillin resistant gene; pBR322 origin, origin of replication of plasmid pBR322; rrnBTl,2, r.,;_ \C transcriptional terminator of rrnB. ~ 00 -...,l 00 =~ =N e• 00 • . cs.. on ~ ~ ~ ··•·· .. , ........·.·(····· •. •.. •.• ~ =~ C7 ·. >IT.····· z t······ L 0 ri ~ ~CIO _ ... A,.···········~~········~~~ ·~···· N 0.... A O'I rJJ ('D=­ ('D..... .i;... 0.... l,O B d rJl. \C FIG. 4. Profile ofheptanoic acid (C7) and hydroxyoctanoic acid (C8-0H) standards using GC-MS. A) GC chromatogram of ~ 00 -....l hydroxyoctanoic acid (C8-0H); B) MS 1 of hydroxyoctanoic acid 00 =~ =N U.S. Patent Nov. 8, 2016 Sheet 5 of 9 US 9,487,804 B2 A cs;.oH B C FIG. 5. Identification of octanoic acid (C8) and hydroxyoctanoic acid (C8-0H) using GC­ MS from samples. Heptanoic acid (C7) was used as internal standard. A) GC chromatogram of sample K272 (pALK + pBDTUM3) with no IPTG; B) GC chromatogram of sample K272 (pALK + pBDTUM3) with 100 mM IPTG; C) MS1 ofhydroxyoctanoic acid from sample K272 (pALK + pBDTUM3) with 100 mM IPTG. U.S. Patent Nov. 8, 2016 Sheet 6 of 9 US 9,487,804 B2 A B ~.uun,.. ~ ...~ten. ······························································································································································1 2,;nmKj 111 .,y,;;;, \T: ::;:.1;: I j ' T. I I 1 C :~{: iUO 1 bt~ ' z(K~ , 2-5n ~ _::;{fU m!;~ FIG. 6 Profile of commercial suberic acid (C8) using LC-MS. A) Extracted ion chromatogram (EIC) of suberic acid (m/z =173.0); B) MSl of suberic acid (m/z = 173.0); (C) MS/MS of suberic acid (m/z 173.0 -> m/z 111.0). U.S. Patent Nov. 8, 2016 Sheet 7 of 9 US 9,487,804 B2 A •).,:, fn:im~ ::::::::,z:,:_J-., '', iii,,,"" i,,, ;:::.~ :p!f;, 1 .-. ~ p::,::w i • \' ••• ,;, t p;::>::~ ~ il , 1 • i , t I • B be i OU ·1 t,O :.?U:J 2 E.,n 3('«) ((V2 l,,,.,,r, -; -----------------------------------------------------------------------------------------------------------------------------------------------------------------, 6Wm}J,7'i()()()j ·1 ·, • \,',,,,,,(·-{~ • '· ..: 2,:·.-,:! .. ..; ..... ~J ::i c~:+:-~-~~~--~~~~~~~~~-~~~--~~~~~___,, C :::n iCO 1:)(: ~·O{t 2C){} 3(W m/Z FTG.
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