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C12) United States Patent (IO) Patent No.: US 9,441,253 B2 San Et Al

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C12) United States Patent (IO) Patent No.: US 9,441,253 B2 San Et Al IIIIII IIIIIIII Ill lllll lllll lllll lllll lllll lllll lllll lllll 111111111111111111 US009441253B2 c12) United States Patent (IO) Patent No.: US 9,441,253 B2 San et al. (45) Date of Patent: Sep.13,2016 (54) METABOLIC TRANSISTOR IN BACTERIA 114/13027 (2013.01); C12Y 205/01001 (2013.01); C12Y 205/01032 (2013.01); C12Y (71) Applicant: William Marsh Rice University, 205/01093 (2013.01); C12Y 305/01022 Houston, TX (US) (2013.01); C12Y 305/99002 (2013.01); C12Y 401/01024 (2013.01); C12Y 602/01011 (72) Inventors: Ka-Yiu San, Houston, TX (US); (2013.01); Y02E 50/17 (2013.01); Y02P 20/52 George N. Bennett, Houston, TX (US); (2015.11) Hui Wu, Houston, TX (US) (58) Field of Classification Search CPC .................................. C12P 7/56; C12N 15/70 (73) Assignee: William Marsh Rice University, Houston, TX (US) USPC .................... 435/108, 111, 115, 116, 252.33 See application file for complete search history. ( *) Notice: Subject to any disclaimer, the term ofthis patent is extended or adjusted under 35 (56) References Cited U.S.C. 154(b) by 130 days. U.S. PATENT DOCUMENTS (21) Appl. No.: 14/176,008 2004/0152159 Al 8/2004 Causey (22) Filed: Feb. 7, 2014 OTHER PUBLICATIONS (65) Prior Publication Data Wu et al. Biotechnology and bioengineering, (Aug. 2015) vol. 112, US 2014/0227745 Al Aug. 14, 2014 No. 8, pp. 1720-1726.* Alper H., Miyaoku K., Stephanopoulos G., (2005) Construction of Related U.S. Application Data lycopene overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nat. Biotechnol. 23,612-616. (60) Provisional application No. 61/763,019, filed on Feb. Blombach B. et al., (2007) I-Valine Production with Pyruvate 11, 2013. Dehydrogenase Complex-Deficient Corynebacterium glutamicum, Appl. Environ. Microbiol. 73(7): 2079-2084. (51) Int. Cl. Borisov VB, Gennis RB, Hemp J, Verkhovsky MI (2011) The C12P 13/22 (2006.01) cytochrome bd respiratory oxygen reductases. Biochim Biophys C12P 7156 (2006.01) Acta 1807: 1398-1413. C12P 7154 (2006.01) Cunningham F.X. Jr., et al., (1994) Molecular structure and enzy­ C12P 13/18 (2006.01) matic function of lycopene cyclase from the cyanobacterium C12P 13/08 (2006.01) Synechococcus sp strain PCC7942. Plant Cell 6: 1107-1121. C12P 13/06 (2006.01) Farmer W.R., Liao J.C., (2000) Improving lycopene production in C12N 15152 (2006.01) Escherichia coli by engineering metabolic control. Nat. Biotechnol. C12N 9/02 (2006.01) 18, 533-537. Hassan BH, Cronan JE (2011) Protein-protein interactions in assem­ C12N 9/10 (2006.01) bly of lipoic acid on the 2-oxoacid dehydrogenases of aerobic C12N 9/80 (2006.01) metabolism. J. of Biol. Chem. 286: 8263-8276. C12N 9/88 (2006.01) Hermes FA, Cronan JE (2009) Scavenging of cytosolic octanoic C12N 9/00 (2006.01) acid by mutant LpIA lipoate ligases allows growth of Escherichia C12P 1/04 (2006.01) coli strains lacking the LipB octanoyltransferase of lipoic acid C12P 7106 (2006.01) synthesis. Journal of bacteriology 191: 6796-6803. C12P 7118 (2006.01) Iram SH, Cronan JE (2006) The beta-oxidation systems of C12P 7140 (2006.01) Escherichia coliand Salmonella enterica are not functionally C12P 7146 (2006.01) equivalent. Journal of bacteriology 188: 599-608. C12P 13/04 (2006.01) Kameda K, Imai Y (1985) Isolation and characterization of the C12P 13/14 (2006.01) multiple charge isoforms of acyl-CoA synthetase from Escherichia coli. Biochim Biophys Acta 832: 343-350. (52) U.S. Cl. (Continued) CPC ............. C12P 13/225 (2013.01); C12N 9/001 (2013.01); C12N 9/0008 (2013.01); C12N 9/0073 (2013.01); C12N 9/1085 (2013.01); C12N 9/80 (2013.01); C12N 9/88 (2013.01); Primary Examiner - Tekchand Saidha C12N 9/93 (2013.01); C12N 15152 (2013.01); (74) Attorney, Agent, or Firm - Boulware & Valoir C12P 1/04 (2013.01); C12P 71065 (2013.01); C12P 7118 (2013.01); C12P 7140 (2013.01); (57) ABSTRACT C12P 7146 (2013.01); C12P 7154 (2013.01); C12P 7156 (2013.01); C12P 13/04 (2013.01); The disclosure relates to a metabolic transistor in bacteria C12P 13/06 (2013.01); C12P 13/08 (2013.01); where a competitive pathway is introduced to compete with C12P 13/14 (2013.01); C12P 13/18 (2013.01); a product pathway for available carbon so as to control the C12P 13/222 (2013.01); C12Y 102/01025 carbon flux in the bacteria. (2013.01); C12Y 102/04004 (2013.01); C12Y 102/07007 (2013.01); C12Y 103/01076 (2013.01); C12Y 103/05005 (2013.01); C12Y 7 Claims, 7 Drawing Sheets US 9,441,253 B2 Page 2 (56) References Cited Stolz M, et al., (2007) Reduced folate supply as a key to enhanced L-serine production by Corynebacterium glutamicum, Appl. OTHER PUBLICATIONS Environ. Microbiol. 73(3):750-5. Taboada, H., et al. (2008) Thiamine limitation determines the transition from aerobic to fermentative-like metabolism in Londer YY (2011) Expression of recombinant cytoclnomes c in E. Rhizobium etli CE3, FEMS Microbiology Letters 279(1): 48-55. coli. Methods Mo! Biol 705: 123-150. Thony-Meyer L (1997) Biogenesis of respiratory cytochromes in Lu P., (2007) Absolute protein expression profiling estimates the bacteria. Microbiol. Mo!. Biol. Rev. 61: 337-376. relative contributions of transcriptional and translational regulation. Tsai C.H., et al., (2000) Novel recombinant hemoglobin, rHb (beta Nature Biotechnol. 25: 117-124. Nl08Q), with low oxygen affinity, high cooperativity, and stability Morris T.W., (1995) Lipoic acid metabolism in Escherichia coli: the against autoxidation. Biochem. 39: 13719-13729. IpIA and lipB genes define redundant pathways for ligation of lipoyl Vadali R.V., et al., (2005) Enhanced lycopene productivity by groups to apoprotein. Journal of bacteriology 177: 1-10. manipulation of carbon flow to isopentenyl diphosphate in Escherichia coli, Biotechnol. Prog. 21, 1558-1561. Perham RN (1991) Domains, motifs, and linkers in 2-oxo acid Varnado C.L., et al., (2013) Development of recombinant hemo­ dehydrogenase multienzyme complexes: a paradigm in the design globin-based oxygen carriers, Antioxid. Redox Signal 18:2314- of a multifunctional protein. Biochem. 30:8501-8512. 2328. Peters-Wendisch P., (2012) Biotin protein ligase from Warren M.J., et al., (1994) Gene dissection demonstrates that the Corynebacterium glutamicum: role for growth and L:-lysine pro­ Escherichia coli cysG gene encodes a multifunctional protein, duction, Appl, Microbiol, Biotechnol. 93(6):2493-502. Biochem J 302(Pt 3): 837-844. Reed K.E., Cronan J.E., Jr. (1993) Lipoic acid metabolism in Warren M.J., et al., (1990) The Escherichia coli cysG gene encodes Escherichia coli: sequencing and functional characterization of the Sadenosylmethionine-dependent uroporphyrinogen III methylase, lipA and lipB genes. J. Bacteriol. 175:1325-1336. Biochem J. 265: 725-729. Schellhorn H.E, Hassan H.M. ( 1988) Isolation and characterization Yazaki K., et al., (2002) Geranyl diphosphate:4-hydroxybenzoate of respiratorydeficient mutants of Escherichia coli K-12. J. geranyltransferase from Lithospermum erytlnorhizon. Cloning and Bacteriol. 170: 78-83. characterization of a key enzyme in shikonin biosynthesis, J. Biol. Spencer JB, Stolowich NJ, Roessner CA, Scott AL (1993) The Chem. 277, 6240-6246. Escherichia coli cysG gene encodes the multifunctional protein, siroheme synthase. FEBS Lett. 335: 57-60. * cited by examiner U.S. Patent Sep.13,2016 Sheet 1 of 7 US 9,441,253 B2 ,.,,,. 4' Metabolic: I \ I ControHer ' I I I I I I I I I I I \ ,,, I ' ' ~ JODODI ODODO JDODOI OODOt IODODC tDODO XIODOt OODOD IODOOC NODt IOODOC MDOt lODODC FIG.1 ETC = electron transfer chain. U.S. Patent Sep.13,2016 Sheet 2 of 7 US 9,441,253 B2 glucose Competitor competes with Product for carbon / /.... / Cofactor Product Competitor FIG. 2A glucose Add Diverting l Geneswhlch use cofactor, thus reducing C J and increasing P Cofactor,, Product )~ Competitor FIG. 2B e• 00 • PPO.,..__..., ... ./-:::._,.._; .. /~ idi PP(),....___.,,/--......__"{""/ ~ CH3 i CH, ~ D'l'lAPP '--..,__ /__.___..--- '- IPP ~ D:H-\.PPx\ /,.- 1 ~ /n'Pxl 1 =~ ( ,~ \- PPO,,_~,-"'"'"Y:,-,,,_,,-_,,,o-,/ , ;.,pll ppo___ //'~~ 1-- _,,,. t_-- -- .... ,, ~~ r~ ~_!~ 1 t rJJ ('D c':1-[_. cu, UL, (H3 '? , GPP. I .... i.,py· '\ /~::-.._':"hI-l ~ j/ ~ ,/ ... - [f ] -----------,,,, N \ ,, ',.,:/ 0.... \ / ,'>H uhiA O'I 7 : \ hpgt' 4-HB cH3 CH, (-.'H-~ c'lI::: \ CO,H L " L, - J, , , L " \ < c£!1_.......- ''.:::,,.,. ... -....,,-" ·---~""....- ',.... ,,. ... ~,, -..._.,.......- ''.::: .... ,, ... l}PP /),"- \ rJJ (:;;<_;pp C02H Q -,+ ...... ;;:--]t--:,~/ ..... ~~~r'~J ~--1, 1 ('D=­ /l, OH CH3 ~ ('D.... mM ~ 1t,r:1f'-./_,.,.~,r<1' il J ub;x, ul,il> ~ CO.:•~/ l 0 OH t...... CH3 ~ CH{ .... G-4-HB -....J (~)t"/"~r/J; H Oll CH, Lyc:-opene 111 uhif:, uh;,:; Abbreviation: idi, IPP isornerase: ispA, geranyl/farnesyl diphosphate (FPP) synthase; ispB, octaprenyl diphosphate synthase; ubiA, 4-hydroxybenzoate octaprenyltransfcrnse; OH ubiD, decarboxylase catalyzing the decarboxylation of 4-hydroxy-3-ociaprenyl­ t'-.1d)_, ____ ,.. ....,-l, .,.,.. .. CJ-£~ d benzoate; uhiX, similar as ubiD, decarboxylase catalyzing the decarboxylation of 4- r.,;_ }.,[e{))L,tJi>./""~,(J~ n hydroxy-3-octaprcnyl-bcnzoate; ubiE, C-methyltransfcrase; ubiG, 0-methyltransfcrase; \C C>H CH_.., r, crrE, gcranylgeranyl diphosphatc synthase; crtB, phytoene synthase; crtl, phytoene ~ dcsaturase; lepgr, gcranyl diphosphatc:4-hydroxyben7oate 3-geranyltransfcrase. TPP, Ubiquinonc Q8 ~ 'N"""' UI w FIG. 3 =N U.S. Patent Sep.13,2016 Sheet 4 of 7 US 9,441,253 B2 pAC-LYC FIG.4 U.S. Patent Sep.13,2016 Sheet 5 of 7 US 9,441,253 B2 Ptrc pTrc-lePGT AmpR FIG. SA pBlePGT .. /Mind Ill CmR FIG. SB e• 00 • ~ ~ ~ Total QS in nmole/mg DCW ~ =~ 2.0 iii Total Coenzyme QB (Q8 + Total QB in nmole/mg DCW rJJ ('D Na adduct (Na)) 11:1 Total Coenzyme Q8 (Q8 + Na '? 10_00 adduct {Na)) .... 1.5 ~ s s ~ C 0 N <.) 1.00 0 u bO ...
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