title: Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system.
abstract: BACKGROUND
  Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroGfbr, tktA, aroB and aroE, on SA synthesis.
  
  
  RESULTS
  Batch cultures were performed to evaluate the effects of genetic modifications on growth, glucose consumption, and aromatic intermediate production. All derivatives showed a two-phase growth behavior with initial high specific growth rate (mu) and specific glucose consumption rate (qs), but low level production of aromatic intermediates. During the second growth phase the mu decreased, whereas aromatic intermediate production reached its maximum. The double aroK- aroL- mutant expressing plasmid-coded genes (strain PB12.SA22) accumulated SA up to 7 g/L with a yield of SA on glucose of 0.29 mol/mol and a total aromatic compound yield (TACY) of 0.38 mol/mol. Single inactivation of pykF or pykA was performed in PB12.SA22 strain. Inactivation of pykF caused a decrease in mu, qs, SA production, and yield; whereas TACY increased by 33% (0.5 mol/mol).
  
  
  CONCLUSIONS
  The effect of increased availability of carbon metabolites, their channeling into the synthesis of aromatic intermediates, and disruption of the SA pathway on SA production was studied. Inactivation of both aroK and aroL, and transformation with plasmid-coded genes resulted in the accumulation of SA up to 7 g/L with a yield on glucose of 0.29 mol/mol PB12.SA22, which represents the highest reported yield. The pykF and pykA genes were inactivated in strain PB12.SA22 to increase the production of aromatic compounds in the PTS- background. Results indicate differential roles of Pyk isoenzymes on growth and aromatic compound production. This study demonstrated for the first time the simultaneous inactivation of PTS and pykF as part of a strategy to improve SA production and its aromatic precursors in E. coli, with a resulting high yield of aromatic compounds on glucose of 0.5 mol/mol.
authors.affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Av, Universidad 2001, Col, Chamilpa, Cuernavaca, Morelos, 62210, México. adelfo@ibt.unam.mx
authors.forename: Adelfo
authors.initials: A
authors.lastname: Escalante
authors.forename: Rocío
authors.initials: R
authors.lastname: Calderón
authors.forename: Araceli
authors.initials: A
authors.lastname: Valdivia
authors.forename: Ramón
authors.initials: R
authors.lastname: de Anda
authors.forename: Georgina
authors.initials: G
authors.lastname: Hernández
authors.forename: Octavio T
authors.initials: OT
authors.lastname: Ramírez
authors.forename: Guillermo
authors.initials: G
authors.lastname: Gosset
authors.forename: Francisco
authors.initials: F
authors.lastname: Bolívar
mesh_terms: D001709:Biotechnology / Q000379:methods*; D004345:Drug Industry / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D016296:Mutagenesis; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000172:deficiency*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D012765:Shikimic Acid / Q000138:chemical synthesis / Q000378:metabolism*
chemical_list: D012765:Shikimic Acid; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D017853:Phosphotransferases (Alcohol Group Acceptor); C022642:shikimate kinase; D005947:Glucose
references.citation: J Ind Microbiol. 1996 Jul;17(1):47-52
references.pmid: 8987689
references.citation: Metab Eng. 2002 Apr;4(2):124-37
references.pmid: 12009792
references.citation: Microb Cell Fact. 2007 Sep 19;6:30
references.pmid: 17880710
references.citation: J Biotechnol. 2006 Dec 1;126(4):528-45
references.pmid: 16828913
references.citation: Nature. 2006 Sep 7;443(7107):45-9
references.pmid: 16915235
references.citation: Appl Environ Microbiol. 1994 Nov;60(11):3903-8
references.pmid: 7993080
references.citation: J Bacteriol. 1995 Oct;177(19):5719-22
references.pmid: 7559366
references.citation: Microb Cell Fact. 2008 Jan 22;7:1
references.pmid: 18211716
references.citation: Gene. 1977;2(2):95-113
references.pmid: 344137
references.citation: Biotechnol Bioeng. 2005 Dec 5;92(5):541-52
references.pmid: 16240440
references.citation: J Bacteriol. 2009 May;191(9):3041-9
references.pmid: 19251844
references.citation: Biotechnol Bioeng. 1999 Jul 5;64(1):61-73
references.pmid: 10397840
references.citation: Biotechnol Bioeng. 2001 Jun 20;73(6):530-5
references.pmid: 11344458
references.citation: Biotechnol Bioeng. 1997 Jan 20;53(2):132-8
references.pmid: 18633957
references.citation: PLoS One. 2009 Oct 19;4(10):e7466
references.pmid: 19838295
references.citation: Biotechnol Prog. 2003 May-Jun;19(3):808-14
references.pmid: 12790643
references.citation: Comp Biochem Physiol B Biochem Mol Biol. 2003 Jun;135(2):197-218
references.pmid: 12798932
references.citation: Mol Syst Biol. 2006;2:2006.0008
references.pmid: 16738554
references.citation: Biosci Biotechnol Biochem. 2006 Oct;70(10):2579-82
references.pmid: 17031026
references.citation: Metab Eng. 2005 Mar;7(2):70-87
references.pmid: 15781417
references.citation: Biotechnol Prog. 2003 Sep-Oct;19(5):1450-9
references.pmid: 14524706
references.citation: J Biol Chem. 1959 Apr;234(4):705-9
references.pmid: 13654246
references.citation: Biotechnol Bioeng. 2003 Sep 20;83(6):687-94
references.pmid: 12889033
references.citation: J Mol Microbiol Biotechnol. 2005;10(1):51-63
references.pmid: 16491026
references.citation: Nature. 2005 May 26;435(7041):407-9
references.pmid: 15917770
references.citation: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5
references.pmid: 10829079
references.citation: Antimicrob Agents Chemother. 2008 Sep;52(9):3284-92
references.pmid: 18625765
references.citation: JAMA. 2009 Mar 11;301(10):1034-41
references.pmid: 19255110
references.citation: Structure. 1998 Feb 15;6(2):195-210
references.pmid: 9519410
references.citation: Eukaryot Cell. 2006 Sep;5(9):1517-31
references.pmid: 16963634
references.citation: Nat Biotechnol. 1996 May;14(5):620-3
references.pmid: 9630954
references.citation: Biotechnol Bioeng. 2004 Aug 20;87(4):516-24
references.pmid: 15286989
references.citation: J Mol Microbiol Biotechnol. 2004;8(4):209-21
references.pmid: 16179798
references.citation: Metab Eng. 2003 Oct;5(4):277-83
references.pmid: 14642355
references.citation: Annu Rev Plant Physiol Plant Mol Biol. 1999 Jun;50:473-503
references.pmid: 15012217
references.citation: Nature. 2006 Sep 7;443(7107):37-8
references.pmid: 16915238
references.citation: J Mol Microbiol Biotechnol. 2007;13(1-3):105-16
references.pmid: 17693718
references.citation: Microb Cell Fact. 2009 Apr 02;8:19
references.pmid: 19341482
references.citation: Biotechnol Prog. 2000 Mar-Apr;16(2):278-86
references.pmid: 10753455
references.citation: Biotechnol Bioeng. 2006 Aug 20;94(6):1164-75
references.pmid: 16718678
references.citation: Metab Eng. 2006 May;8(3):281-90
references.pmid: 16517196
references.citation: Appl Environ Microbiol. 2008 May;74(10):3284-90
references.pmid: 18344329
references.citation: Biotechnol Bioeng. 1995 May 20;46(4):361-70
references.pmid: 18623323
references.citation: J Am Chem Soc. 2001 Oct 24;123(42):10173-82
references.pmid: 11603966