title: Production of hydroxycinnamoyl-shikimates and chlorogenic acid in Escherichia coli: production of hydroxycinnamic acid conjugates.
abstract: BACKGROUND
  Hydroxycinnamates (HCs) are mainly produced in plants. Caffeic acid (CA), p-coumaric acid (PA), ferulic acid (FA) and sinapic acid (SA) are members of the HC family. The consumption of HC by human might prevent cardiovascular disease and some types of cancer. The solubility of HCs is increased through thioester conjugation to various compounds such as quinic acid, shikimic acid, malic acid, anthranilic acid, and glycerol. Although hydroxycinnamate conjugates can be obtained from diverse plant sources such as coffee, tomato, potato, apple, and sweet potato, some parts of the world have limited availability to these compounds. Thus, there is growing interest in producing HC conjugates as nutraceutical supplements.
  
  
  RESULTS
  Hydroxycinnamoyl transferases (HCTs) including hydroxycinnamate-CoA shikimate transferase (HST) and hydroxycinnamate-CoA quinate transferase (HQT) were co-expressed with 4-coumarateCoA:ligase (4CL) in Escherichia coli cultured in media supplemented with HCs. Two hydroxycinnamoyl conjugates, p-coumaroyl shikimates and chlorogenic acid, were thereby synthesized. Total 29.1 mg/L of four different p-coumaroyl shikimates (3-p-coumaroyl shikimate, 4-p-coumaroyl shikimate, 3,4-di-p-coumaroyl shikimate, 3,5-di-p-coumaroyl shikimate, and 4,5-di-p-coumaroyl shikimate) was obtained and 16 mg/L of chlorogenic acid was synthesized in the wild type E. coli strain. To increase the concentration of endogenous acceptor substrates such as shikimate and quinate, the shikimate pathway in E. coli was engineered. A E. coli aroL and aroK gene were mutated and the resulting mutants were used for the production of p-coumaroyl shikimate. An E. coli aroD mutant was used for the production of chlorogenic acid. We also optimized the vector and cell concentration optimization.
  
  
  CONCLUSIONS
  To produce p-coumaroyl-shikimates and chlorogenic acid in E. coli, several E. coli mutants (an aroD mutant for chlorogenic acid production; an aroL, aroK, and aroKL mutant for p-coumaroyl-shikimates production) were made and each mutant was tested using an optimized construct. Using this strategy, we produced 235 mg/L of p-coumaroyl-shikimates and 450 mg/L of chlorogenic acid.
authors: Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea.
authors: Bong-Gyu
authors: BG
authors: Kim
authors: Woo Dam
authors: WD
authors: Jung
authors: Hyejung
authors: H
authors: Mok
authors: Joong-Hoon
authors: JH
authors: Ahn
mesh_terms: D002726:Chlorogenic Acid / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism; D003373:Coumaric Acids / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D011801:Quinic Acid / Q000378:metabolism; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D014166:Transferases / Q000235:genetics / Q000378:metabolism
chemical_list: D003373:Coumaric Acids; D029968:Escherichia coli Proteins; D011801:Quinic Acid; D012765:Shikimic Acid; D002726:Chlorogenic Acid; D014166:Transferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022642:shikimate kinase; D003066:Coenzyme A Ligases
references: Appl Microbiol Biotechnol. 2013 Apr;97(8):3333-41
references: 23179615
references: Redox Rep. 1997 Oct-Dec;3(5-6):319-23
references: 9754331
references: Appl Microbiol Biotechnol. 2009 Jul;83(5):799-808
references: 19475406
references: J Biol Chem. 2003 Jan 3;278(1):95-103
references: 12381722
references: Metab Eng. 2010 Mar;12(2):96-104
references: 19628048
references: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5
references: 10829079
references: Nat Prod Rep. 2009 Aug;26(8):1001-43
references: 19636448
references: BMC Plant Biol. 2007 Mar 20;7:14
references: 17374149
references: Appl Microbiol Biotechnol. 2006 Feb;69(6):615-26
references: 16374633
references: Metab Eng. 2003 Oct;5(4):277-83
references: 14642355
references: J Biol Chem. 2003 May 23;278(21):19463-72
references: 12637497
references: Microb Cell Fact. 2010 Apr 12;9:21
references: 20385022
references: Crit Rev Food Sci Nutr. 1989;28(4):315-47
references: 2690858
references: Plant Cell. 2004 Jun;16(6):1446-65
references: 15161961
references: Plant Cell. 1995 Jul;7(7):1085-1097
references: 12242399
references: Microbiol Mol Biol Rev. 2001 Dec;65(4):523-69, table of contents
references: 11729263
references: J Antibiot (Tokyo). 2008 Dec;61(12):709-28
references: 19194030
references: Appl Environ Microbiol. 2012 Jan;78(1):89-98
references: 22020510
references: Proc Natl Acad Sci U S A. 2007 Jul 10;104(28):11856-61
references: 17609384
references: J Biol Chem. 2005 Feb 25;280(8):7162-9
references: 15596430
references: J Microbiol Biotechnol. 2011 Aug;21(8):854-7
references: 21876376
references: Curr Opin Biotechnol. 2008 Dec;19(6):597-605
references: 18992815
references: J Bacteriol. 2006 Apr;188(7):2666-73
references: 16547054
references: Nat Biotechnol. 2004 Jun;22(6):746-54
references: 15107863
references: J Agric Food Chem. 2012 Nov 7;60(44):10877-95
references: 22931195
references: J Agric Food Chem. 2010 Aug 11;58(15):8722-37
references: 20681662
references: Appl Environ Microbiol. 2012 Jun;78(12):4256-62
references: 22492444
references: Anal Biochem. 2002 Mar 15;302(2):305-12
references: 11878812
references: Curr Opin Biotechnol. 2009 Dec;20(6):651-8
references: 19875279