title: CRISPRi-mediated metabolic engineering of E. coli for O-methylated anthocyanin production.
abstract: BACKGROUND
  Anthocyanins are a class of brightly colored, glycosylated flavonoid pigments that imbue their flower and fruit host tissues with hues of predominantly red, orange, purple, and blue. Although all anthocyanins exhibit pH-responsive photochemical changes, distinct structural decorations on the core anthocyanin skeleton also cause dramatic color shifts, in addition to improved stabilities and unique pharmacological properties. In this work, we report for the first time the extension of the reconstituted plant anthocyanin pathway from (+)-catechin to O-methylated anthocyanins in a microbial production system, an effort which requires simultaneous co-option of the endogenous metabolites UDP-glucose and S-adenosyl-L-methionine (SAM or AdoMet).
  
  
  RESULTS
  Anthocyanin O-methyltransferase (AOMT) orthologs from various plant sources were co-expressed in Escherichia coli with Petunia hybrida anthocyanidin synthase (PhANS) and Arabidopsis thaliana anthocyanidin 3-O-glucosyltransferase (At3GT). Vitis vinifera AOMT (VvAOMT1) and fragrant cyclamen 'Kaori-no-mai' AOMT (CkmOMT2) were found to be the most effective AOMTs for production of the 3'-O-methylated product peonidin 3-O-glucoside (P3G), attaining the highest titers at 2.4 and 2.7 mg/L, respectively. Following modulation of plasmid copy number and optimization of VvAOMT1 and CkmOMT2 expression conditions, production was further improved to 23 mg/L using VvAOMT1. Finally, CRISPRi was utilized to silence the transcriptional repressor MetJ in order to deregulate the methionine biosynthetic pathway and improve SAM availability for O-methylation of cyanidin 3-O-glucoside (C3G), the biosynthetic precursor to P3G. MetJ repression led to a final titer of 51 mg/L (56 mg/L upon scale-up to shake flask), representing a twofold improvement over the non-targeting CRISPRi control strain and 21-fold improvement overall.
  
  
  CONCLUSIONS
  An E. coli strain was engineered for production of the specialty anthocyanin P3G using the abundant and comparatively inexpensive flavonol precursor, (+)-catechin. Furthermore, dCas9-mediated transcriptional repression of metJ alleviated a limiting SAM pool size, enhancing titers of the methylated anthocyanin product. While microbial production of P3G and other O-methylated anthocyanin pigments will likely be valuable to the food industry as natural food and beverage colorants, we expect that the strain constructed here will also prove useful to the ornamental plant industry as a platform for evaluating putative anthocyanin O-methyltransferases in pursuit of bespoke flower pigment compositions.
authors.affiliation: Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Biotech 4005D, 110 8th Street, Troy, NY, 12180, USA.
authors.forename: Brady F
authors.identifier: http://orcid.org/0000-0002-2948-2846
authors.initials: BF
authors.lastname: Cress
authors.affiliation: Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Biotech 4005D, 110 8th Street, Troy, NY, 12180, USA.
authors.forename: Quentin D
authors.identifier: http://orcid.org/0000-0001-7280-0777
authors.initials: QD
authors.lastname: Leitz
authors.affiliation: Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Biotech 4005D, 110 8th Street, Troy, NY, 12180, USA.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Kim
authors.affiliation: Department of Tropical Plant and Soil Sciences, University of Hawaii, 3190 Maile Way, Honolulu, HI, 96822, USA.
authors.forename: Teresita D
authors.initials: TD
authors.lastname: Amore
authors.affiliation: U.S. Department of Agriculture, Agricultural Research Service, Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center, Hilo, HI, 96720, USA.
authors.forename: Jon Y
authors.initials: JY
authors.lastname: Suzuki
authors.affiliation: Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Biotech 4005D, 110 8th Street, Troy, NY, 12180, USA.|Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.|Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
authors.affiliation: Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Biotech 4005D, 110 8th Street, Troy, NY, 12180, USA. koffam@rpi.edu.|Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA. koffam@rpi.edu.
authors.forename: Mattheos A G
authors.initials: MA
authors.lastname: Koffas
mesh_terms: D000872:Anthocyanins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D053898:Biosynthetic Pathways; D064113:CRISPR-Cas Systems; D002392:Catechin / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D005960:Glucosides / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D060847:Metabolic Engineering / Q000379:methods*; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D032306:Petunia / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D012436:S-Adenosylmethionine / Q000378:metabolism
chemical_list: D000872:Anthocyanins; D005960:Glucosides; D010940:Plant Proteins; C480521:peonidin 3-glucoside; D012436:S-Adenosylmethionine; D002392:Catechin; D008780:Methyltransferases; D005947:Glucose
keywords: AdoMet; Anthocyanin O-methyltransferase; CRISPRi; Deregulation; MetJ; Peonidin 3-O-glucoside; S-Adenosyl methionine; SAM; Transcriptional repression; dCas9