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@PHDTHESIS{Kohl:954643,
      author       = {Kohl, Anna Christiane},
      othercontributors = {Commandeur, Ulrich Heinrich and Blank, Lars M.},
      title        = {{S}ynthetic phenylpropanoid pathway for the in vivo
                      production of coniferyl alcohol in {E}scherichia coli},
      school       = {RWTH Aachen University},
      type         = {Dissertation},
      address      = {Aachen},
      publisher    = {RWTH Aachen University},
      reportid     = {RWTH-2023-03313},
      pages        = {1 Online-Ressource : Illustrationen, Diagramme},
      year         = {2023},
      note         = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
                      University; Dissertation, RWTH Aachen University, 2023},
      abstract     = {Lignans constitute a valuable substance group exhibiting
                      many health-promoting effects on the human body as potential
                      pharmaceuticals and nutraceuticals. One of the most popular
                      lignans is (−) podophyllotoxin, which is applied in its
                      semi-synthetic derivatives for the treatment of various
                      types of cancer. The industrial demand for alternative
                      production routes remains high, as the main extraction
                      method still depends on endangered natural plant resources.
                      Thus, the controlled microbial fermentation could provide
                      the important lignans in sufficient and scalable amounts in
                      the future. As one of the preconditions, the precursor for
                      lignan synthesis coniferyl alcohol needs to be produced in
                      industrial bulk. For a foremost cost-efficient and
                      economically sustainable green synthesis of coniferyl
                      alcohol, the fermentation from inexpensive resources such as
                      glucose carried out in well-examined production strains like
                      Escherichia coli (E. coli) can be advantageous also with
                      respect to a possible combinable synthesis of lignans. The
                      objective of this dissertation was to establish the
                      microbial synthesis of coniferyl alcohol in E. coli from
                      L-tyrosine or L-phenylalanine according to the plant
                      phenylpropanoid pathway. In the context of metabolic
                      engineering, different enzyme candidates for crucial central
                      reaction steps concerning deamination, C3- and
                      C4-hydroxylation and methylation were first evaluated. By
                      division of the phenylpropanoid pathway into upstream and
                      downstream pathway, the best enzyme combinations were then
                      investigated for both modules. Furthermore, the complete and
                      partly plasmid-based expression of the phenylpropanoid
                      pathway was realized in E. coli BL21(DE3) or its
                      genome-integrated derivatives containing variable gene
                      copies and promoters. At last, two different cell assays
                      were applied and compared.This thesis describes the first
                      successful application of the TALs from Flavobacterium
                      johnsoniae (FjTAL) and Saccharothrix espanaensis (Sesam8),
                      the cytochrome P450 enzyme from Rhodopseudomonas palustris
                      (CYP199A2) as C3H and the two methyltransferases from Zea
                      mays for the production of coniferyl alcohol from L-tyrosine
                      in E. coli. Noteworthily, caffeic acid was obtained from
                      L-phenylalanine by using the bifunctionality of the CYP199A2
                      F185L mutant as C3- and C4-hydroxylase (C3H/C4H) and the TAL
                      from Rhodotorula glutinis (RgTAL) for the first time. In the
                      end, the highest amount of coniferyl alcohol (~ 850 µM) was
                      achieved in growing E. coli cells of a genome-integrated
                      strain. This coniferyl alcohol titer, produced under
                      non-optimized conditions and in a prototype strain, is
                      comparable to a value in one literature report, in which an
                      L-tyrosine overproduction strain and adjusted cultivation
                      conditions were used. Thus, the results from this
                      dissertation lay a substantial foundation for the further
                      development of a microbial coniferyl alcohol production
                      platform.},
      cin          = {162910 / 160000},
      ddc          = {570},
      cid          = {$I:(DE-82)162910_20140620$ / $I:(DE-82)160000_20140620$},
      typ          = {PUB:(DE-HGF)11},
      doi          = {10.18154/RWTH-2023-03313},
      url          = {https://publications.rwth-aachen.de/record/954643},
}