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@PHDTHESIS{Wohlers:906180,
      author       = {Wohlers, Karen},
      title        = {{S}train development of $\textit{{G}luconobacter oxydans}$
                      and $\textit{{P}seudomonas putida}$ for production of the
                      sweetener 5-ketofructose},
      volume       = {252},
      school       = {Heinrich-Heine-Univesität Düsseldorf},
      type         = {Dissertation},
      address      = {Jülich},
      publisher    = {Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag},
      reportid     = {FZJ-2022-01279},
      isbn         = {978-3-95806-612-0},
      series       = {Schriften des Forschungszentrums Jülich. Reihe
                      Schlüsseltechnologien / Key Technologies},
      pages        = {118 S.},
      year         = {2022},
      note         = {IBT-1; Heinrich-Heine-Universität Düsseldorf, Diss.,
                      2021},
      abstract     = {Consumption of added sugar is a health threat since it can
                      cause obesity and type 2 diabetes. Consequently, there is an
                      increasing demand for sugar substitutes. Available
                      sweeteners, however, have different drawbacks resulting in a
                      need for alternative sugar substitutes. The natural
                      metabolite 5-ketofructose (5-KF) is a promising sweetener
                      candidate. It is not metabolized by the human body and
                      probably not metabolized by the human gut microbiome while
                      having a comparable sweet taste as fructose. 5-KF can be
                      produced from fructose via oxidation by the membrane-bound
                      fructose dehydrogenase (Fdh) of $\textit{Gluconbacter
                      japonicus}$, encoded by the $\textit{fdhSCL}$ genes. Recent
                      studies showed the production of the sweetener with
                      heterologous strains of the industrially relevant acetic
                      acid bacterium $\textit{Gluconobacter oxydans}$. As
                      $\textit{G. oxydans}$ possesses no Fdh, plasmid-based
                      $\textit{fdhSCL}$ expression was applied in previous
                      studies. For production of a food additive, however,
                      antibiotic-free production is desirable. Aiming at plasmid-
                      and antibiotic-free 5-KF production, in this study the
                      $\textit{fdhSCL}$ genes were integrated into the chromosome
                      of engineered $\textit{G. oxydans}$ IK003.1. Four different
                      genomic integration sites were selected, including three
                      intergenic regions and one gene replacement, to compare the
                      effects of the genomic environment. The four integration
                      strains were successfully constructed, and all allowed
                      functional expression of the $\textit{fdhSCL}$ genes with
                      minor differences in 5-KF production. However, the
                      efficiency and velocity of 5-KF production was lower
                      compared to plasmid-based $\textit{fdhSCL}$ expression. To
                      improve the plasmid-free production of the sweetener, the
                      two best integration sites were combined in a double
                      integration strain, $\textit{G. oxydans}$
                      IK003.1::$\textit{fdhSCL}^{2}$} containing two chromosomal
                      $\textit{fdhSCL}$ copies. This strain showed accelerated
                      5-KF production, approaching that of the strain with
                      plasmid-based $\textit{fdhSCL}$ expression. Methods for
                      genetic engineering and expression systems for $\textit{G.
                      oxydans}$ are still limited. $\textit{G. oxydans}$ needs
                      complex medium components for good growth and has a low
                      biomass yield. Hence, in the second part of this study, the
                      well-established organism $\textit{Pseudomonas putida}$ was
                      selected as alternative 5-KF production host. Tn7-based
                      chromosomal integration of the $\textit{fdhSCL}$ genes
                      enabled $\textit{P. putida}$ to produce 5-KF from fructose
                      in mineral salts medium. In a batch fermentation with 150
                      g/L fructose, a product concentration of 129 ± 5 g/L 5-KF
                      was reached. Overall, shake flask experiments, bioreactor
                      cultivations and whole-cell biotransformations demonstrated
                      a competitive ability of $\textit{P.
                      putida}$::$\textit{fdhSCL}$ to produce 5-KF when compared to
                      a $\textit{G. oxydans fdhSCL}$ integration strain. The
                      substrate spectrum of $\textit{P.
                      putida}$::$\textit{fdhSCL}$ was expanded by plasmid-based
                      expression of $\textit{inv1417}$, encoding a periplasmic
                      invertase of $\textit{G. japonicus}$. Inv1417 enabled 5-KF
                      production from sucrose as cheaper substrate at rates
                      comparable to productionfrom fructose.},
      cin          = {IBG-1},
      cid          = {I:(DE-Juel1)IBG-1-20101118},
      pnm          = {2172 - Utilization of renewable carbon and energy sources
                      and engineering of ecosystem functions (POF4-217)},
      pid          = {G:(DE-HGF)POF4-2172},
      typ          = {PUB:(DE-HGF)3 / PUB:(DE-HGF)11},
      urn          = {urn:nbn:de:0001-2022040647},
      url          = {https://juser.fz-juelich.de/record/906180},
}