Book/Dissertation / PhD Thesis

FZJ-2022-01279

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Strain development of $\textit{Gluconobacter oxydans}$ and $\textit{Pseudomonas putida}$ for production of the sweetener 5-ketofructose

Wohlers, K. (Corresponding author)FZJ*

2022
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich
ISBN: 978-3-95806-612-0

Please use a persistent id in citations: http://hdl.handle.net/2128/30972  urn:nbn:de:0001-2022040647

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.

Note: IBT-1
Note: Heinrich-Heine-Universität Düsseldorf, Diss., 2021

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Contributing Institute(s):

1. Biotechnologie (IBG-1)

Research Program(s):

1. 2172 - Utilization of renewable carbon and energy sources and engineering of ecosystem functions (POF4-217) (POF4-217)

Appears in the scientific report 2022

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Database coverage:
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