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  1. Article ; Online: Engineering a tunable bicistronic TetR autoregulation expression system in Gluconobacter oxydans

    Monica Bertucci / Ky Ariano / Meg Zumsteg / Paul Schweiger

    PeerJ, Vol 10, p e

    2022  Volume 13639

    Abstract: Acetic acid bacteria are well-known for their ability to incompletely oxidize their carbon sources. Many of the products of these oxidations find industrial uses. Metabolic engineering of acetic acid bacteria would improve production efficiency and yield ...

    Abstract Acetic acid bacteria are well-known for their ability to incompletely oxidize their carbon sources. Many of the products of these oxidations find industrial uses. Metabolic engineering of acetic acid bacteria would improve production efficiency and yield by allowing controllable gene expression. However, the molecular tools necessary for regulating gene expression have only recently started being explored. To this end the ability of the activation-dependent Plux system and two constitutive repression Ptet systems were examined for their ability to modulate gene expression in Gluconobacter oxydans. The activation-dependent Plux system increased gene expression approximately 5-fold regardless of the strength of the constitutive promoter used to express the luxR transcriptional activator. The Ptet system was tunable and had a nearly 20-fold induction when the tetR gene was expressed from the strong constitutive promoters P0169 and P264, but only had a 4-fold induction when a weak constitutive promoter (P452) was used for tetR expression. However, the Ptet system was somewhat leaky when uninduced. To mitigate this background activity, a bicistronic TetR expression system was constructed. Based on molecular modeling, this system is predicted to have low background activity when not induced with anhydrotetracycline. The bicistronic system was inducible up to >3,000-fold and was highly tunable with almost no background expression when uninduced, making this bicistronic system potentially useful for engineering G. oxydans and possibly other acetic acid bacteria. These expression systems add to the newly growing repertoire of suitable regulatable promoter systems in acetic acid bacteria.
    Keywords Promoter ; Induction ; Expression ; Plasmid ; β-D-Glucuronidase UidA ; GFP ; Medicine ; R ; Biology (General) ; QH301-705.5
    Subject code 570
    Language English
    Publishing date 2022-07-01T00:00:00Z
    Publisher PeerJ Inc.
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Surface display for metabolic engineering of industrially important acetic acid bacteria

    Marshal Blank / Paul Schweiger

    PeerJ, Vol 6, p e

    2018  Volume 4626

    Abstract: Acetic acid bacteria have unique metabolic characteristics that suit them for a variety of biotechnological applications. They possess an arsenal of membrane-bound dehydrogenases in the periplasmic space that are capable of regiospecific and ... ...

    Abstract Acetic acid bacteria have unique metabolic characteristics that suit them for a variety of biotechnological applications. They possess an arsenal of membrane-bound dehydrogenases in the periplasmic space that are capable of regiospecific and enantioselective partial oxidations of sugars, alcohols, and polyols. The resulting products are deposited directly into the medium where they are easily recovered for use as pharmaceutical precursors, industrial chemicals, food additives, and consumer products. Expression of extracytoplasmic enzymes to augment the oxidative capabilities of acetic acid bacteria is desired but is challenging due to the already crowded inner membrane. To this end, an original surface display system was developed to express recombinant enzymes at the outer membrane of the model acetic acid bacterium Gluconobacter oxydans. Outer membrane porin F (OprF) was used to deliver alkaline phosphatase (PhoA) to the cell surface. Constitutive high-strength p264 and moderate-strength p452 promoters were used to direct expression of the surface display system. This system was demonstrated for biocatalysis in whole-cell assays with the p264 promoter having a twofold increase in PhoA activity compared to the p452 promoter. Proteolytic cleavage of PhoA from the cell surface confirmed proper delivery to the outer membrane. Furthermore, a linker library was constructed to optimize surface display. A rigid (EAAAK)1 linker led to the greatest improvement, increasing PhoA activity by 69%. This surface display system could be used both to extend the capabilities of acetic acid bacteria in current biotechnological processes, and to broaden the potential of these microbes in the production of value-added products.
    Keywords Surface display ; Fusion linkers ; Biocatalysis ; Outer membrane proteins ; Gluconobacter oxydans ; Medicine ; R ; Biology (General) ; QH301-705.5
    Subject code 570
    Language English
    Publishing date 2018-04-01T00:00:00Z
    Publisher PeerJ Inc.
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Horizontal gene transfer-mediated bacterial strain variation affects host fitness in Drosophila

    Yun Wang / Franz Baumdicker / Paul Schweiger / Sven Kuenzel / Fabian Staubach

    BMC Biology, Vol 19, Iss 1, Pp 1-

    2021  Volume 12

    Abstract: Abstract Background How microbes affect host fitness and environmental adaptation has become a fundamental research question in evolutionary biology. To better understand the role of microbial genomic variation for host fitness, we tested for ... ...

    Abstract Abstract Background How microbes affect host fitness and environmental adaptation has become a fundamental research question in evolutionary biology. To better understand the role of microbial genomic variation for host fitness, we tested for associations of bacterial genomic variation and Drosophila melanogaster offspring number in a microbial Genome Wide Association Study (GWAS). Results We performed a microbial GWAS, leveraging strain variation in the genus Gluconobacter, a genus of bacteria that are commonly associated with Drosophila under natural conditions. We pinpoint the thiamine biosynthesis pathway (TBP) as contributing to differences in fitness conferred to the fly host. While an effect of thiamine on fly development has been described, we show that strain variation in TBP between bacterial isolates from wild-caught D. melanogaster contributes to variation in offspring production by the host. By tracing the evolutionary history of TBP genes in Gluconobacter, we find that TBP genes were most likely lost and reacquired by horizontal gene transfer (HGT). Conclusion Our study emphasizes the importance of strain variation and highlights that HGT can add to microbiome flexibility and potentially to host adaptation.
    Keywords Drosophila ; Microbiome ; GWAS ; Horizontal gene transfer ; Lateral gene transfer ; Biology (General) ; QH301-705.5
    Subject code 572
    Language English
    Publishing date 2021-09-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article: Production of 5-ketofructose from fructose or sucrose using genetically modified Gluconobacter oxydans strains

    Siemen, Anna / Konrad Kosciow / Paul Schweiger / Uwe Deppenmeier

    Applied microbiology and biotechnology. 2018 Feb., v. 102, no. 4

    2018  

    Abstract: The growing consumer demand for low-calorie, sugar-free foodstuff motivated us to search for alternative non-nutritive sweeteners. A promising sweet-tasting compound is 5-keto-D-fructose (5-KF), which is formed by membrane-bound fructose dehydrogenases ( ... ...

    Abstract The growing consumer demand for low-calorie, sugar-free foodstuff motivated us to search for alternative non-nutritive sweeteners. A promising sweet-tasting compound is 5-keto-D-fructose (5-KF), which is formed by membrane-bound fructose dehydrogenases (Fdh) in some Gluconobacter strains. The plasmid-based expression of the fdh genes in Gluconobacter (G.) oxydans resulted in a much higher Fdh activity in comparison to the native host G. japonicus. Growth experiments with G. oxydans fdh in fructose-containing media indicated that 5-KF was rapidly formed with a conversion efficiency of 90%. 5-KF production from fructose was also observed using resting cells with a yield of about 100%. In addition, a new approach was tested for the production of the sweetener 5-KF by using sucrose as a substrate. To this end, a two-strain system composed of the fdh-expressing strain and a G. oxydans strain that produced the sucrose hydrolyzing SacC was developed. The strains were co-cultured in sucrose medium and converted 92.5% of the available fructose units into 5-KF. The glucose moiety of sucrose was converted to 2-ketogluconate and acetate. With regard to the development of a sustainable and resource-saving process for the production of 5-KF, sugar beet extract was used as substrate for the two-strain system. Fructose as product from sucrose cleavage was mainly oxidized to 5-KF which was detected in a concentration of over 200 mM at the end of the fermentation process. In summary, the two-strain system was able to convert fructose units of sugar beet extract to 5-KF with an efficiency of 82 ± 5%.
    Keywords Gluconobacter oxydans ; acetates ; coculture ; consumer demand ; fermentation ; fructose ; genes ; glucose ; moieties ; nonnutritive sweeteners ; sucrose ; sugar beet ; sweet-tasting compounds
    Language English
    Dates of publication 2018-02
    Size p. 1699-1710.
    Publishing place Springer Berlin Heidelberg
    Document type Article
    ZDB-ID 392453-1
    ISSN 1432-0614 ; 0171-1741 ; 0175-7598
    ISSN (online) 1432-0614
    ISSN 0171-1741 ; 0175-7598
    DOI 10.1007/s00253-017-8699-1
    Database NAL-Catalogue (AGRICOLA)

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  5. Article ; Online: Influence of CNTRENE® C100LM carbon nanotube material on the growth and regulation of Escherichia coli

    Brittany Twibell / Kalie Somerville / Geoffrey Manani / Molly Duszynski / Adam Wanekaya / Paul Schweiger

    PeerJ, Vol 5, p e

    2017  Volume 3721

    Abstract: The growing use of carbon nanotubes (CNTs) in industrial and consumer products raises important questions about their environmental fate and impact on prokaryotes. In the environment, CNTs are exposed to a variety of conditions (e.g., UV light) that ... ...

    Abstract The growing use of carbon nanotubes (CNTs) in industrial and consumer products raises important questions about their environmental fate and impact on prokaryotes. In the environment, CNTs are exposed to a variety of conditions (e.g., UV light) that could lead to decomposition and changes in their chemical properties. Therefore, the potential cytotoxic effect of both pristine and artificially aged carboxyl functionalized CNTRENE® C100LM CNTmaterial at neutral and acidic conditions on Escherichia coli K12 was analyzed using a minimal inhibitory concentration (MIC) assay, which also allowed monitoring of non-lethal growth effects. However, there were no observable MIC or significant changes in growth behavior in E. coli K12 when exposed to pristine or aged CNTs. Exposure to pristine CNTRENE® C100LM CNT material did not appear to influence cell morphology or damage the cells when examined by electron microscopy. In addition, RNA sequencing revealed no observable regulatory changes in typical stress response pathways. This is surprising considering that previous studies have claimed high cytotoxicity of CNTs, including carboxyl functionalized single-walled CNTs, and suggest that other factors such as trace heavy metals or other impurities are likely responsible for many of the previously reported cytotoxicity in E. coli and possibly other microorganisms.
    Keywords Cytotoxicity ; Nanoparticles ; Minimum inhibitory concentration ; Gene expression ; Life cycle assessment ; Medicine ; R ; Biology (General) ; QH301-705.5
    Subject code 500
    Language English
    Publishing date 2017-08-01T00:00:00Z
    Publisher PeerJ Inc.
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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