Production of a periplasmic trehalase in Gluconobacter oxydans and growth on trehalose

Kosciow, Konrad; Zahid, Nageena; Schweiger, Paul; Deppenmeier, Uwe

doi:10.1016/j.jbiotec.2014.08.029

Cited by 10 publications

(3 citation statements)

References 57 publications

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“…Generally, microorganisms respond to the environmental stress conditions by accumulation or lowering amount of some compounds within the cells. 15 , 34 Increasing the osmotic condition of the media may cause to reduce the content of the osmoregulated periplasmic glucans. 35 This phenomenon may be a driving force to increase the periplasmic content of the expressed scFv antibody that could be a subject for the further studies.…”

Section: Discussionmentioning

confidence: 99%

Osmotic conditions could promote scFv antibody production in the Escherichia coli HB2151

Mesgari-Shadi¹,

Sarrafzadeh²

2017

Bioimpacts

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Introduction: Single chain variable fragment (scFv) antibodies are reduced forms of the whole antibodies that could be regarded as an alternative tool for diagnostic and therapeutic purposes. The optimization of processes and environmental conditions is necessary to increase the production yields and enhance the productivity. This can result in a cost-effective process and respond to the high demand for these antibodies. Methods: In this research, physical and chemical factors influencing the batch fermentation was investigated in 50 mL batch tubes using minimum media to find the optimum conditions for production of a single chain variable fragment antibody in the Escherichia coli HB2151. Experimental designs were used to screen the effective parameters and to optimize the main factors. Results: Arabinose was used instead of IPTG as a cheaper and nontoxic inducer and its optimum concentration was determined 0.1% (w/w). Induction duration time and filling volume fraction were set on the relatively better states 24 hours and 1/10 respectively. Regarding our previous study, stationary phase of the cell growth was selected as induction start time that showed higher specific scFv production yields (YP/X) in the minimum media. Finally, a statistical experimental design was extended to a central composite design (CCD) and analysis was performed based on sucrose and sorbitol concentrations producing osmotic condition for induction. The optimum region in the contour plot for the periplasmic scFv production was an osmotic circle area with total sugar molarity 0.8 to 0.9. Conclusion: Sugars such as sucrose and sorbitol producing osmotic conditions could lead to periplasmic scFv concentrations up to 2.85 mg/L of culture media improving scFv concentration near to five times of the average of the screening step (0.59 mg/L).

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Section: Discussionmentioning

confidence: 99%

Osmotic conditions could promote scFv antibody production in the Escherichia coli HB2151

Mesgari-Shadi¹,

Sarrafzadeh²

2017

Bioimpacts

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“…Alkaline phosphatase assays were done using a modified method of Kosciow et al (2014) . Experimental cultures were inoculated 1:100 from overnight cultures and grown to mid-late exponential phase (OD 600 of 0.9–1.1 for E. coli and 0.6–0.9 for G. oxydans ).…”

Section: Methodsmentioning

confidence: 99%

“…The ability to produce recombinant enzymes for the modification of extracellular substrates has the potential to expand and compliment the natural incomplete-oxidative metabolism of acetic acid bacteria. Signal peptides that allow for periplasmic export are known ( Kosciow et al, 2014 ), but the ability to produce additional enzymes bound to the cytoplasmic membrane is limited because this space is already crowded by the large number of native dehydrogenases ( Guigas & Weiss, 2016 ). Production of extracellular enzymes is also challenging because G. oxydans lacks the machinery to secrete proteins across the outer membrane ( Prust et al, 2005 ).…”

Section: Introductionmentioning

confidence: 99%

Surface display for metabolic engineering of industrially important acetic acid bacteria

Blank

Schweiger

2018

PeerJ

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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.

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Production of 5-ketofructose from fructose or sucrose using genetically modified Gluconobacter oxydans strains

Siemen¹,

Kosciow²,

Schweiger

et al. 2017

Appl Microbiol Biotechnol

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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%.

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Production of a periplasmic trehalase in Gluconobacter oxydans and growth on trehalose

Cited by 10 publications

References 57 publications

Osmotic conditions could promote scFv antibody production in the Escherichia coli HB2151

Osmotic conditions could promote scFv antibody production in the Escherichia coli HB2151

Surface display for metabolic engineering of industrially important acetic acid bacteria

Production of 5-ketofructose from fructose or sucrose using genetically modified Gluconobacter oxydans strains

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