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      13C Tracers for Glucose Degrading Pathway Discrimination in Gluconobacter oxydans 621H

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          Abstract

          Gluconobacter oxydans 621H is used as an industrial production organism due to its exceptional ability to incompletely oxidize a great variety of carbohydrates in the periplasm. With glucose as the carbon source, up to 90% of the initial concentration is oxidized periplasmatically to gluconate and ketogluconates. Growth on glucose is biphasic and intracellular sugar catabolism proceeds via the Entner–Doudoroff pathway (EDP) and the pentose phosphate pathway (PPP). Here we studied the in vivo contributions of the two pathways to glucose catabolism on a microtiter scale. In our approach we applied specifically 13C labeled glucose, whereby a labeling pattern in alanine was generated intracellularly. This method revealed a dynamic growth phase-dependent pathway activity with increased activity of EDP in the first and PPP in the second growth phase, respectively. Evidence for a growth phase-independent decarboxylation-carboxylation cycle around the pyruvate node was obtained from 13C fragmentation patterns of alanine. For the first time, down-scaled microtiter plate cultivation together with 13C-labeled substrate was applied for G. oxydans to elucidate pathway operation, exhibiting reasonable labeling costs and allowing for sufficient replicate experiments.

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          The oxidative pentose phosphate pathway: structure and organisation.

          Nicholas J Kruger, Antje von Schaewen (2003)
          The oxidative pentose phosphate pathway is a major source of reducing power and metabolic intermediates for biosynthetic processes. Some, if not all, of the enzymes of the pathway are found in both the cytosol and plastids, although the precise distribution of their activities varies. The apparent absence of sections of the pathway from the cytosol potentially complicates metabolism. These complications are partly offset, however, by exchange of intermediates between the cytosol and the plastids through the activities of a family of plastid phosphate translocators. Molecular analysis is confirming the widespread presence of multiple genes encoding each of the enzymes of the oxidative pentose phosphate pathway. Differential expression of these isozymes may ensure that the kinetic properties of the activity that catalyses a specific reaction match the metabolic requirements of a particular tissue. This hypothesis can be tested thanks to recent developments in the application of 13C-steady-state labelling strategies. These strategies make it possible to quantify flux through metabolic networks and to discriminate between pathways of carbohydrate oxidation in the cytosol and plastids.
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            Biotechnological applications of acetic acid bacteria.

            P Raspor, Dušan Goranovič (2007)
            The acetic acid bacteria (AAB) have important roles in food and beverage production, as well as in the bioproduction of industrial chemicals. In recent years, there have been major advances in understanding their taxonomy, molecular biology, and physiology, and in methods for their isolation and identification. AAB are obligate aerobes that oxidize sugars, sugar alcohols, and ethanol with the production of acetic acid as the major end product. This special type of metabolism differentiates them from all other bacteria. Recently, the AAB taxonomy has been strongly rearranged as new techniques using 16S rRNA sequence analysis have been introduced. Currently, the AAB are classified in ten genera in the family Acetobacteriaceae. AAB can not only play a positive role in the production of selected foods and beverages, but they can also spoil other foods and beverages. AAB occur in sugar- and alcohol-enriched environments. The difficulty of cultivation of AAB on semisolid media in the past resulted in poor knowledge of the species present in industrial processes. The first step of acetic acid production is the conversion of ethanol from a carbohydrate carried out by yeasts, and the second step is the oxidation of ethanol to acetic acid carried out by AAB. Vinegar is traditionally the product of acetous fermentation of natural alcoholic substrates. Depending on the substrate, vinegars can be classified as fruit, starch, or spirit substrate vinegars. Although a variety of bacteria can produce acetic acid, mostly members of Acetobacter, Gluconacetobacter, and Gluconobacter are used commercially. Industrial vinegar manufacturing processes fall into three main categories: slow processes, quick processes, and submerged processes. AAB also play an important role in cocoa production, which represents a significant means of income for some countries. Microbial cellulose, produced by AAB, possesses some excellent physical properties and has potential for many applications. Other products of biotransformations by AAB or their enzymes include 2-keto-L-gulonic acid, which is used for the production of vitamin C; D-tagatose, which is used as a bulking agent in food and a noncalorific sweetener; and shikimate, which is a key intermediate for a large number of antibiotics. Recently, for the first time, a pathogenic acetic acid bacterium was described, representing the newest and tenth genus of AAB.
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              Gluconobacter oxydans: its biotechnological applications.

              A. Kumar, Neha A Gupta, Ghulam Qazi … (2001)
              Gluconobacter oxydans is a gram-negative bacterium belonging to the family Acetobacteraceae. G. oxydans is an obligate aerobe, having a respiratory type of metabolism using oxygen as the terminal electron acceptor. Gluconobacter strains flourish in sugary niches e.g. ripe grapes, apples, dates, garden soil, baker's soil, honeybees, fruit, cider, beer, wine. Gluconobacter strains are non-pathogenic towards man and other animals but are capable of causing bacterial rot of apples and pears accompanied by various shades of browning. Several soluble and particulate polyol dehydrogenases have been described. The organism brings about the incomplete oxidation of sugars, alcohols and acids. Incomplete oxidation leads to nearly quantitative yields of the oxidation products making G. oxydans important for industrial use. Gluconobacter strains can be used industrially to produce L-sorbose from D-sorbitol; D-gluconic acid, 5-keto- and 2-ketogluconic acids from D-glucose; and dihydroxyacetone from glycerol. It is primarily known as a ketogenic bacterium due to 2,5-diketogluconic acid formation from D-glucose. Extensive fermentation studies have been performed to characterize its direct glucose oxidation, sorbitol oxidation, and glycerol oxidation. The enzymes involved have been purified and characterized, and molecular studies have been performed to understand these processes at the molecular level. Its possible application in biosensor technology has also been worked out. Several workers have explained its basic and applied aspects. In the present paper, its different biotechnological applications, basic biochemistry and molecular biology studies are reviewed.
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                Author and article information

                Contributors
                Kazuyuki Shimizu: Role: Academic Editor
                Journal
                Journal ID (nlm-ta): Metabolites
                Journal ID (iso-abbrev): Metabolites
                Journal ID (publisher-id): metabolites
                Title: Metabolites
                Publisher: MDPI
                ISSN (Electronic): 2218-1989
                Publication date (Electronic): 02 September 2015
                Publication date Collection: September 2015
                Volume: 5
                Issue: 3
                Pages: 455-474
                Affiliations
                Institute of Bio- and Geosciences—IBG-1: Biotechnology, Leo-Brandt-Straße, 52428 Jülich, Germany; E-Mails: s.ostermann@ 123456fz-juelich.de (S.O.); j.richhardt@ 123456fz-juelich.de (J.R.); st.bringer-meyer@ 123456fz-juelich.de (S.B.); m.bott@ 123456fz-juelich.de (M.B.); w.wiechert@ 123456fz-juelich.de (W.W.)
                Author notes
                [* ]Author to whom correspondence should be addressed; E-Mail: m.oldiges@ 123456fz-juelich.de ; Tel.: +49-2461-61-3951; Fax: +49-2461-61-3870.
                Article
                Publisher ID: metabolites-05-00455
                DOI: 10.3390/metabo5030455
                PMC ID: 4588806
                PubMed ID: 26404385
                SO-VID: b5ab8577-c14b-4230-8901-9bebf7312fc6
                Copyright © © 2015 by the authors; licensee MDPI, Basel, Switzerland.
                License:

                This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 10 July 2015
                Date accepted : 24 August 2015
                Categories
                Subject: Article

                Keywords: microtiter cultivation,13c tracer,pentose phosphate pathway,entner–doudoroff pathway,glucose
                Data availability:
                Keywords: microtiter cultivation, 13c tracer, pentose phosphate pathway, entner–doudoroff pathway, glucose

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