L-sorbose dissimilation in 2-keto-L-gulonic acid-producing mutant UV10 derived from Gluconobacter melanogenus IFO 3293.

Shinjoh, Masako; Setoguchi, Yutaka; Hoshino, Tatsuo; Fujiwara, Akiko

doi:10.1271/bbb1961.54.2257

Cited by 11 publications

(7 citation statements)

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“…Strain U13 is a derivative of UV10 and exhibits higher SDH activity than UV10. In strains exhibiting weaker SDH activities, there is stronger carbon flow toward final assimilation of the substrate to CO 2 , mainly via the pentose cycle, as reported previously (21).…”

Section: Formation Of 2kga From L-sorbosone L-sorbose and Dsorbitolsupporting

confidence: 80%

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Cloning and nucleotide sequencing of the membrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO 12258 and its expression in Gluconobacter oxydans

Shinjoh

Tomiyama

Asakura

et al. 1995

Appl Environ Microbiol

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Cloning and expression of the gene encoding Acetobacter liquefaciens IFO 12258 membrane-bound L-sorbosone dehydrogenase (SNDH) were studied. A genomic library of A. liquefaciens IFO 12258 was constructed with the mobilizable cosmid vector pVK102 (mob ؉) in Escherichia coli S17-1 (Tra ؉). The library was transferred by conjugal mating into Gluconobacter oxydans OX4, a mutant of G. oxydans IFO 3293 that accumulates L-sorbosone in the presence of L-sorbose. The transconjugants were screened for SNDH activity by performing a direct expression assay. One clone harboring plasmid p7A6 converted L-sorbosone to 2-keto-L-gulonic acid (2KGA) more rapidly than its host did and also converted L-sorbose to 2KGA with no accumulation of L-sorbosone. The insert (25 kb) of p7A6 was shortened to a 3.1-kb fragment, in which one open reading frame (1,347 bp) was found and was shown to encode a polypeptide with a molecular weight of 48,222. The SNDH gene was introduced into the 2KGA-producing strain G. oxydans IFO 3293 and its derivatives, which contained membrane-bound L-sorbose dehydrogenase. The cloned SNDH was correctly located in the membrane of the host. The membrane fraction of the clone exhibited almost stoichiometric formation of 2KGA from L-sorbosone and L-sorbose. Resting cells of the clones produced 2KGA very efficiently from L-sorbosone and L-sorbose, but not from D-sorbitol; the conversion yield from L-sorbosone was improved from approximately 25 to 83%, whereas the yield from L-sorbose was increased from 68 to 81%. Under fermentation conditions, cloning did not obviously improve the yield of 2KGA from L-sorbose.

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Section: Formation Of 2kga From L-sorbosone L-sorbose and Dsorbitolsupporting

confidence: 80%

“…Most of the carbon loss was found to be in the form of CO 2 gas that evolved mainly from the pentose phosphate pathway in the cytoplasm (cSNDH in Fig. 1) (21). In addition, we found that Acetobacter liquefaciens (formerly G. melanogenus) IFO 12258 converted L-sorbosone to 2KGA stoichiometrically (21a) and contained the membrane-bound SNDH.…”

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confidence: 88%

Cloning and nucleotide sequencing of the membrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO 12258 and its expression in Gluconobacter oxydans

Shinjoh

Tomiyama

Asakura

et al. 1995

Appl Environ Microbiol

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“…This involves an initial phosphorylation of glucose or gluconate to glucose-6-phosphate or 6-phosphogluconate, respectively, and subsequent metabolism of these intermediates via the PPP or EDP (29,31,40). The fact that the majority of the carbon source is incompletely oxidized to ketoacids as products rather than being metabolized via the PPP and EDP results in very low biomass yields, in the range of 0.10 g cell dry weight (CDW) per g of glucose consumed (29).…”

Section: And N-formyl-1-amino-1-deoxy-d-sorbitolmentioning

confidence: 99%

Metabolic Engineering of Gluconobacter oxydans for Improved Growth Rate and Growth Yield on Glucose by Elimination of Gluconate Formation

Krajewski

Šimić²,

Mouncey³

et al. 2010

Appl Environ Microbiol

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Gluconobacter oxydans N44-1, an obligatory aerobic acetic acid bacterium, oxidizes glucose primarily in the periplasm to the end products 2-ketogluconate and 2,5-diketogluconate, with intermediate formation of gluconate. Only a minor part of the glucose (less than 10%) is metabolized in the cytoplasm after conversion to gluconate or after phosphorylation to glucose-6-phosphate via the only functional catabolic routes, the pentose phosphate pathway and the Entner-Doudoroff pathway. This unusual method of glucose metabolism results in a low growth yield. In order to improve it, we constructed mutants of strain N44-1 in which the gene encoding the membrane-bound glucose dehydrogenase was inactivated either alone or together with the gene encoding the cytoplasmic glucose dehydrogenase. The growth and product formation from glucose of the resulting strains, N44-1 mgdH::kan and N44-1 ⌬mgdH sgdH::kan, were analyzed. Both mutant strains completely consumed the glucose but produced neither gluconate nor the secondary products 2-ketogluconate and 2,5-diketogluconate. Instead, carbon dioxide formation of the mutants increased by a factor of 4 (N44-1 mgdH::kan) or 5.5 (N44-1 ⌬mgdH sgdH::kan), and significant amounts of acetate were produced, presumably by the activities of pyruvate decarboxylase and acetaldehyde dehydrogenase. Most importantly, the growth yields of the two mutants increased by 110% (N44-1 mgdH::kan) and 271% (N44-1 ⌬mgdH sgdH::kan). In addition, the growth rates improved by 39% (N44-1 mgdH::kan) and 78% (N44-1 ⌬mgdH sgdH::kan), respectively, compared to the parental strain. These results show that the conversion of glucose to gluconate and ketogluconates has a strong negative impact on the growth of G. oxydans.As the Gram-negative acetic acid bacterium Gluconobacter oxydans is able to oxidize sugars and sugar alcohols regioselectively, it is a valuable and versatile biocatalyst and has been used in industry for a long time, e.g., for the production of vitamin C via Reichstein synthesis (32) and of 1-deoxynojirimycin, a precursor of the antidiabetic drug miglitol (35). Both processes are combined biotechnological-chemical syntheses carried out on a large scale (10,35). The key biotechnological reactions in these two examples are the regioselective oxidation of D-sorbitol to L-sorbose and N-formyl-1-amino-1-deoxy-D-sorbitol to N-formyl-6-amino-6-deoxy-L-sorbose, respectively. The latter conversion is performed in whole-cell biotransformations with resting cells of G. oxydans as a catalyst.A characteristic trait of G. oxydans is the presence of parallel but spatially separated pathways for the oxidation of nonphosphorylated substrates and intermediates in both the periplasmic and cytoplasmic compartments (Fig.

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“…In either case, an NAD(P)H-dependent reductase catalyzing the reduction of L-sorbose or L-sorbose-1-phosphate is essential for L-sorbose utilization in these microorganisms (8). The metabolic pathways of D-sorbitol, Lsorbose, and their metabolites in Gluconobacter strains have been studied previously (9,20,22), and these C sources were shown to be utilized through a nonphosphorylating pathway; however, no transcriptional analysis has yet been performed.…”

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confidence: 99%

l -Sorbose Reductase and Its Transcriptional Regulator Involved in l -Sorbose Utilization of Gluconobacter frateurii

Soemphol¹,

Toyama

Moonmangmee

et al. 2007

J Bacteriol

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Upstream of the gene for flavin adenine dinucleotide (FAD)-dependent D-sorbitol dehydrogenase (SLDH),sldSLC, a putative transcriptional regulator was found in Gluconobacter frateurii THD32 (NBRC 101656). In this study, the whole sboR gene and the adjacent gene, sboA, were cloned and analyzed. sboR mutation did not affect FAD-SLDH activity in the membrane fractions. The SboA enzyme expressed and purified from an Escherichia coli transformant showed NADPH-dependent L-sorbose reductase (NADPH-SR) activity, and the enzyme was different from the NADPH-SR previously reported for Gluconobacter suboxydans IFO 3291 in molecular size and amino acid sequence. A mutant defective in sboA showed significantly reduced growth on L-sorbose, indicating that the SboA enzyme is required for efficient growth on L-sorbose. The sboR mutant grew on L-sorbose even better than the wild-type strain did, and higher NADPH-SR activity was detected in cytoplasm fractions. Reverse transcription-PCR experiments indicated that sboRA comprises an operon. These data suggest that sboR is involved in the repression of sboA, but not in the induction of sldSLC, on D-sorbitol and that another activator is required for the induction of these genes by D-sorbitol or L-sorbose.

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L-sorbose dissimilation in 2-keto-L-gulonic acid-producing mutant UV10 derived from Gluconobacter melanogenus IFO 3293.

Cited by 11 publications

References 0 publications

Cloning and nucleotide sequencing of the membrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO 12258 and its expression in Gluconobacter oxydans

Cloning and nucleotide sequencing of the membrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO 12258 and its expression in Gluconobacter oxydans

Metabolic Engineering of Gluconobacter oxydans for Improved Growth Rate and Growth Yield on Glucose by Elimination of Gluconate Formation

l -Sorbose Reductase and Its Transcriptional Regulator Involved in l -Sorbose Utilization of Gluconobacter frateurii

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