Cloning of genes coding for L-sorbose and L-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of 2-keto-L-gulonate, a precursor of L-ascorbic acid, in a recombinant G. oxydans strain

Saito, Yoshimasa; Ishii, Yoshinori; Hayashi, Hiromi; Imao, Y; Akashi, Tetuyuki; Yoshikawa, Kunio; Noguchi, Yuji; Soeda, Sinsuke; Yoshida, Masaru; Niwa, Mineo; Hosoda, Junji; Shimomura, Kyoichi

doi:10.1128/aem.63.2.454-460.1997

Cited by 121 publications

(36 citation statements)

References 33 publications

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“…4) and active sites (E 262 and C 296 , bold italic in Fig. 4) are well conserved with those of NAD(P) dependent BADH (Lamark et al, 1991;Saito et al, 1997). This data is in accordance with the enzymatic character, and suggests that SNDH is a NAD(P) dependent enzyme.…”

Section: Discussionsupporting

confidence: 75%

“…Gluconobacter oxydans T-100 (FERM BP-4188) and G. oxydans G624 (FERM BP-4415) are 2-keto-L-gulonic acid (2-KLGA) producing and L-sorbose accumulating strains, respectively. Preparation of plasmids pF4, pFG15A, pUC19SD5, pSDH155 and pSDH-tufB1 were described previously (Saito et al, 1997). SNDH was purified from the cytosolic fraction by QAE-TOYOPEARL 550C (Tosoh) and Blue-Sepharose (Pharmacia) column chromatography, successively.…”

Section: Methodsmentioning

confidence: 99%

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Direct fermentation of 2‐keto‐l‐gulonic acid in recombinant Gluconobacter oxydans

Saito

Ishii

Hayashi³

et al. 1998

Biotechnol. Bioeng.

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We isolated Gluconobacter oxydans T-100 that had an activity to produce 2-KLGA from D-sorbitol; however, the yield of 2-KLGA was quite insufficient. Therefore, enzymes involved in the biosynthesis of L-sorbosone and 2-KLGA, L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH), respectively, were purified from G. oxydans T-100. A genomic library of G. oxydans T-100 was screened to clone both genes for SDH and SNDH based on their amino acid sequences. SNDH and SDH were encoded in sequential open reading frames with 1497 and 1596 nucleotides, respectively, which were verified by the expression in Escherichia coli. The amino acid sequence of SDH and SNDH showed close similarity with E. coli choline dehydrogenase (CDH) and betaine-aldehyde dehydrogenase (BADH), respectively, which cooperatively play a key role for conferring osmotic tolerance. Because the yield of 2-KLGA by G. oxydans introduced with the genes for SDH and SNDH were insufficient, replacement of the promoter with that of Escherichia coli tufB1 in combination with chemical mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine resulted in improvement of the production level.

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Section: Discussionsupporting

confidence: 75%

Section: Methodsmentioning

confidence: 99%

Direct fermentation of 2‐keto‐l‐gulonic acid in recombinant Gluconobacter oxydans

Saito

Ishii

Hayashi³

et al. 1998

Biotechnol. Bioeng.

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“…The new strain was an improved producer of 2-KGA. 64 Further mutation to suppress the L-idonate pathway and to improve the promoter led to production of 130 g l -1 of 2-KGA from 150 g l -1 sorbitol.…”

Section: Genome-wide Transcript Expression Analysis Riboflavinmentioning

confidence: 99%

Recombinant organisms for production of industrial products

Adrio¹,

Demain²

2010

Bioengineered Bugs

177

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A revolution in industrial microbiology was sparked by the discoveries of ther double-stranded structure of DNA and the development of recombinant DNA technology. Traditional industrial microbiology was merged with molecular biology to yield improved recombinant processes for the industrial production of primary and secondary metabolites, protein biopharmaceuticals and industrial enzymes. Novel genetic techniques such as metabolic engineering, combinatorial biosynthesis and molecular breeding techniques and their modifications are contributing greatly to the development of improved industrial processes. In addition, functional genomics, proteomics and metabolomics are being exploited for the discovery of novel valuable small molecules for medicine as well as enzymes for catalysis. The sequencing of industrial microbal genomes is being carried out which bodes well for future process improvement and discovery of new industrial products.

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“…A mixed culture of G. oxydans strain DSM4025 (which converts L-sorbose to 2-KLGA) and Gluconobacter suboxydans IFO 3255 (which converts D-sorbitol to L-sorbose) was able to convert 138 g l -1 D-sorbitol to 112 g l -1 2-KLGA, with a molecular conversion yield of 75% in 2 days (Hoshino, 2000). A recombinant strain of G. oxydans containing genes encoding L-sorbose dehydrogenase and L-sorbosone dehydrogenase from G. oxydans T-100 was able to produce 2-KLGA effectively from D-sorbitol (Saito et al, 1997). Mutation to suppress the L-idonate pathway and improvement of the promoter led to production of 130 g l -1 2-KLGA from 150 g l -1 D-sorbitol.…”

Section: Riboflavinmentioning

confidence: 99%

Metabolic regulation and overproduction of primary metabolites

Sánchez

Demain

2008

Microbial Biotechnology

155

103

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SummaryOverproduction of microbial metabolites is related to developmental phases of microorganisms. Inducers, effectors, inhibitors and various signal molecules play a role in different types of overproduction. Biosynthesis of enzymes catalysing metabolic reactions in microbial cells is controlled by well‐known positive and negative mechanisms, e.g. induction, nutritional regulation (carbon or nitrogen source regulation), feedback regulation, etc. The microbial production of primary metabolites contributes significantly to the quality of life. Fermentative production of these compounds is still an important goal of modern biotechnology. Through fermentation, microorganisms growing on inexpensive carbon and nitrogen sources produce valuable products such as amino acids, nucleotides, organic acids and vitamins which can be added to food to enhance its flavour, or increase its nutritive values. The contribution of microorganisms goes well beyond the food and health industries with the renewed interest in solvent fermentations. Microorganisms have the potential to provide many petroleum‐derived products as well as the ethanol necessary for liquid fuel. Additional applications of primary metabolites lie in their impact as precursors of many pharmaceutical compounds. The roles of primary metabolites and the microbes which produce them will certainly increase in importance as time goes on. In the early years of fermentation processes, development of producing strains initially depended on classical strain breeding involving repeated random mutations, each followed by screening or selection. More recently, methods of molecular genetics have been used for the overproduction of primary metabolic products. The development of modern tools of molecular biology enabled more rational approaches for strain improvement. Techniques of transcriptome, proteome and metabolome analysis, as well as metabolic flux analysis. have recently been introduced in order to identify new and important target genes and to quantify metabolic activities necessary for further strain improvement.

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Cloning of genes coding for L-sorbose and L-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of 2-keto-L-gulonate, a precursor of L-ascorbic acid, in a recombinant G. oxydans strain

Cited by 121 publications

References 33 publications

Direct fermentation of 2‐keto‐l‐gulonic acid in recombinant Gluconobacter oxydans

Direct fermentation of 2‐keto‐l‐gulonic acid in recombinant Gluconobacter oxydans

Recombinant organisms for production of industrial products

Metabolic regulation and overproduction of primary metabolites

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