Molecular cloning and functional expression of d-arabitol dehydrogenase gene from<i>Gluconobacter oxydans</i>in<i>Escherichia coli</i>

Cheng, Hairong; Jiang, Ning; Shen, An; Feng, Youjun

doi:10.1016/j.femsle.2005.08.023

Cited by 16 publications

(14 citation statements)

References 33 publications

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“…The optimal values are near pH 7 (i.e., 6 for reduction and 7.5 for dehydrogenation), which is similar to one of the pK a values of the amine groups in the imidazole ring of histidine. In the case of other SDR family enzymes, such as mannitol dehydrogenase from Candida magnolia or D-arabitol dehydrogenase from Gluconobacter oxydans, the optimal pHs were around 6.5 to 7.5 for reduction and 8.5 to 10 for dehydrogenation (36,37). Therefore, the histidine residue would lower the pH optimum for dehydrogenase activity.…”

Section: Discussionmentioning

confidence: 99%

P212A Mutant of Dihydrodaidzein Reductase Enhances ( S )-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

Lee

Kim

et al. 2016

Appl Environ Microbiol

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d(S)-Equol, a gut bacterial isoflavone derivative, has drawn great attention because of its potent use for relieving female postmenopausal symptoms and preventing prostate cancer. Previous studies have reported on the dietary isoflavone metabolism of several human gut bacteria and the involved enzymes for conversion of daidzein to (S)-equol. However, the anaerobic growth conditions required by the gut bacteria and the low productivity and yield of (S)-equol limit its efficient production using only natural gut bacteria. In this study, the low (S)-equol biosynthesis of gut microorganisms was overcome by cloning the four enzymes involved in the biosynthesis from Slackia isoflavoniconvertens into Escherichia coli BL21(DE3). The reaction conditions were optimized for (S)-equol production from the recombinant strain, and this recombinant system enabled the efficient conversion of 200 M and 1 mM daidzein to (S)-equol under aerobic conditions, achieving yields of 95% and 85%, respectively. Since the biosynthesis of trans-tetrahydrodaidzein was found to be a rate-determining step for (S)-equol production, dihydrodaidzein reductase (DHDR) was subjected to rational site-directed mutagenesis. The introduction of the DHDR P212A mutation increased the (S)-equol productivity from 59.0 mg/liter/h to 69.8 mg/liter/h in the whole-cell reaction. The P212A mutation caused an increase in the (S)-dihydrodaidzein enantioselectivity by decreasing the overall activity of DHDR, resulting in undetectable activity for (R)-dihydrodaidzein, such that a combination of the DHDR P212A mutant with dihydrodaidzein racemase enabled the production of (3S,4R)-tetrahydrodaidzein with an enantioselectivity of >99%.

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Section: Discussionmentioning

confidence: 99%

P212A Mutant of Dihydrodaidzein Reductase Enhances ( S )-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

Lee

Kim

et al. 2016

Appl Environ Microbiol

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“…The G. oxydans arabitol dehydrogenase gene gox2181, previously reported by Cheng et al (2005), was homologously expressed and protein production was compared to E. coli production to investigate the usefulness of these vectors as a protein production platform in G. oxydans. After 24 h expression, Gox2181 was purified from the cell free lysate by StrepTactin affinity chromatography as described in Schweiger et al (2010).…”

Section: Protein Productionmentioning

confidence: 99%

“…The genome sequence of G. oxydans 621H is known and it was found to contain over 70 uncharacterized oxidoreductases (Prust et al, 2005). Some of these proteins have been functionally overproduced heterologously in Escherichia coli and characterized (Cheng et al, 2005;Saichana et al, 2007;Schweiger et al, 2008Schweiger et al, , 2010. However, protein production in the natural host is preferred, especially for membrane-bound dehydrogenases, which are of major importance for industrial bioconversions as the products are excreted into the medium to almost quantitative yields.…”

Section: Introductionmentioning

confidence: 99%

Construction of expression vectors for protein production in Gluconobacter oxydans

Kallnik

Meyer

Deppenmeier

et al. 2010

Journal of Biotechnology

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“…Bioinformatics analysis revealed that 75 open reading frames are putative dehydrogenases/oxidoreductases, which may have potential abilities in production of chemical intermediates . Recently, more than 40 of them have been cloned and overexpressed in Escherichia coli , and several putative oxidoreductases have been characterized . Among them, Gox0499 preferentially oxidizes long‐chained aliphatic and aromatic aldehydes, while Gox1122 shows substrate preference towards short‐chain aliphatic aldehydes, in addition Gox0644, an NADPH‐dependent aldehyde reductase, favors aromatic aldehydes …”

Section: Introductionmentioning

confidence: 99%

Structural insights into substrate and coenzyme preference by SDR family protein Gox2253 from Gluconobater oxydans

et al. 2014

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Gox2253 from Gluconobacter oxydans belongs to the short-chain dehydrogenases/reductases family, and catalyzes the reduction of heptanal, octanal, nonanal, and decanal with NADPH. To develop a robust working platform to engineer novel G. oxydans oxidoreductases with designed coenzyme preference, we adopted a structure based rational design strategy using computational predictions that considers the number of hydrogen bonds formed between enzyme and docked coenzyme. We report the crystal structure of Gox2253 at 2.6 Å resolution, ternary models of Gox2253 mutants in complex with NADH/short-chain aldehydes, and propose a structural mechanism of substrate selection. Molecular dynamics simulation shows that hydrogen bonds could form between 2'-hydroxyl group in the adenosine moiety of NADH and the side chain of Gox2253 mutant after arginine at position 42 is replaced with tyrosine or lysine. Consistent with the molecular dynamics prediction, Gox2253-R42Y/K mutants can use both NADH and NADPH as a coenzyme. Hence, the strategies here could provide a practical platform to engineer coenzyme selectivity for any given oxidoreductase and could serve as an additional consideration to engineer substrate-binding pockets.

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Molecular cloning and functional expression of d-arabitol dehydrogenase gene fromGluconobacter oxydansinEscherichia coli

Cited by 16 publications

References 33 publications

P212A Mutant of Dihydrodaidzein Reductase Enhances ( S )-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

P212A Mutant of Dihydrodaidzein Reductase Enhances ( S )-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

Construction of expression vectors for protein production in Gluconobacter oxydans

Structural insights into substrate and coenzyme preference by SDR family protein Gox2253 from Gluconobater oxydans

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