Characterisation of Glycerol Dehydrogenase from a Methylotrophic Yeast, Hansenula polymorpha Dl-1, and its Gene Cloning

Yamada‐Onodera, Keiko; Yamamoto, Hidenori; Emoto, E.; Kawahara, Nobuhiro; Tani, Yoshiki

doi:10.1002/1521-3846(200207)22:3/4<337::aid-abio337>3.0.co;2-6

Cited by 23 publications

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“…Characterization and complementation of the gdh -deficient mutant B. licheniformis WX-02Δ gdh further confirmed the catalytic role of WX-02 GDH in the conversion of D -AC to D -2,3-BD. A similar substrate range was observed for the GDH from other strains, such as Serratia , Klebsiella and Hansenula species [ 20 , 23 , 28 ].…”

Section: Discussionsupporting

confidence: 64%

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Engineering Bacillus licheniformis for the production of meso-2,3-butanediol

Qiu

Zhang

et al. 2016

Biotechnol Biofuels

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Background2,3-Butanediol (2,3-BD) can be used as a liquid fuel additive to replace petroleum oil, and as an important platform chemical in the pharmaceutical and plastic industries. Microbial production of 2,3-BD by Bacillus licheniformis presents potential advantages due to its GRAS status, but previous attempts to use this microorganism as a chassis strain resulted in the production of a mix of D-2,3-BD and meso-2,3-BD isomers.ResultsThe aim of this work was to develop an engineered strain of B. licheniformis suited to produce the high titers of the pure meso-2,3-BD isomer. Glycerol dehydrogenase (Gdh) was identified as the catalyst for D-2,3-BD biosynthesis from its precursor acetoin in B. licheniformis. The gdh gene was, therefore, deleted from the wild-type strain WX-02 to inhibit the flux of acetoin to D-2,3-BD biosynthesis. The acoR gene involved in acetoin degradation through AoDH ES was also deleted to provide adequate flux from acetoin towards meso-2,3-BD. By re-directing the carbon flux distribution, the double-deletion mutant WX-02ΔgdhΔacoR produced 28.2 g/L of meso-2,3-BD isomer with >99 % purity. The titer was 50 % higher than that of the wide type. A bench-scale fermentation by the double-deletion mutant was developed to further improve meso-2,3-BD production. In a fed-batch fermentation, meso-2,3-BD titer reached 98.0 g/L with a purity of >99.0 % and a productivity of 0.94 g/L–h.ConclusionsThis work demonstrates the potential of producing meso-2,3-BD with high titer and purity through metabolic engineering of B. licheniformis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0522-1) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 64%

“…Its catalytic activity on glycerol was only around 5 % of that for D -2,3-BD. The preference on D -2,3-BD as the substrate by GDH was also reported in Hansenula polymorpha [ 23 ]. These results indicated that the GDH from B. licheniformis WX-02 possessed a substrate specific catalytic activity towards D -2,3-BD.…”

Section: Resultsmentioning

confidence: 68%

Engineering Bacillus licheniformis for the production of meso-2,3-butanediol

Qiu

Zhang

et al. 2016

Biotechnol Biofuels

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“…Glycerol is a vicinal diol with a similar structure to 2,3-BD except the first hydroxyl group. It was reported that GDH in O. angusta had the ability to catalyze interconversion between (3 R )-AC and (2 R ,3 R )-2,3-BD and this enzyme showed greater activity towards (2 R ,3 R )-2,3-BD than towards glycerol [25]. This indicated that GDH might play a role in 2,3-BD formation.…”

Section: Discussionmentioning

confidence: 97%

“…These enzymes exhibited the abilities in interconversion of (3 S )-AC/ meso -2,3-BD and (3 R )-AC/(2 R ,3 R )-2,3-BD [19,20,24]. On the other hand, it was reported that glycerol dehydrogenase (GDH) in Ogataea angusta could catalyze interconversion between (3 R )-AC and (2 R ,3 R )-2,3-BD [25]. It might be another enzyme for 2,3-BD production.…”

Section: Introductionmentioning

confidence: 99%

A newly isolated Bacillus licheniformisstrain thermophilically produces 2,3-butanediol, a platform and fuel bio-chemical

Zhang

et al. 2013

Biotechnol Biofuels

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Background2,3-Butanediol (2,3-BD), a platform and fuel bio-chemical, can be efficiently produced by Klebsiella pneumonia, K. oxytoca, and Serratia marcescens. However, these strains are opportunistic pathogens and not favorable for industrial application. Although some generally regarded as safe (GRAS) microorganisms have been isolated in recent years, there is still a demand for safe 2,3-BD producing strains with high productivity and yield under thermophilic fermentation.ResultsBacillus licheniformis strain 10-1-A was newly isolated for 2,3-BD production. The optimum temperature and medium pH were 50°C and pH 7.0 for 2,3-BD production by strain 10-1-A. The medium composition was optimized through Plackett–Burman design and response surface methodology techniques. With a two-stage agitation speed control strategy, 115.7 g/L of 2,3-BD was obtained from glucose by fed-batch fermentation in a 5-L bioreactor with a high productivity (2.4 g/L·h) and yield (94% of its theoretical value). The 2,3-BD produced by strain 10-1-A comprises (2R,3R)-2,3-BD and meso-2,3-BD with a ratio of nearly 1:1. The bdh and gdh genes encoding meso-2,3-butanediol dehydrogenase (meso-BDH) and glycerol dehydrogenase (GDH) of strain 10-1-A were expressed in Escherichia coli and the proteins were purified. meso-2,3-BD and (2R,3R)-2,3-BD were transformed from racemic acetoin by meso-BDH and GDH with NADH, respectively.ConclusionsCompared with the reported GRAS 2,3-BD producers, B. licheniformis 10-1-A could thermophilically produce 2,3-BD with a high concentration, productivity and yield. Thus, the newly isolated GRAS strain 10-1-A might be a promising strain for industrial production of 2,3-BD. Two key enzymes for meso-2,3-BD and (2R,3R)-2,3-BD production were purified and further studied, and this might be helpful to understand the mechanism for 2,3-BD stereoisomers forming in B. licheniformis.

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“…As such, we presently remain unable to comment on the identity and activity of these specific gene products. It is worth noting that 2,3-BD dehydrogenase activity has previously been demonstrated by glycerol dehydrogenase of the yeast Hansenula polymorpha (Pichia angusta) [29,30]. Whereas E. coli also possesses a glycerol dehydrogenase (encoded by gldA), its substrate specificity with respect to the reduction of acetoin to meso-2,3-BD has not yet been examined.…”

Section: Resultsmentioning

confidence: 99%

Metabolic engineering of acetoin and meso‐2, 3‐butanediol biosynthesis in E. coli

Nielsen

Yoon

Yuan

et al. 2010

Biotechnology Journal

108

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The functional reconstruction of acetoin and meso-2,3-butanediol (meso-2,3-BD) biosynthetic pathways in Escherichia coli have been explored systematically. Pathway construction involved the in vivo screening of prospective pathway isozymes of yeast and bacterial origin. After substantial engineering of the host background to increase pyruvate availability, E. coli YYC202(DE3) ldhA − ilvC − expressing ilvBN from E. coli and aldB from L. lactis (encoding acetolactate synthase and acetolactate decarboxylase activities, respectively) was able to produce up to 870 mg/L acetoin, with no coproduction of diacetyl observed. These strains were also found to produce small quantities of meso-2,3-BD, suggesting the existence of endogenous 2,3-BD dehydrogenase activity. Finally, the coexpression of bdh1 from S. cerevisiae, encoding 2,3-BD dehydrogenase, in this strain resulted in the production of up to 1120 mg/L meso-2,3-BD, with glucose a yield of 0.29 g/g. While disruption of the native lactate biosynthesis pathway increased pyruvate precursor availability to this strain, increased availability of NADH for acetoin reduction to meso-2,3-BD was found to be the most important consequence of ldhA deletion.

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Characterisation of Glycerol Dehydrogenase from a Methylotrophic Yeast, Hansenula polymorpha Dl-1, and its Gene Cloning

Cited by 23 publications

References 0 publications

Engineering Bacillus licheniformis for the production of meso-2,3-butanediol

Engineering Bacillus licheniformis for the production of meso-2,3-butanediol

A newly isolated Bacillus licheniformisstrain thermophilically produces 2,3-butanediol, a platform and fuel bio-chemical

Metabolic engineering of acetoin and meso‐2, 3‐butanediol biosynthesis in E. coli

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