Dataset Information

Draft Genome Sequence of Gluconobacter oxydans NL71, a Strain That Efficiently Biocatalyzes Xylose to Xylonic Acid at a High Concentration.

ABSTRACT: Gluconobacter oxydans NL71, a selected strain in the crude lignocellulosic hydrolysate, catalyzed 600 g/liter xylose to 586.3 g/liter xylonic acid at 95.1% yield. The biocatalysis of xylose yielded three times higher than the best previous output, providing a possibility of the industrial scale utilization of lignocellulosic xylose. Due to its promising industrial applications, we sequenced the complete genome of strain G. oxydans NL71 to further our understanding of its overall metabolism.

SUBMITTER: Miao Y

PROVIDER: S-EPMC4472894 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Draft Genome Sequence of Gluconobacter oxydans NL71, a Strain That Efficiently Biocatalyzes Xylose to Xylonic Acid at a High Concentration.

Miao Yuanyuan Y Zhou Xin X Xu Yong Y Yu Shiyuan S

Genome announcements 20150618 3

Gluconobacter oxydans NL71, a selected strain in the crude lignocellulosic hydrolysate, catalyzed 600 g/liter xylose to 586.3 g/liter xylonic acid at 95.1% yield. The biocatalysis of xylose yielded three times higher than the best previous output, providing a possibility of the industrial scale utilization of lignocellulosic xylose. Due to its promising industrial applications, we sequenced the complete genome of strain G. oxydans NL71 to further our understanding of its overall metabolism. ...[more]

PMID: 26089417

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Project description:The acetic acid bacterium (AAB) Gluconobacter oxydans incompletely oxidizes a wide variety of carbohydrates and is therefore used industrially for oxidative biotransformations. For G. oxydans, no system was available that allows regulatable plasmid-based expression. We found that the L-arabinose-inducible PBAD promoter and the transcriptional regulator AraC from Escherichia coli MC4100 performed very well in G. oxydans. The respective pBBR1-based plasmids showed very low basal expression of the reporters ?-glucuronidase and mNeonGreen, up to 480-fold induction with 1% L-arabinose, and tunability from 0.1 to 1% L-arabinose. In G. oxydans 621H, L-arabinose was oxidized by the membrane-bound glucose dehydrogenase, which is absent in the multi-deletion strain BP.6. Nevertheless, AraC-PBAD performed similar in both strains in the exponential phase, indicating that a gene knockout is not required for application of AraC-PBAD in wild-type G. oxydans strains. However, the oxidation product arabinonic acid strongly contributed to the acidification of the growth medium in 621H cultures during the stationary phase, which resulted in drastically decreased reporter activities in 621H (pH 3.3) but not in BP.6 cultures (pH 4.4). These activities could be strongly increased quickly solely by incubating stationary cells in D-mannitol-free medium adjusted to pH 6, indicating that the reporters were hardly degraded yet rather became inactive. In a pH-controlled bioreactor, these reporter activities remained high in the stationary phase (pH 6). Finally, we created a multiple cloning vector with araC-PBAD based on pBBR1MCS-5. Together, we demonstrated superior functionality and good tunability of an AraC-PBAD system in G. oxydans that could possibly also be used in other AAB. KEY POINTS: • We found the AraC-PBAD system from E. coli MC4100 was well tunable in G. oxydans. •??In the absence of AraC or l-arabinose, expression from PBAD was extremely low. •?This araC-PBAD system could also be fully functional in other acetic acid bacteria.

Project description:For the acetic acid bacterium (AAB) Gluconobacter oxydans only recently the first tight system for regulatable target gene expression became available based on the heterologous repressor-activator protein AraC from Escherichia coli and the target promoter ParaBAD. In this study, we tested pure repressor-based TetR- and LacI-dependent target gene expression in G. oxydans by applying the same plasmid backbone and construction principles that we have used successfully for the araC-ParaBAD system. When using a pBBR1MCS-5-based plasmid, the non-induced basal expression of the Tn10-based TetR-dependent expression system was extremely low. This allowed calculated induction ratios of up to more than 3500-fold with the fluorescence reporter protein mNeonGreen (mNG). The induction was highly homogeneous and tunable by varying the anhydrotetracycline concentration from 10 to 200 ng/mL. The already strong reporter gene expression could be doubled by inserting the ribosome binding site AGGAGA into the 3' region of the Ptet sequence upstream from mNG. Alternative plasmid constructs used as controls revealed a strong influence of transcription terminators and antibiotics resistance gene of the plasmid backbone on the resulting expression performance. In contrast to the TetR-Ptet-system, pBBR1MCS-5-based LacI-dependent expression from PlacUV5 always exhibited some non-induced basal reporter expression and was therefore tunable only up to 40-fold induction by IPTG. The leakiness of PlacUV5 when not induced was independent of potential read-through from the lacI promoter. Protein-DNA binding simulations for pH 7, 6, 5, and 4 by computational modeling of LacI, TetR, and AraC with DNA suggested a decreased DNA binding of LacI when pH is below 6, the latter possibly causing the leakiness of LacI-dependent systems hitherto tested in AAB. In summary, the expression performance of the pBBR1MCS-5-based TetR-Ptet system makes this system highly suitable for applications in G. oxydans and possibly in other AAB. KEY POINTS: • A pBBR1MCS-5-based TetR-Ptet system was tunable up to more than 3500-fold induction. • A pBBR1MCS-5-based LacI-PlacUV5 system was leaky and tunable only up to 40-fold. • Modeling of protein-DNA binding suggested decreased DNA binding of LacI at pH < 6.

Dataset Information

Draft Genome Sequence of Gluconobacter oxydans NL71, a Strain That Efficiently Biocatalyzes Xylose to Xylonic Acid at a High Concentration.

Publications

Draft Genome Sequence of Gluconobacter oxydans NL71, a Strain That Efficiently Biocatalyzes Xylose to Xylonic Acid at a High Concentration.

Similar Datasets

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