Identification and Characterization of a Novel N- and O-Glycosyltransferase from Saccharopolyspora erythraea
Abstract
:1. Introduction
2. Results
2.1. In Vivo Biotransformation of Anthraquinone Derivatives
2.2. Genes Encoding for Glycosyltransferases in the Genome of S. erythraea
2.3. Heterologous Expression of Sace_3599 in S. albus Gluc
2.4. Gene Deletion and Complementation Experiments
2.5. Structure Elucidation of U3G and U3DG
2.6. Production, Purification and In Vitro Activity Assay of Sace_3599
2.7. Genome Sequence Analysis
2.8. Homologous Proteins to Sace_3599
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains and Growth Conditions
4.2. Plasmid Construction
4.3. Whole-Cell Feeding Experiments
4.4. Heterologous Test System
4.5. Inactivation of Sace_3599 in the Genome of S. erythraea
4.6. Complementation of S. erythraea Δsace_3599 with an Intact Copy of Sace_3599
4.7. Purification of Biotransformation Products
4.8. Sample Analysis by HPLC-MS
4.9. Structure Elucidation Using NMR Spectroscopy
4.10. Separation of Mono-Glucosylated Products Using Capillary Electrophoresis
4.11. Expression of Sace_3599 in E. coli and Purification of the Protein
4.12. In Vitro Glycosylation Activity Assay
4.12.1. Variation of Time and Temperature
4.12.2. Variation of Sugar Donor
4.12.3. Variation of Buffer
4.12.4. Variation of Metal Ions as Putative Cofactors
4.12.5. Variation of Substrate
4.12.6. Calculation of Conversion of Substrate to Glucosylated Product
4.13. In Vitro Glycohydrolase Assay
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample Availability: Samples of the compounds are available from the authors. |
Strain or Plasmid | Description 1 | Source or Reference (s) |
---|---|---|
Strains | ||
S. albus J1074 | WT strain, heterologous host | [58] |
S. albus Gluc | WT expressing the biosynthetic gene for dTDP-d-glucose (oleS) | This study |
S. albus Gluc x pUWL-A-sace_3599 | S. albus Gluc expressing the gene sace_3599 | This study |
S. erythraea NRRL2338 | WT strain, biotransformation host | [57] |
S. erythraea Δsace_3599 | WT with deletion of the gene sace_3599 | This study |
S. erythraea Δsace_3599 x pTOS(z)-sace_3599 | Deletion mutant of sace_3599 complemented with sace_3599 | This study |
E. coli Turbo | General cloning host | NEB, Frankfurt am Main, Germany |
E. coli ET12567 x pUZ8002 | Strain for intergeneric conjugation | [64,65] |
E. coli Rosetta™ 2 | Heterologous expression host containing seven tRNAs for rarely used codons | Novagen, Darmstadt, Germany |
Plasmids | ||
pTOS-Rham | pTOS derivative with oleS, oleE, oleL, and oleU under ermE* promoter | [66] |
pTOS-Gluc | pTOS derivative with oleS under ermE* promoter | This study |
pUWL-A [pUWL-oriT-aac(3)IV] | replicative vector for actinomycetes; oriT, bla, aac(3)IV, ermE (pUWL201) | [67] |
pUWL-A-sace_1884 | pUWL-A derivative with sace_1884 under ermE promoter | This study |
pUWL-A-sace_3599 | pUWL-A derivative with sace_3599 under ermE promoter | This study |
pUWL-A-sace_4470 | pUWL-A derivative with sace_4470 under ermE promoter | This study |
pUWL-Dre | Replicative vector for actinomycetes; oriT, bla, tsr, and dre gene under ermE promoter (pUWL201) | [61] |
pKC1132 | Replicative vector in E. coli, non-replicative in actinomycetes; lacZa, aac(3)IV, and oriT | [68] |
pLERE-spec-oriT | Cloning vector with bla, aadA, and oriT flanked by two loxLE sites and two loxRE sites | [66] |
pKCΔsace_3599 | Vector for deletion of sace_3599, based on pKC1132 | This study |
pTOS(z) | integrative vector, containing oriT, int, and attP (VWB), aac(3)IV, lacZ gene, and ermEp1* promoter | [69] |
pTOS(z)-sace_3599 | pTOS(z) derivative with sace_3599 | This study |
pBluescript II SK(−) | cloning vector for E. coli, with a MCS (SacI to KpnI) flanked by T3 and T7 RNA polymerase promoters; lacZ gene, lac promoter, pUC origin, bla | Agilent Technologies, Santa Clara, CA, USA |
pET28a(+) | Expression vector with aphII and N-terminal hexahistidine affinity tag | Novagen, Darmstadt, Germany |
pET28a-sace_3599-N-his6 | pET28a(+) derivative for expression of sace_3599 | This study |
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Gutacker, F.; Schmidt-Bohli, Y.-I.; Strobel, T.; Qiu, D.; Jessen, H.; Paululat, T.; Bechthold, A. Identification and Characterization of a Novel N- and O-Glycosyltransferase from Saccharopolyspora erythraea. Molecules 2020, 25, 3400. https://doi.org/10.3390/molecules25153400
Gutacker F, Schmidt-Bohli Y-I, Strobel T, Qiu D, Jessen H, Paululat T, Bechthold A. Identification and Characterization of a Novel N- and O-Glycosyltransferase from Saccharopolyspora erythraea. Molecules. 2020; 25(15):3400. https://doi.org/10.3390/molecules25153400
Chicago/Turabian StyleGutacker, Fabienne, Yvonne-Isolde Schmidt-Bohli, Tina Strobel, Danye Qiu, Henning Jessen, Thomas Paululat, and Andreas Bechthold. 2020. "Identification and Characterization of a Novel N- and O-Glycosyltransferase from Saccharopolyspora erythraea" Molecules 25, no. 15: 3400. https://doi.org/10.3390/molecules25153400