GLORIA

GEOMAR Library Ocean Research Information Access

X

Your email was sent successfully. Check your inbox.

An error occurred while sending the email. Please try again.

Proceed reservation?

Export
Filter
  • Braun, Andreas  (3)
  • 2010-2014  (3)
  • 1
    Online Resource
    Online Resource
    Steroid biotransformations in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes
    Springer Science and Business Media LLC ; 2012
    In:  Microbial Cell Factories Vol. 11, No. 1 ( 2012-12)
    Details
    In: Microbial Cell Factories, Springer Science and Business Media LLC, Vol. 11, No. 1 ( 2012-12)
    Abstract: Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase. Results For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems. Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system. Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains. Conclusions Alkane-assimilating yeast Y. lipolytica , coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared to aqueous systems and even enable simple, continuous or at least high yield long time processes.
    Type of Medium: Online Resource
    ISSN: 1475-2859
    URL: Article
    DOI: 10.1186/1475-2859-11-106
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2012
    detail.hit.zdb_id: 2091377-1
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 2
    Online Resource
    Online Resource
    Production of human cytochrome P450 2D6 drug metabolites with recombinant microbes – a comparative study
    Wiley ; 2012
    In:  Biotechnology Journal Vol. 7, No. 11 ( 2012-11), p. 1346-1358
    Details
    In: Biotechnology Journal, Wiley, Vol. 7, No. 11 ( 2012-11), p. 1346-1358
    Abstract: The processes of drug development require efficient strategies to produce the respective drug metabolites, which are often difficult to obtain. Biotransformations employing recombinant microorganisms as whole‐cell biocatalysts have become an attractive alternative to the chemical syntheses of such metabolites. For the first time, the potential of four different microbial systems expressing the human cytochrome P450 2D6 (CYP2D6), which is one of the most important drug‐metabolizing enzymes, were compared and evaluated for such applications. The microbial host Pichia pastoris was the most efficient at expressing CYP2D6. Without additional over‐expression of chaperons, the achieved yield of CYP2D6 was the highest of microbial hosts reported so far. Therefore, the system described in this study outperformed the previously reported expression of the N‐terminally modified enzyme. It was also shown that the activities of the whole‐cell conversions of bufuralol in recombinant P. pastoris were significantly higher than the Escherichia coli catalyst, which expressed the same unmodified gene.
    Type of Medium: Online Resource
    ISSN: 1860-6768 , 1860-7314
    URL: Issue
    URL: Article
    DOI: 10.1002/biot.v7.11
    DOI: 10.1002/biot.201200187
    Language: English
    Publisher: Wiley
    Publication Date: 2012
    detail.hit.zdb_id: 2214038-4
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 3
    Online Resource
    Online Resource
    Double site saturation mutagenesis of the human cytochrome P450 2D6 results in regioselective steroid hydroxylation
    Wiley ; 2013
    In:  The FEBS Journal Vol. 280, No. 13 ( 2013-07), p. 3094-3108
    Details
    In: The FEBS Journal, Wiley, Vol. 280, No. 13 ( 2013-07), p. 3094-3108
    Abstract: The human cytochrome P450 2D6 ( CYP 2D6) is one of the major human drug metabolizing enzymes and acts preferably on substrates containing a basic nitrogen atom. Testosterone − just as other steroids − is an atypical substrate and only poorly metabolized by CYP 2D6. The present study intended to investigate the influence of the two active site residues 216 and 483 on the capability of CYP 2D6 to hydroxylate steroids such as for example testosterone. All 400 possible combinatorial mutations at these two positions have been generated and expressed individually in P ichia pastoris . Employing whole‐cell biotransformations coupled with HPLC ‐ MS analysis the testosterone hydroxylase activity and regioselectivity of every single CYP 2D6 variant was determined. Covering the whole sequence space, CYP 2D6 variants with improved activity and so far unknown regio‐preference in testosterone hydroxylation were identified. Most intriguingly and in contrast to previous literature reports about mutein F483I, the mutation F483G led to preferred hydroxylation at the 2β‐position, while the slow formation of 6β‐hydroxytestosterone, the main product of wild‐type CYP 2D6, was further reduced. Two point mutations have already been sufficient to convert CYP 2D6 into a steroid hydroxylase with the highest ever reported testosterone hydroxylation rate for this enzyme, which is of the same order of magnitude as for the conversion of the standard substrate bufuralol by wild‐type CYP 2D6. Furthermore, this study is also an example for efficient human CYP engineering in P . pastoris for biocatalytic applications and to study so far unknown pharmacokinetic effects of individual and combined mutations in these key enzymes of the human drug metabolism.
    Type of Medium: Online Resource
    ISSN: 1742-464X , 1742-4658
    URL: Issue
    URL: Article
    DOI: 10.1111/febs.2013.280.issue-13
    DOI: 10.1111/febs.12270
    Language: English
    Publisher: Wiley
    Publication Date: 2013
    detail.hit.zdb_id: 2172518-4
    SSG: 12
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
Close ⊗
This website uses cookies and the analysis tool Matomo. More information can be found here...