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Light‐Dependent and Aeration‐Independent Gram‐Scale Hydroxylation of Cyclohexane to Cyclohexanol by CYP450 Harboring Synechocystis sp. PCC 6803
Author(s) -
Hoschek Anna,
Toepel Jörg,
Hochkeppel Adrian,
Karande Rohan,
Bühler Bruno,
Schmid Andreas
Publication year - 2019
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201800724
Subject(s) - chemistry , substrate (aquarium) , hydroxylation , synechocystis , biotransformation , monooxygenase , photochemistry , organic chemistry , biochemistry , biology , cytochrome p450 , enzyme , mutant , ecology , gene
Oxygenase‐containing cyanobacteria constitute promising whole‐cell biocatalysts for oxyfunctionalization reactions. Photosynthetic water oxidation thereby delivers the required cosubstrates, that is activated reduction equivalents and O 2 , sustainably. A recombinant Synechocystis sp. PCC 6803 strain showing unprecedentedly high photosynthesis‐driven oxyfunctionalization activities is developed, and its technical applicability is evaluated. The cells functionally synthesize a heterologous cytochrome P450 monooxygenase enabling cyclohexane hydroxylation. The biocatalyst‐specific reaction rate is found to be light‐dependent, reaching 26.3 ± 0.6 U g CDW −1 (U = μmol min −1 and cell dry weight [CDW]) at a light intensity of 150 µmol photons m −2 s −1 . In situ substrate supply via a two‐liquid phase system increases the initial specific activity to 39.2 ± 0.7 U g CDW −1 and stabilizes the biotransformation by preventing cell toxification. This results in a tenfold increased specific product yield of 4.5 g cyclohexanol g CDW −1 as compared to the single aqueous phase system. Subsequently, the biotransformation is scaled from a shake flask to a 3 L stirred‐tank photobioreactor setup. In situ O 2 generation via photosynthetic water oxidation allows a nonaerated process operation, thus circumventing substrate evaporation as the most critical factor limiting the process performance and stability. This study for the first time exemplifies the technical applicability of cyanobacteria for aeration‐independent light‐driven oxyfunctionalization reactions involving highly toxic and volatile substrates.