Three-steps in one-pot: whole-cell biocatalytic synthesis of enantiopure (+)- and (−)-pinoresinol via kinetic resolution

Additional file 5. HPLC chromatograms of enantiomeric separations of reaction products. a Application of AtPrR2; b application of FiPLR. [3a] = (+)-pinoresinol 3a, [3b] = (−)-pinoresinol 3b, [4a] = (+)-lariciresinol 4a, [4b] = (−)-lariciresinol 4b, [5a] = (−)-secoisolariciresinol 5a

P450BM3-Catalyzed Oxidations Employing Dual Functional Small Molecules

A set of dual functional small molecules (DFSMs) containing different amino acids has been synthesized and employed together with three different variants of the cytochrome P450 monooxygenase P450BM3 from Bacillus megaterium in H2O2-dependent oxidation reactions. These DFSMs enhance P450BM3 activity with hydrogen peroxide as an oxidant, converting these enzymes into formal peroxygenases. This system has been employed for the catalytic epoxidation of styrene and in the sulfoxidation of thioanisole. Various P450BM3 variants have been evaluated in terms of activity and selectivity of the peroxygenase reactions.MINECO-CTQ2016-76908-C2-1,2-PComisión Europea de Investigación-ERC-648026Unión Europea-H2020-BBI-PPP-2015-2-1-720297Organización Holandesa de Investigación Científica (VICI)-724.014.00

Regioselective biooxidation of (+)-valencene by recombinant E. coli expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system

<p>Abstract</p> <p>Background</p> <p>(+)-Nootkatone (<b>4</b>) is a high added-value compound found in grapefruit juice. Allylic oxidation of the sesquiterpene (+)-valencene (<b>1</b>) provides an attractive route to this sought-after flavoring. So far, chemical methods to produce (+)-nootkatone (<b>4</b>) from (+)-valencene (<b>1</b>) involve unsafe toxic compounds, whereas several biotechnological approaches applied yield large amounts of undesirable byproducts. In the present work 125 cytochrome P450 enzymes from bacteria were tested for regioselective oxidation of (+)-valencene (<b>1</b>) at allylic C2-position to produce (+)-nootkatone (<b>4</b>) via <it>cis</it>- (<b>2</b>) or <it>trans</it>-nootkatol (<b>3</b>). The P450 activity was supported by the co-expression of putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from <it>Pseudomonas putida </it>in <it>Escherichia coli</it>.</p> <p>Results</p> <p>Addressing the whole-cell system, the cytochrome CYP109B1 from <it>Bacillus subtilis </it>was found to catalyze the oxidation of (+)-valencene (<b>1</b>) yielding nootkatol (<b>2 </b>and <b>3</b>) and (+)-nootkatone (<b>4</b>). However, when the <it>in vivo </it>biooxidation of (+)-valencene (<b>1</b>) with CYP109B1 was carried out in an aqueous milieu, a number of undesired multi-oxygenated products has also been observed accounting for approximately 35% of the total product. The formation of these byproducts was significantly reduced when aqueous-organic two-liquid-phase systems with four water immiscible organic solvents – isooctane, <it>n</it>-octane, dodecane or hexadecane – were set up, resulting in accumulation of nootkatol (<b>2 </b>and <b>3</b>) and (+)-nootkatone (<b>4</b>) of up to 97% of the total product. The best productivity of 120 mg l^-1of desired products was achieved within 8 h in the system comprising 10% dodecane.</p> <p>Conclusion</p> <p>This study demonstrates that the identification of new P450s capable of producing valuable compounds can basically be achieved by screening of recombinant P450 libraries. The biphasic reaction system described in this work presents an attractive way for the production of (+)-nootkatone (<b>4</b>), as it is safe and can easily be controlled and scaled up.</p

Spotlight on CYP4B1

The mammalian cytochrome P450 monooxygenase CYP4B1 can bioactivate a wide range of xenobiotics, such as its defining/hallmark substrate 4-ipomeanol leading to tissue-specific toxicities. Similar to other members of the CYP4 family, CYP4B1 has the ability to hydroxylate fatty acids and fatty alcohols. Structural insights into the enigmatic role of CYP4B1 with functions in both, xenobiotic and endobiotic metabolism, as well as its unusual heme-binding characteristics are now possible by the recently solved crystal structures of native rabbit CYP4B1 and the p.E310A variant. Importantly, CYP4B1 does not play a major role in hepatic P450-catalyzed phase I drug metabolism due to its predominant extra-hepatic expression, mainly in the lung. In addition, no catalytic activity of human CYP4B1 has been observed owing to a unique substitution of an evolutionary strongly conserved proline 427 to serine. Nevertheless, association of CYP4B1 expression patterns with various cancers and potential roles in cancer development have been reported for the human enzyme. This review will summarize the current status of CYP4B1 research with a spotlight on its roles in the metabolism of endogenous and exogenous compounds, structural properties, and cancer association, as well as its potential application in suicide gene approaches for targeted cancer therapy