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

Heterologous Lignan Production in Stirred-Tank Reactors—Metabolomics-Assisted Bioprocess Development for an In Vivo Enzyme Cascade

Towards establishing a prospective industrial microbial lignan production process, we set up and investigated the biotransformation of coniferyl alcohol to secoisolariciresinol with recombinant Escherichia coli in a stirred-tank reactor (STR). Initially, we tested different cofactor concentrations and antifoam additions in shake flasks. Next, we designed an STR batch bioprocess and tested aeration rates, pH regulation, and substrate-feeding strategies. Targeted metabolomics of phenylpropanoids and lignans assisted the bioprocess development by monitoring the lignan pathway activity. We found that the copper concentration and the substrate-feeding strategy had considerable impact on lignan production. Furthermore, time-resolved monitoring of pathway metabolites revealed two maximal intracellular lignan concentrations, the first shortly after induction of gene expression and the second after the cells entered the stationary growth phase. During STR cultivation, a maximal intracellular titer of 130.4 mg L−1 secoisolariciresinol was achieved, corresponding to a yield coefficient of 26.4 mg g−1 and a space–time yield of 2.6 mg L−1 h−1. We report for the first time the in-depth evaluation of microbially produced lignans in a well-controlled STR bioprocess. Monitoring of the lignan pathway activity showed that lignan accumulation is highly dynamic during the cultivation and points towards the need for a more efficient coniferyl alcohol dimerization system for optimal microbial production conditions

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.BT/Biocatalysi

Structural insights into oxidation of medium-chain fatty acids and flavanone by myxobacterial cytochrome P450 CYP267B1

Oxidative biocatalytic reactions performed by cytochrome P450 enzymes (P450s) are of high interest for the chemical and pharmaceutical industries. CYP267B1 is a P450 enzyme from myxobacterium Sorangium cellulosum So ce56 displaying a broad substrate scope. In this work, a search for new substrates was performed, combined with product characterization, and a structural analysis of substrate-bound complexes using X-ray crystallography and computational docking. The results demonstrate the ability of CYP267B1 to perform in-chain hydroxylations of medium-chain saturated fatty acids (decanoic acid, dodecanoic acid and tetradecanoic acid) and a regioselective hydroxylation of flavanone. The fatty acids are mono-hydroxylated at different in-chain positions, with decanoic acid displaying the highest regioselectivity towards ω-3 hydroxylation. Flavanone is preferably oxidized to 3-hydroxyflavanone. High-resolution crystal structures of CYP267B1 revealed a very spacious active site pocket, similarly to other P450s able to convert macrocyclic compounds. The pocket becomes more constricted near to the heme, and is closed off from solvent by residues of the FG helices and the B-C loop. The crystal structure of the tetradecanoic acid-bound complex displays the fatty acid bound near to the heme, but in a non-productive conformation. Molecular docking allowed modeling of the productive binding modes for the four investigated fatty acids and flavanone, as well as of two substrates identified in a previous study (diclofenac and ibuprofen), explaining the observed product profiles. The obtained structures of CYP267B1 thus serve as a valuable prediction tool for substrate hydroxylations by this highly versatile enzyme and will encourage future selectivity changes by rational protein engineering