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

Recent advances in oxygenase-catalyzed biotransformations

Oxygenases continue to be widely studied for selective biooxidation of organic compounds. Protein engineering has resulted in heme and flavin monooxygenases with widely altered substrate specificities, and attempts have been reported to scale up reactions catalyzed by these enzymes. Cofactor regeneration is still a key issue in these developments. Protein engineering contributed to understanding of structure vs. function in dioxygenases

Biotransformations using prokaryotic P450 monooxygenases

Recent studies on microbial cytochrome P450 enzymes cover several new areas. Advances have been made in structure-function analysis. New non-enzymatic/electrochemical systems for the replacement of NAD(P)H have been developed. The properties of some enzymes have been re-engineered by site-directed mutagenesis or by methods of directed evolution. New P450s have been functionally expressed and characterized. A combination of these approaches is believed to facilitate the use of isolated P450 monooxygenases in biocatalysis

Protein engineering of the cytochrome P450 monooxygenase from bacillus megaterium

The role and importance of cytochrome P450 enzymes (CYP) in drug development, biodegradation processes and biocatalysis has been widely acknowledged. P450 monooxygenases exhibit an extremely wide substrate spectrum which is the basis of their ability to activate or detoxify a large variety of target molecules. P450 monooxygenases have been isolated from bacteria, yeasts, insects, as well as mammalian and plant tissues. Currently, the enzyme family is one of the best known gene subfamilies with over 1000 characterized members (http://drnelson.utmem.edu/CytochromeP450.html). Many studies have been dedicated to structural models of cytochrome P450 in order to improve our understanding of the mechanistic details of the enzymes, their substrate specificity and their pronounced stereo- and regiospecificity. In addition, homology modeling of mammalian P450s and QSAR analyses using chemicals which are metabolized by P450s, have added considerably to our understanding of the metabolic variations and functions of the enzyme. Cytochrome P450 enzymes are of considerable interest to pharmaceutical and chemical industry and have thus become targets for protein engineering approaches. Protein engineering is generally defined as the modification of an enzyme by site-directed or random mutagenesis with the aim of altering its properties. Rational design requires a solid structural basis and profound knowledge of the catalytic mechanism of the enzyme which was provided by determining the structures of CYPs using X-ray crystallography at high-resolution. Ten of the twelve crystallized cytochrome P450s are of prokaryotic origin and water-soluble. From a technical point of view, microbial P450s are easier to handle than P450 enzymes from plants and animals. They are not membrane-associated and exhibit a relatively high stability. Eukaryotic cytochrome P450 enzymes are membrane-associated proteins and are hence more difficult to crystallize. Currently, only the X-ray structures of two membrane-bound mammalian P450s, rabbit CYP2C5 and human CYP2C9 are known. Models of other mammalian P450s were built based on the structure of CYP2C5 and its bacterial analogues. P450cam, the cytochrome P450 monooxygenase from Pseudomonas putida, is the best characterized microbial P450 enzyme. In the last fifteen years, a large number of other soluble prokaryotic P450 enzymes have been identified, isolated, subcloned in Escherichia coli, overexpressed and characterized. Cytochrome P450 BM-3 from Bacillus megaterium is catalytically self-sufficient. It contains a P450-heme domain of 54 kDa and an FAD/FMN-reductase domain of 64kDa on a single polypeptide chain. The enzyme catalyzes the subterminal oxidation of saturated and unsaturated fatty acids with a chain length of 12 to 20 carbons. High-resolution X-ray crystal structures are available for substrate-free, palmitic acid-bound and N-palmitoylglycine-bound wild-type and mutant P450 BM-3 enzymes. The structure resolved by NMR is also available. The well-known structure, the availability of the CYP102A1 gene which encodes the protein, and the possibility of expressing the protein in E. coli have encouraged a number of research groups to undertake site-directed mutagenesis studies in order to identify key amino acids. Insights into the mechanisms of P450 BM-3 have been gained and transferred to eukaryotic P450 enzymes. Since P450 BM-3 is an excellent model for addressing questions on the wide substrate specificity of P450s in general and techniques involving the mutagenesis of P450 BM-3 have led to a variety of biocatalysts with features of industrial interests. This review will summarize the recent research on this particular P450 enzyme

Effect of detergents, salts and organic co-solvents on Ssl1.

<p>A: Relative activity of Ssl1 in presence of organic solvents, salts and detergents towards 2,6-dimethoxyphenol oxidation. Water immiscible solvents did not affect Ssl1 activity, with water miscible co-solvents the activity dropped to 20–40%. Ssl1 tolerated the addition of 10 mM sodium azide, with the detergents SDS and Triton-X-100 the activity was reduced to 60 or 80%. B: Residual activity of Ssl1 after 20 h incubation with organic solvents, salts and deteregents. DMSO stabilized Ssl1 whereas acetonitrile lead to a reduction in residual activity. All other studied additives did not lead to major changes of Ssl1 stability.</p