Selective hydroxylation of highly branched fatty acids and their derivatives by CYP102A1 from Bacillus megaterium

Highly branched fatty acids, the main components of the preen-gland waxes of the domestic goose and the Muscovy duck, and their derivatives are promising chiral precursors for the synthesis of macrolide antibiotics. The key step in the utilisation of these compounds is their regioselective hydroxylation, which cannot be achieved in a classical chemical approach. Three P450 monooxygenases, CYP102A1, CYP102A2 and CYP102A3, demonstrating high turnover numbers in the hydroxylation of iso and anteiso fatty acids (>400 min(-1)), were tested for their activity towards these substrates. CYP102A1 from Bacillus megaterium and its A74G F87V L188Q triple mutant hydroxylate a variety of these substrates with high activity and regioselectivity. In all cases, the triple mutant showed much higher activities than the wild-type enzyme. The binding constants, determined for wild-type CYP102A1 and the triple mutant with tetramethylnonanol as substrate, were >200 microM and approximately 23 microM, respectively. Data derived from binding analysis support the differences in activity found for the wild-type CYP102A1 and the triple mutant. Surprisingly, CYP102A2 and CYP102A3 from Bacillus subtilis did not show any activity. Substrate binding spectra, recorded to investigate substrate accessibility to the enzyme's active sites, revealed that the substrates either could not access the active site of the Bacillus subtilis monooxygenases, or did not come into proximity with the heme.     

Regioselectivity and Activity of Cytochrome P450 BM‐3 and Mutant F87A in Reactions Driven by Hydrogen Peroxide

Cytochrome P450 BM‐3 (EC 1.14.14.1) is a monooxygenase that utilizes NADPH and dioxygen to hydroxylate fatty acids at subterminal positions. The enzyme is also capable of functioning as a peroxygenase in the same reaction, by utilizing hydrogen peroxide in place of the reductase domain, cofactor and oxygen. As a starting point for developing a practically useful hydroxylation biocatalyst, we compare the activity and regioselectivity of wild‐type P450 BM‐3 and its F87A mutant on various fatty acids. Neither enzyme catalyzes terminal hydroxylation under any of the conditions studied. While significantly enhancing peroxygenase activity, the F87A mutation also shifts hydroxylation further away from the terminal position. The H₂O₂‐driven reactions with either the full‐length BM‐3 enzyme or the heme domain are slow, but yield product distributions very similar to those generated when using NADPH and O₂.     

An efficient route to selective bio-oxidation catalysts: an iterative approach comprising modeling, diversification, and screening, based on CYP102A1

Perillyl alcohol is the terminal hydroxylation product of the cheap and readily available terpene, limonene. It has high potential as an anti‐tumor substance, but is of limited availability. In principle, cytochrome P450 monooxygenases, such as the self‐sufficient CYP102A1, are promising catalysts for the oxidation of limonene or other inert hydrocarbons. The wild‐type enzyme converts (4R)‐limonene to four different oxidation products; however, terminal hydroxylation at the allylic C7 is not observed. Here we describe a generic strategy to engineer this widely used enzyme to hydroxylate exclusively the exposed, but chemically less reactive, primary C7 in the presence of other reactive positions. The approach presented here turns CYP102A1 into a highly selective catalyst with a shifted product spectra by successive rounds of modeling, the design of small focused libraries, and screening. In the first round a minimal CYP102A1 mutant library was rationally designed. It contained variants with improved or strongly shifted regio‐, stereo‐ and chemoselectivity, compared to wild‐type. From this library the variant with the highest perillyl alcohol ratio was fine‐tuned by two additional rounds of molecular modeling, diversification, and screening. In total only 29 variants needed to be screened to identify the triple mutant A264V/A238V/L437F that converts (4R)‐limonene to perillyl alcohol with a selectivity of 97 %. Focusing mutagenesis on a small number of relevant positions identified by computational approaches is the key for efficient screening for enzyme selectivity.     

Characterisation of CYP102A25 from Bacillus marmarensis and CYP102A26 from Pontibacillus halophilus: P450 Homologues of BM3 with Preference towards Hydroxylation of Medium‐Chain Fatty Acids

Cytochrome P450 monooxygenases are highly desired biocatalysts owing to their ability to catalyse a wide variety of chemically challenging C?H activation reactions. The CYP102A subfamily of enzymes are natural catalytically self‐sufficient proteins consisting of a haem and FMN‐FAD reductase domain fused in a single‐component system. They catalyse the oxygenation of saturated and unsaturated fatty acids to produce primarily ω?1, ω?2 and ω?3 hydroxy acids. These monooxygenases have potential applications in biotechnology; however, their substrate range is still limited and there is a continued need to add diversity to this class of biocatalysts. Herein, we present the characterisation of two new members of this class of enzymes, CYP102A25 (BMar) from Bacillus marmarensis and CYP102A26 (PHal) from Pontibacillus halophilus, both of which express readily in a recombinant bacterial host. BMar exhibits the highest activity toward myristic acid and shows moderate activity towards unsaturated fatty acids. PHal exhibits broader activity towards mid‐chain‐saturated (C₁₄–C₁₈) and unsaturated fatty acids. Furthermore, PHal shows good regioselectivity for the hydroxylation of myristic acid, targeting the ω?2 position for C?H activation.     

Engineering Cytochrome P450 BM3 for Terminal Alkane Hydroxylation

Enzymes that catalyze the terminal hydroxylation of alkanes could be used to produce more valuable chemicals from hydrocarbons. Cytochrome P450 BM3 from Bacillus megaterium hydroxylates medium‐chain fatty acids at subterminal positions at high rates. To engineer BM3 for terminal alkane hydroxylation, we performed saturation mutagenesis at selected active‐site residues of a BM3 variant that hydroxylates alkanes. Recombination of beneficial mutations generated a library of BM3 mutants that hydroxylate linear alkanes with a wide range of regioselectivities. Mutant 77‐9H exhibits 52% selectivity for the terminal position of octane. This regioselectivity is octane‐specific and does not transfer to other substrates, including shorter and longer hydrocarbons or fatty acids. These results show that BM3 can be readily molded for regioselective oxidation.     

Revisiting Cytochrome P450‐Mediated Oxyfunctionalization of Linear and Cyclic Alkanes

Cytochrome P450 monooxygenases (CYPs) of the CYP153 family catalyse terminal hydroxylation of n‐alkanes. Alkane hydroxylating mutants of self‐sufficient CYP102A1 have also been described. We evaluated two CYP153s (a three‐component system and a fused self‐sufficient CYP), wild‐type CYP102A1 and nine CYP102A1 mutants, for the conversion of three cycloalkanes (C₆, C₇ and C₈) and three n‐alkanes (C₆, C₈ and C₁₀) using whole cells (WCs) and crude cell‐free extracts (CFEs). The aim was to identify substrate–enzyme combinations that give high product titres and space‐time yields (STYs). Comparisons were made using total turnover numbers (TTNs) and turnover frequencies (TOFs) to normalize for CYP expression. Reactions were carried out using high enzyme and substrate concentrations compatible with high STYs. Under these conditions CYP102A1 and the double R47L,Y51F mutant, although not regioselective, performed better on all substrates in terms of product titres over 8 h, and thus STYs and TTNs, than heavily mutated variants that have been reported to give very high TOFs. CYP153A6, with its ferredoxin (Fdx) and ferredoxin reductase (FdR), emerged as the superior catalyst for conversion of n‐alkanes. In addition to its excellent regioselectivity it also gave the highest final product titres and STYs in WC conversions of hexane and octane. Interaction with FdR and Fdx initially limited performance in CFEs, but with additional FdR and Fdx gave 1‐octanol titres of 50 mmol⋅L_BRM⁻¹ and TTNs exceeding 12,000 over 18 h, rivalling results reported with self‐sufficient CYPs. Selecting biocatalysts for application requires caution, since experimental conditions such as amount of substrate added and solubility as well as cofactor dependence and regeneration can have a profound effect on catalyst performance, while stability and efficiency with regard to cofactor usage (coupling efficiency) are at least as important as TOFs when high product titres and STYs are the target.

     

Selective and Sustainable Production of Sub-terminal Hydroxy Fatty Acids by a Self-Sufficient CYP102 Enzyme from Bacillus Amyloliquefaciens

Enzymatic hydroxylation of fatty acids by Cytochrome P450s (CYPs) offers an eco-friendly route to hydroxy fatty acids (HFAs), high-value oleochemicals with various applications in materials industry and with potential as bioactive compounds. However, instability and poor regioselectivity of CYPs are their main drawbacks. A newly discovered self-sufficient CYP102 enzyme, BAMF0695 from Bacillus amyloliquefaciens DSM 7, exhibits preference for hydroxylation of sub-terminal positions (ω-1, ω-2, and ω-3) of fatty acids. Our studies show that BAMF0695 has a broad temperature optimum (over 70 % of maximal enzymatic activity retained between 20 to 50 °C) and is highly thermostable (T₅₀ >50 °C), affording excellent adaptive compatibility for bioprocesses. We further demonstrate that BAMF0695 can utilize renewable microalgae lipid as a substrate feedstock for HFA production. Moreover, through extensive site-directed and site-saturation mutagenesis, we isolated variants with high regioselectivity, a rare property for CYPs that usually generate complex regioisomer mixtures. BAMF0695 mutants were able to generate a single HFA regiosiomer (ω-1 or ω-2) with selectivities from 75 % up to 91 %, using C12 to C18 fatty acids. Overall, our results demonstrate the potential of a recent CYP and its variants for sustainable and green production of high-value HFAs.     

Semirational Protein Engineering of CYP153AM.aq.‐CPRBM3 for Efficient Terminal Hydroxylation of Short‐ to Long‐Chain Fatty Acids

The regioselective terminal hydroxylation of alkanes and fatty acids is of great interest in a variety of industrial applications, such as in cosmetics, in fine chemicals, and in the fragrance industry. The chemically challenging activation and oxidation of non‐activated C?H bonds can be achieved with cytochrome P450 enzymes. CYP153A_M.aq.‐CPR_BM3 is an artificial fusion construct consisting of the heme domain from Marinobacter aquaeolei and the reductase domain of CYP102A1 from Bacillus megaterium. It has the ability to hydroxylate medium‐ and long‐chain fatty acids selectively at their terminal positions. However, the activity of this interesting P450 construct needs to be improved for applications in industrial processes. For this purpose, the design of mutant libraries including two consecutive steps of mutagenesis is demonstrated. Targeted positions and residues chosen for substitution were based on semi‐rational protein design after creation of a homology model of the heme domain of CYP153A_M.aq., sequence alignments, and docking studies. Site‐directed mutagenesis was the preferred method employed to address positions within the binding pocket, whereas diversity was created with the aid of a degenerate codon for amino acids located at the substrate entrance channel. Combining the successful variants led to the identification of a double variant—G307A/S233G—that showed alterations of one position within the binding pocket and one position located in the substrate access channel. This double variant showed twofold increased activity relative to the wild type for the terminal hydroxylation of medium‐chain‐length fatty acids. This variant furthermore showed improved activity towards short‐ and long‐chain fatty acids and enhanced stability in the presence of higher concentrations of fatty acids.     

Self-sufficient Cytochrome P450s and their potential applications in biotechnology

Cytochrome P450s(CYPs) are ubiquitously found in all kingdoms of life, playing important role in various biosynthetic pathways as well as degradative pathways; accordingly find applications in a vast variety of areas from organic synthesis and drug metabolite production to modification of biomaterials and bioremediation.Significantly, CYPs catalyze chemically challenging C—H and C—C activation reactions using a reactive high-valent iron-oxo intermediate generated upon dioxygen activation at their heme center,while the other oxygen atom is reduced to the level of water by electrons provided through a reductase partner protein.Self-sufficient CYPs, encoding their heme domain and reductase protein in a single polypeptide, facilitate increased catalytic efficiency and render a less complicated system to work with.The self-sufficient CYP enzyme from CYP102A family(CYP102A1, BM3) is among the earliest and most-investigated model enzymes for mechanistic and structural studies as well as for biotechnological applications.An increasing number of self-sufficient CYPs from the same CYP102 family and from other families have also been reported in last decade.In this review, we introduce chemistry and biology of CYPs, followed by an overview of the characteristics of self-sufficient CYPs and representative reactions.Enzyme engineering efforts leading to novel self-sufficient CYP variants that can catalyze synthetically useful natural and non-natural(nature-mimicking) reactions are highlighted.Lastly, the strategy and efforts that aim to circumvent the challenges for improved thermostability, regio-and enantioselectivity,and total turnover number; associated with practical use of self-sufficient CYPs are reviewed.     

Species-Dependent Metabolism of Benzo[c]Chrysene Mediated by c-DNA-Expressed Human,Rodent and Fish Cytochrome P450 Enzymes

The metabolism of benzo[c]chrysene (B[c]Ch) with various cytochrome P450 (CYP) enzymes including rat 1A1, 1A2, 2B1 and 2E1, human 1A1, 1A2, 2A6, 1B1, 3A4 and 2E1, mouse 1B1, and scup fish 1A1 expressed in Chinese hamster V79 cells has been investigated to clarify the role of individual enzymes in the regioselective oxidation of B[c]Ch and the species dependency. In six cell lines expressing individual CYP enzymes from four different species B[c]Ch was metabolized to several isomeric phenols and trans?dihydrodiols. However, cell lines expressing human 3A4, 2A6 and 2E1 or rat 1A2, 2B1 and 2E1 were metabolically in-competent towards B[c]Ch. Among the trans?dihydrodiols the 9,10-isomer could be detected in cells expressing human, rat and fish CYP 1A1 and to a minor extent in cells with human 1A2, but not in cells expressing human and mouse CYP 1B1. The latter two cell lines produced high amounts of the bay region 3,4-dihydrodiol, whereas the K-region 7,8-dihydrodiol was a minor metabolite. Oxidation of B[c]Ch to the 1,2-dihydrodiol could not be catalyzed by any of the CYP enzymes investigated except fish 1A1. Our results suggest that metabolic activation of B[c]Ch is initiated predominantly by CYP 1A1 to result selectively in the formation of fjord region 9,10-dihydrodiol 11,12-epoxides regardless of the species involved. The activation of B[c]Ch appears to be limited by a low regioselectivity for the 9,10-oxidation.     

CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering**

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.     

Enhancing the Efficiency and Regioselectivity of P450 Oxidation Catalysts by Unnatural Amino Acid Mutagenesis

The development of effective strategies for modulating the reactivity and selectivity of cytochrome P450 enzymes represents a key step toward expediting the use of these biocatalysts for synthetic applications. We have investigated the potential of unnatural amino acid mutagenesis to aid efforts in this direction. Four unnatural amino acids with diverse aromatic side chains were incorporated at 11 active‐site positions of a substrate‐promiscuous CYP102A1 variant. The resulting “uP450s” were then tested for their catalytic activity and regioselectivity in the oxidation of two representative substrates: a small‐molecule drug and a natural product. Large shifts in regioselectivity resulted from these single mutations, and in particular, for para‐acetyl‐Phe substitutions at positions close to the heme cofactor. Screening this mini library of uP450s enabled us to identify P450 catalysts for the selective hydroxylation of four aliphatic positions in the target substrates, including a C(sp³)?H site not oxidized by the parent enzyme. Furthermore, we discovered a general activity‐enhancing effect of active‐site substitutions involving the unnatural amino acid para‐amino‐Phe, which resulted in P450 catalysts capable of supporting the highest total turnover number reported to date on a complex molecule (34 650). The functional changes induced by the unnatural amino acids could not be reproduced by any of the 20 natural amino acids. This study thus demonstrates that unnatural amino acid mutagenesis constitutes a promising new strategy for improving the catalytic activity and regioselectivity of P450 oxidation catalysts.     

顶空气相色谱法测定爱罗苏中醋酸乙酯残留量

研究了顶空气相色谱法测定爱罗苏中醋酸乙酯残留量的方法，该法使用FID检测器、GDX-102固定相，不仅避免高沸点，非挥发性组分对色谱柱的污染，而且准确，灵敏，色谱分离好。方法的标准加入回收率为97.5%-102.1%。相对标准偏差为1.12%-2.52%。     

Cluster Screening: An Effective Approach for Probing the Substrate Space of Uncharacterized Cytochrome P450s

Cytochrome P450 monooxygenases (P450s) are versatile enzymes with high potential for biocatalysis. The number of newly annotated P450 genes has been increasing constantly, and these thus represent a rich resource for new biocatalysts. However, the substrate scopes of newly identified P450s are often not known, and thus their exploitation is difficult. Herein we describe an approach, named “cluster screening”, and its application for the primary characterization of two P450s: CYP154E1 and CYP154A8. A library comprising 51 compounds was designed and organized into nine groups according to their chemical properties. The activities of both P450s in vitro were maintained with suitable nonphysiological redox partners, and the cluster library was screened with these enzymes for product formation. From this library, 30 compounds tested positive for CYP154E1 and 23 were positive for CYP154A8. Cluster screening distinguishes subtle differences in activity and selectivity of enzymes as closely related as those of the same P450 family. For example, the alkaloid pergolide mesylate was converted by CYP154E1 (4 %) but not by CYP154A8. A building block of vitamin D₃, Grundmann's ketone, was converted by both enzymes, although conversion was higher with CYP154E1 (100 vs 53 %).     

Identification of CYP106A2 as a regioselective allylic bacterial diterpene hydroxylase

The cytochrome P450 monooxygenase CYP106A2 from Bacillus megaterium ATCC 13368 catalyzes hydroxylations of a variety of 3-oxo-Δ(4) -steroids such as progesterone and deoxycorticosterone (DOC), mainly in the 15β-position. We combined a high-throughput screening and a rational approach for identifying new substrates of CYP106A2. The diterpene resin acid abietic acid was found to be a substrate and was docked into the active site of a CYP106A2 homology model to provide further inside into the structural basis of the regioselectivity of hydroxylation. The products of the hydroxylation reaction were analyzed by HPLC and the V(max) and K(m) values were calculated. The corresponding reaction products were analyzed by NMR spectroscopy and identified as 12α- and 12β-hydroxyabietic acid. CYP106A2 was therefore identified as the first reported bacterial cytochrome P450 diterpene hydroxylase. Furthermore, an effective whole-cell catalyst for the selective allylic 12α- and 12β-hydroxylation was applied to produce the hydroxylated products.     

Carving the Active Site of CYP153A7 Monooxygenase for Improving Terminal Hydroxylation of Medium-Chain Fatty Acids

The P450-mediated terminal hydroxylation of non-activated C−H bonds is a chemically challenging reaction. CYP153A7 monooxygenase, discovered in Sphingomonas sp. HXN200, belongs to the CYP153A subfamily and shows a pronounced terminal selectivity. Herein, we report the significantly improved terminal hydroxylation activity of CYP153A7 by redesign of the substrate binding pocket based on molecular docking of CYP153A7−C_8:0 and sequence alignments. Some of the resultant single mutants were advantageous over the wild-type enzyme with higher reaction rates, achieving a complete conversion of n-octanoic acid (C_8:0, 1 mM) in a shorter time period. Especially, a single-mutation variant, D258E, showed 3.8-fold higher catalytic efficiency than the wild type toward the terminal hydroxylation of medium-chain fatty acid C_8:0 to the high value-added product 8-hydroxyoctanoic acid.     

Characterization of a Self-Sufficient Cytochrome P450 Monooxygenase from Deinococcus apachensis for Enantioselective Benzylic Hydroxylation

A self-sufficient cytochrome P450 monooxygenase from Deinococcus apachensis (P450DA) was identified and successfully overexpressed in Escherichia coli BL21(DE3). P450DA would be a member of the CYP102D subfamily and assigned as CYP102D2 according to the phylogenetic tree and sequence alignment. Purification and characterization of the recombinant P450DA indicated both NADH and NADPH could be used by P450DA as a reducing cofactor. The recombinant E. coli (P450DA) strain was functionally active, showing excellent enantioselectivity for benzylic hydroxylation of methyl 2-phenylacetate. Further substrate scope studies revealed that P450DA is able to catalyze benzylic hydroxylation of a variety of compounds, affording the corresponding chiral benzylic alcohols in 86–99 % ee and 130–1020 total turnover numbers.     

Alternating copolymerization of carbon monoxide and α-olefins

Summary Pd⁺⁺ catalysts with bis(diphenylphosphino)alkane ligand and methanol as coinitiator were found to be very active for the alternating copolymerization of carbon monoxide with propylene, butene-1 and hexene-1. The catalysts with bis(diphenylphosphino) propane and butane have the highest polymerization activities but only modest regiospecificity. The chiral (-)-2, 3-0-isopropylidene-2,3-dihydroxy-1,4-bis(diphenylphosphino)butane-Pd catalyst has good activity as well as regioselectivity to produce semi-crystalline CO/propylene alternating copolymer.     

Structural basis for the properties of two single-site proline mutants of CYP102A1 (P450BM3)

The crystal structures of the haem domains of Ala330Pro and Ile401Pro, two single‐site proline variants of CYP102A1 (P450_BM3) from Bacillus megaterium, have been solved. In the A330P structure, the active site is constricted by the relocation of the Pro329 side chain into the substrate access channel, providing a basis for the distinctive C? H bond oxidation profiles given by the variant and the enhanced activity with small molecules. I401P, which is exceptionally active towards non‐natural substrates, displays a number of structural similarities to substrate‐bound forms of the wild‐type enzyme, notably an off‐axial water ligand, a drop in the proximal loop, and the positioning of two I‐helix residues, Gly265 and His266, the reorientation of which prevents the formation of several intrahelical hydrogen bonds. Second‐generation I401P variants gave high in vitro oxidation rates with non‐natural substrates as varied as fluorene and propane, towards which the wild‐type enzyme is essentially inactive. The substrate‐free I401P haem domain had a reduction potential slightly more oxidising than the palmitate‐bound wild‐type haem domain, and a first electron transfer rate that was about 10 % faster. The electronic properties of A330P were, by contrast, similar to those of the substrate‐free wild‐type enzyme.