Cytochrome P450 enzymes (P450s) are broadly distributed among living organisms and play crucial roles in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, and drug metabolism. P450s are considered as the most versatile biocatalysts in nature because of the vast variety of substrate structures and the types of reactions they catalyze. In particular, P450s can catalyze regio- and stereoselective oxidations of nonactivated C–H bonds in complex organic molecules under mild conditions, making P450s useful biocatalysts in the production of commodity pharmaceuticals, fine or bulk chemicals, bioremediation agents, flavors, and fragrances. Major efforts have been made in engineering improved P450 systems that overcome the inherent limitations of the native enzymes. In this review, we focus on recent progress of different strategies, including protein engineering, redox-partner engineering, substrate engineering, electron source engineering, and P450-mediated metabolic engineering, in efforts to more efficiently produce pharmaceuticals and other chemicals. We also discuss future opportunities for engineering and applications of the P450 systems.
The irreversibly diminishing reserves and the fluctuating price of crude oil, together with the serious concerns on the global environment and energy security, all suggests an unpleasant future of fossil fuel. Within this context, it is imperative to find alternative eco-friendly fuel with properties that highly mimic fossil fuel. Aliphatic alka(e)nes generated from renewable resources via biosynthetic pathways perfectly suits the criteria and represents the ideal ″drop-in″ biofuels. α-Alkenes are special microbial natural products. These biohydrocarbons have attracted much attention due to their potential application as biofuels and biomaterials. By coupling the activity of lipase and P450 decarboxylase, lipids (i.e. triglycerides) can be transformed into α-alkenes via free fatty acid intermediates. To improve the conversion efficiency of lipids to α-alkenes, in this study, four different versions of fusion proteins of the lipase TlL from Thermomyces lanuginosus and the P450 decarboxylase OleTJE from Jeotgalicoccus sp. ATCC 8456, including FusA (His6-TlL-linker-OleTJE), FusB (TlL-Linker-OleTJE-His6), FusC (His6-OleTJE-linker-TlL) and FusD (OleTJE- linker-TlL-His6), were constructed and comparatively evaluated. The optimal fusion enzyme FusC was able to convert a number of triacylglycerols including trilaurin (C12), trimyristin (C14) and tripalmitin (C16) into corresponding α-alkenes via the coupled hydrolysis and decarboxylation. The 31.7% yield of 1-tridecene from trimyristin demonstrates good substrate channeling effect. Due to the higher catalytic efficiency compared to the mixed TlL and OleTJE, as well as the simplified enzyme purification and hence the lower cost, this fusion strategy may hold significant potential of application.
The lake bottom scattering function depends, in general, on the grazing angles and the azimuthal angles of the incident and scattered energy. However, most measurements were for backscatter only. Little was understood about forward bottom scattering in shallow water. In order to address this issue, an experiment was conducted in Qiandao Lake using 16 element vertical array and 8 element horizontal array. The measurement result indicated that there was relatively strong scattering in the forward direction near the specularly reflected ray, while the scattering was much weaker and relatively isotropic away from the specular ray. The Ellis and Crowe’s three-dimensional scattering function was used to predict the forward scattered energy. Comparsion between model and measured data has shown that the model can explain the lake bottom scattering well, and the frequency dependence of bottom scattering function from 5 to 9 kHz can be neglected. [Work supported by the NSFC.]
ABSTRACT The cytochrome P450 enzyme CYP-sb21 from Sebekia benihana is capable of catalyzing the site-specific hydroxylation of the immunosuppressant cyclosporine (CsA), leading to the single product γ-hydroxy- N -methyl- l -Leu4-CsA (CsA-4-OH). Unlike authentic CsA, this hydroxylated CsA shows significantly reduced immunosuppressive activity while it retains a side effect of CsA, the hair growth stimulation effect. Although CYP-sb21 was previously identified to be responsible for CsA-specific hydroxylation in vivo , the in vitro activity of CYP-sb21 has yet to be established for a deeper understanding of this P450 enzyme and further reaction optimization. In this study, we reconstituted the in vitro activity of CYP-sb21 by using surrogate redox partner proteins of bacterial and cyanobacterial origins. The highest CsA site-specific hydroxylation activity by CYP-sb21 was observed when it was partnered with the cyanobacterial redox system composed of se Fdx and se FdR from Synechococcus elongatus PCC 7942. The best bioconversion yields were obtained in the presence of 10% methanol as a cosolvent and an NADPH regeneration system. A heterologous whole-cell biocatalyst using Escherichia coli was also constructed, and the permeability problem was solved by using N -cetyl- N , N , N -trimethylammonium bromide (CTAB). This work provides a useful example for reconstituting a hybrid P450 system and developing it into a promising biocatalyst for industrial application.
Selective oxidation of C-H bonds in alkylphenols holds great significance for not only structural derivatization in pharma- and biomanufacturing but also biological degradation of these toxic chemicals in environmental protection. A unique chemomimetic biocatalytic system using enzymes from a p-cresol biodegradation pathway has recently been developed. As the central biocatalyst, the cytochrome P450 monooxygenase CreJ oxidizes diverse p- and m-alkylphenol phosphates with perfect stereoselectivity at different efficiencies. However, the mechanism of regio- and stereoselectivity of this chemomimetic biocatalytic system remained unclear. Here, using p- and m-ethylphenol substrates, we elucidate the CreJ-catalyzed key steps for selective oxidations. The crystal structure of CreJ in complex with m-ethylphenol phosphate was solved and compared with its complex structure with p-ethylphenol phosphate isomer. The results indicate that the conformational changes of substrate-binding residues are slight, while the substrate promiscuity is achieved mainly by the available space in the catalytic cavity. Moreover, the catalytic preferences of regio- and stereoselective hydroxylation for the two ethylphenol substrates is explored by molecular dynamics simulations. The ethyl groups in the complexes display different flexibilities, and the distances of the active oxygen to H pro-S and H pro-R of methylene agree with the experimental stereoselectivity. The regioselectivity can be explained by the distances and bond dissociation energy. These results provide not only the mechanistic insights into CreJ regio- and stereoselectivity but also the structural basis for further P450 enzyme design and engineering.IMPORTANCE The key cytochrome P450 monooxygenase CreJ showed excellent regio- and stereoselectivity in the oxidation of various alkylphenol substrates. C-H bond functionalization of these toxic alkylphenols holds great significance for both biological degradation of these environmental chemicals and production of value-added structural derivatives in pharmaceutical and biochemical industries. Our results, combined with in vitro enzymatic assays, crystal structure determination of enzyme-substrate complex, and molecular dynamics simulations, provide not only significant mechanism elucidation of the regio- and stereoselective catalyzation mediated by CreJ but also the promising directions for future engineering efforts of this enzyme toward more useful products. It also has great extendable potential to couple this multifunctional P450 enzyme with other biocatalysts (e.g., hydroxyl-based glycosylase) to access more alkylphenol-derived high-value chemicals through environment-friendly biocatalysis and biotransformation.
The drastically increasing consumption of petroleum-derived plastics hasserious environmental impacts and raises public concerns. Poly(ethylene terephthalate) (PET) is amongst the most extensively produced synthetic polymers. Enzymatic hydrolysis of PET recently emerged as an enticing path for plastic degradation and recycling. In-lab directed evolution has revealed the great potential of PET hydrolases (PETases). However, the time-consuming and laborious PETase assays hinder the identification of effective variants in large mutant libraries. Herein, we devise and validate a dual fluorescence-based high-throughput screening (HTS) assay for a representative IsPETase. The two-round HTS of a pilot library consisting of 2850 IsPETase variants yields six mutant IsPETases with 1.3-4.9 folds improved activities. Compared to the currently used structure- or computational redesign-based PETase engineering, this HTS approach provides a new strategy for discovery of new beneficial mutation patterns of PETases.
The economic identity and the utility function of the stateowned enterprise operators are the key of the institutional arrangement. By the serious research, this paper argues that the stateowned enterprises were short of real enterpreneurs, and the operators were merely the combination of bureaucracies and agents. Based on the bureaucracy utility function model, the paper makes mechanism arrangement and tries to find the rational market equilibrium in market game by employing the backward induction.
Cytochrome P450 enzymes (P450s) are broadly distributed among living organisms and play crucial roles in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, and drug metabolism. P450s are considered as the most versatile biocatalysts in nature because of the vast variety of substrate structures and the types of reactions they catalyze. In particular, P450s can catalyze regio- and stereoselective oxidations of nonactivated C–H bonds in complex organic molecules under mild conditions, making P450s useful biocatalysts in the production of commodity pharmaceuticals, fine or bulk chemicals, bioremediation agents, flavors, and fragrances. Major efforts have been made in engineering improved P450 systems that overcome the inherent limitations of the native enzymes. In this review, we focus on recent progress of different strategies, including protein engineering, redox-partner engineering, substrate engineering, electron source engineering, and P450-mediated metabolic engineering, in efforts to more efficiently produce pharmaceuticals and other chemicals. We also discuss future opportunities for engineering and applications of the P450 systems. Cytochrome P450 enzymes (P450s) are broadly distributed among living organisms and play crucial roles in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, and drug metabolism. P450s are considered as the most versatile biocatalysts in nature because of the vast variety of substrate structures and the types of reactions they catalyze. In particular, P450s can catalyze regio- and stereoselective oxidations of nonactivated C–H bonds in complex organic molecules under mild conditions, making P450s useful biocatalysts in the production of commodity pharmaceuticals, fine or bulk chemicals, bioremediation agents, flavors, and fragrances. Major efforts have been made in engineering improved P450 systems that overcome the inherent limitations of the native enzymes. In this review, we focus on recent progress of different strategies, including protein engineering, redox-partner engineering, substrate engineering, electron source engineering, and P450-mediated metabolic engineering, in efforts to more efficiently produce pharmaceuticals and other chemicals. We also discuss future opportunities for engineering and applications of the P450 systems.
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