Directed evolution of Baeyer-Villiger monooxygenase for highly secretory expressed in Pichia pastoris and efficient preparation of chiral pyrazole sulfoxide.

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a highly distinguished expression platform for the excellent synthesis of various heterologous proteins in recent years. With the advantages of high-density fermentation, P. pastoris can produce gram amounts of recombinant proteins. While not every protein of interest can be expressed to such high titers, such as Baeyer-Villiger monooxygenase (BVMO) (AcPSMO) which is responsible for pyrazole sulfide asymmetric oxidation. In this work, an excellent yeast expression system was established to facilitate efficient AcPSMO expression, which exhibited 9.5-fold enhanced secretion. Subsequently, an ultrahigh throughput screening method based on fluorescence-activated cell sorting by fusing super folder green fluorescent protein (sfGFP) in the C-terminal of AcPSMO was developed, and directed evolution was performed. The protein expression level of the superior mutant AcPSMOP1 (S58T/T252P/E336N/H456D) reached 84.6 mg/L at 100 mL shaking flask, which was 4.7 times higher than the levels obtained with the wild-type. Finally, the optimized chassis cells were used for high-density fermentation on a 5-L scale, and AcPSMOP1 protein yield of 3.4 g/L was achieved, representing approximately 85% of the total protein secreted. By directly employing the pH-adjusted supernatant as a biocatalyst, 20 g/L pyrmetazole sulfide was completely transformed into the corresponding (S)-sulfoxide, with a 78.8% isolated yield. This work confers dramatic benefits for efficient secretion of other BVMOs in P. pastoris.

Rational Design of Taxadiene Hydroxylase by Ancestral Enzyme Construction and the Elucidation of Key Amino Acids.

Cytochrome P450 monooxygenases (CYP450s) play an important role in the biosynthesis of natural products by activating inert C-H bonds and inserting hydroxyl groups. However, the activities of most plant-derived CYP450s are extremely low, limiting the heterologous biosynthesis of natural products. Traditional enzyme engineering methods, either rational or screening-based, are not suitable for CYP450s because of the lack of crystal structures and high-throughput screening methods for this class of enzymes. CYP725A4 is the first hydroxylase involved in the biosynthesis pathway of Taxol. Its low activity, promiscuity, and multispecificity make it a bottleneck in Taxol biosynthesis. Here, we identified key amino acids that affect the in vivo activity of CYP725A4 by constructing the ancestral enzymes of CYP725A4. We obtained positive mutants that showed an improved yield of hydroxylated products based on the key amino acids identified, providing guidance for the modification of other CYP450s involved in the biosynthesis of natural products.

Engineering a Formate Dehydrogenase for NADPH Regeneration.

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) constitute major hydrogen donors for oxidative/reductive bio-transformations. NAD(P)H regeneration systems coupled with formate dehydrogenases (FDHs) represent a dreamful method. However, most of the native FDHs are NAD+ -dependent and suffer from insufficient reactivity compared to other enzymatic tools, such as glucose dehydrogenase. An efficient and competitive NADP+ -utilizing FDH necessitates the availability and robustness of NADPH regeneration systems. Herein, we report the engineering of a new FDH from Candida dubliniensis (CdFDH), which showed no strict NAD+ preference by a structure-guided rational/semi-rational design. A combinatorial mutant CdFDH-M4 (D197Q/Y198R/Q199N/A372S/K371T/▵Q375/K167R/H16L/K159R) exhibited 75-fold intensification of catalytic efficiency (kcat /Km ). Moreover, CdFDH-M4 has been successfully employed in diverse asymmetric oxidative/reductive processes with cofactor total turnover numbers (TTNs) ranging from 135 to 986, making it potentially useful for NADPH-required biocatalytic transformations.

Improving the Enantioselectivity of CHMOBrevi1 for Asymmetric Synthesis of Podophyllotoxin Precursor.

(R)-ß-piperonyl-γ-butyrolactones are key building blocks for the synthesis of podophyllotoxin, which have demonstrated remarkable potential in cancer treatment. Baeyer-Villiger monooxygenases (BVMOs)-mediated asymmetric oxidation is a green approach to produce chiral lactones. While several BVMOs were able to oxidize the corresponding cyclobutanone, most BVMOs gave the (S) enantiomer while Cyclohexanone monooxygenase (CHMO) from Brevibacterium sp. HCU1 gave (R) enantiomer, but with a low enantioselectivity (75 % ee). In this study, we use a strategy called "focused rational iterative site-specific mutagenesis" (FRISM) at residues ranging from 6 Šfrom substrate. The mutations by using a restricted set of rationally chosen amino acids allow the formation of a small mutant library. By generating and screening less than 60 variants, we achieved a high ee of 96.8 %. Coupled with the cofactor regeneration system, 9.3 mM substrate was converted completely in a 100-mL scale reaction. Therefore, our work reveals a promising synthetic method for (R)-ß-piperonyl-γ-butyrolactone with the highest enantioselectivity, and provides a new opportunity for the chem-enzymatic synthesis of podophyllotoxin.

Virtual screening of carboxylic acid reductases for biocatalytic synthesis of 6-aminocaproic acid and 1,6-hexamethylenediamine.

The key precursors for nylon synthesis, that is, 6-aminocaproic acid (6-ACA) and 1,6-hexamethylenediamine (HMD), are produced from petroleum-based feedstocks. A sustainable biocatalytic alternative method from bio-based adipic acid has been demonstrated recently. However, the low efficiency and specificity of carboxylic acid reductases (CARs) used in the process hampers its further application. Herein, we describe a highly accurate protein structure prediction-based virtual screening method for the discovery of new CARs, which relies on near attack conformation frequency and the Rosetta Energy Score. Through virtual screening and functional detection, five new CARs were selected, each with a broad substrate scope and the highest activities toward various di- and ω-aminated carboxylic acids. Compared with the reported CARs, KiCAR was highly specific with regard to adipic acid without detectable activity to 6-ACA, indicating a potential for 6-ACA biosynthesis. In addition, MabCAR3 had a lower Km with regard to 6-ACA than the previously validated CAR MAB4714, resulting in twice conversion in the enzymatic cascade synthesis of HMD. The present work highlights the use of structure-based virtual screening for the rapid discovery of pertinent new biocatalysts.

Prokaryotic expression and characterization of artificial self-sufficient CYP120A monooxygenases.

Cytochrome P450 monooxygenases CYP120As are the unique non-membrane P450s, which are extensively involved in retinoid biodegradation. As the O-functionalized 1,3,3-trimethylcyclohex-1-ene moiety exists in many bioactive compounds which could only be catalyzed by Class II P450s, exploration of the catalytic repertoire of CYP120As is therefore highly attractive. However, up to date, only one bacteriogenic candidate (CYP120A1) was demonstrated for the hydroxylation of C16 and C17 of retinoic acid, by utilizing the integral membrane protein cytochrome P450 reductase redox partner for the electron transfer. Herein, we provided an efficient prokaryotic functional expression system of CYP120As in E. coli by expression of the CYP120A1 coupled with several reductase partners. Fusion redox partners to the C-terminal of the heme-domain are also working on other CYP120A members. Among them, the fusion protein of CYP120A29 and FAD/FMN reductase from Bacillus megaterium P450BM3 (CYP101A2) showed the highest expression level. Based on the available translational fusion systems, the regioselectivity and the substrate scope of the CYP120As have also been explored. This work represents a good starting point for further expanding the catalytic potential of CYP120 family. KEY POINTS: • Characterization of CYP120As in E. coli is firstly achieved by constructing fusion proteins. • The feasibility of three P450 reductase domains to CYP120As was evaluated. • Hydroxylated products of retinoic acid by six CYP120As were sorted and analyzed.

Colorimetric High-Throughput Screening Method for Directed Evolution of Prazole Sulfide Monooxygenase.

Baeyer-Villiger monooxygenases (BVMOs) are important biocatalysts for the enzymatic synthesis of chiral sulfoxides, including chiral sulfoxide-type proton pump inhibitors for the treatment of gastrointestinal diseases. However, native BVMOs are not yet suitable for practical application due to their unsatisfactory activity and thermostability. Although protein engineering approaches can help address these issues, few feasible high-throughput methods are available for the engineering of such enzymes. Herein, a colorimetric detection method to distinguish sulfoxides from sulfides and sulfones was developed for prazole sulfide monooxygenases. Directed evolution enabled by this method has identified a prazole sulfide monooxygenase CbBVMO variant with improved activity and thermostability that catalyzes the asymmetric oxidation of lansoprazole sulfide. A 71.3 % increase in conversion and 6 °C enhancement in the melting point were achieved compared with the wild-type enzyme. This new method is feasible for high-throughput screening of prazole sulfide monooxygenase variants with improved activity, thermostability, and/or substrate specificity.

A High-Throughput Screening Method for the Directed Evolution of Hydroxynitrile Lyase towards Cyanohydrin Synthesis.

Chiral cyanohydrins are useful intermediates in the pharmaceutical and agricultural industries. In nature, hydroxynitrile lyases (HNLs) are a kind of elegant tool for enantioselective hydrocyanation of carbonyl compounds. However, currently available methods for demonstrating hydrocyanation are still stalled at precise, but low-throughput, GC or HPLC analyses. Herein, we report a chromogenic high-throughput screening (HTS) method that is feasible for the cyanohydrin synthesis reaction. This method was highly anti-interference and sensitive, and could be used to directly profile the substrate scope of HNLs either in cell-free extract or fermentation clear broth. This HTS method was also validated by generating new variants of PcHNL5 that presented higher catalytic efficiency and stronger acidic tolerance in variant libraries.

Identification two key residues at the intersection of domains of a thioether monooxygenase for improving its sulfoxidation performance.

AcCHMO, a cyclohexanone monooxygenase from Acinetobacter calcoaceticus, is a typical Type I Baeyer-Villiger monooxygenase (BVMO). We previously obtained the AcCHMOM6 mutant, which oxidizes omeprazole sulfide (OPS) to the chiral sulfoxide drug esomeprazole. To further improve the catalytic efficiency of the AcCHMOM6 mutant, a focused mutagenesis strategy was adopted at the intersections of the FAD-binding domain, NADPH-binding domain, and α-helical domain based on structural characteristics of AcCHMO. By using focused mutagenesis and subsequent global evolution two key residues (L55 and P497) at the intersections of the domains were identified. Mutant of L55Y improved catalytic efficiency significantly, whereas the P497S mutant alleviated substrate inhibition remarkably. AcCHMOM7 (L55Y/P497S) was obtained by combining the two mutations, which increased the specific activity from 18.5 (M6) to 108 U/g, and an increase in the Ki of the substrate OPS from 34 to 265 µM. The results indicate that catalytic performance can be elevated by modification of the sensitive sites at the intersection of the domains of AcCHMO. The results also provided some insights for the engineering of other Type I BVMOs or other multidomain proteins.

A Baeyer-Villiger monooxygenase from Cupriavidus basilensis catalyzes asymmetric synthesis of (R)-lansoprazole and other pharmaco-sulfoxides.

Biocatalytic synthesis of pharmaco-chiral sulfoxides has gained interest in recent years for its environmental friendliness. However, only a few natural biocatalysts can be used for the efficient synthesis of pharmaco-sulfoxides, including (R)-lansoprazole, a chiral proton pump inhibitor used to treat gastrointestinal diseases. In this study, the sequence of BoBVMO (Baeyer-Villiger monooxygenase from Bradyrhizobium oligotrophicum) was used as a probe to identify BVMOs via genomic mining for the highly efficient synthesis of (R)-lansoprazole and other pharmaco-sulfoxides. After virtual sequence filtering, target gene cloning, heterologous expression, and activity screening for lansoprazole sulfide (LPS) monooxygenation, seven new BVMOs were identified among more than 10,000 homologous BVMOs. According to the conserved sequence and phylogenetic tree analysis, these discovered enzymes belong to the family of type I BVMOs and the ethionamide monooxygenase subtype. Among them, CbBVMO, Baeyer-Villiger monooxygenase from Cupriavidus basilensis, showed the highest efficiency and excellent enantioselectivity for converting LPS into (R)-lansoprazole. Moreover, CbBVMO showed a wide substrate spectrum toward other bulky prazole-family sulfides. The results indicate that CbBVMO is a potential enzyme for extending the application of BVMOs in pharmaceutical industry. KEY POINTS: • CbBVMO is the most efficient biocatalyst for (R)-lansoprazole biosynthesis. • CbBVMO catalyzes the conversion of various bulky prazole sulfides. • CbBVMO is a promising enzyme for the biosynthesis of pharmaco-sulfoxides.

Site-directed mutagenesis of coenzyme-independent carotenoid oxygenase CSO2 to enhance the enzymatic synthesis of vanillin.

Vanillin is a popular flavoring compound and an important food additive. Owing to the consumer preference for inexpensive natural aroma flavors, vanillin production through a biotechnological pathway has become of great interest and commercial value in recent years. In this study, an enzymatic synthetic system for vanillin using a coenzyme-independent decarboxylase (FDC) and oxygenase (CSO2) cascade was reconstituted and optimized. This system produces a slightly higher production yield (40.20%) than the largest yield reported for immobilized FDC and CSO2 (35.00%) with ferulic acid as a substrate. It was previously reported that the low catalytic activity and thermal instability of CSO2 restrict the overall productivity of vanillin. In present study, site-directed mutagenesis was applied to rate-limiting oxygenase CSO2 to generate positive mutants. The production yields of mutants A49P (58.44%) and Q390A (65.29%) were 1.45- and 1.62-fold that of CSO2 wild type, respectively. The potential mechanism for enhanced vanillin production using A49P involved increased thermostability and catalytic efficiency, while that using Q390A was probably associated with a better thermostable performance and increased catalytic efficiency resulting from a larger entrance channel.

Bioamination of alkane with ammonium by an artificially designed multienzyme cascade.

Biocatalytic C-H amination is one of the most challenging tasks. C-H amination reaction can hardly be driven efficiently by solely one enzyme so far. Thus, enzymatic synergy represents an alternative strategy. Herein, we report an "Artificially Bioamination Pathway" for C-H amination of cyclohexane as a model substrate. Three enzymes, a monooxygenase P450BM3 mutant, an alcohol dehydrogenase ScCR from Streptomyces coelicolor and an amine dehydrogenase EsLeuDH from Exiguobacterium sibiricum, constituted a clean cascade reaction system with easy product isolation. Two independent cofactor regeneration systems were optimized to avoid interference from the endogenous NADH oxidases in the host E. coli cells. Based on a stepwise pH adjustment and ammonium supplement strategy, and using an in vitro mixture of cell-free extracts of the three enzymes, cyclohexylamine was produced in a titer of 14.9 mM, with a product content of up to 92.5%. Furthermore, designer cells coexpressing the three required enzymes were constructed and their capability of alkane bio-amination was examined. This artificially designed bioamination paves an attractive approach for enzymatic synthesis of amines from accessible and cheap alkanes.

Discovery of Two Native Baeyer-Villiger Monooxygenases for Asymmetric Synthesis of Bulky Chiral Sulfoxides.

Two Baeyer-Villiger monooxygenases (BVMOs), designated BoBVMO and AmBVMO, were discovered from Bradyrhizobium oligotrophicum and Aeromicrobium marinum, respectively. Both monooxygenases displayed novel features for catalyzing the asymmetric sulfoxidation of bulky and pharmaceutically relevant thioethers. Evolutionary relationship and sequence analysis revealed that the two BVMOs belong to the family of typical type I BVMOs and the subtype ethionamide monooxygenase. Both BVMOs are active toward medium- and long-chain aliphatic ketones as well as various thioether substrates but are ineffective toward cyclohexanone, aromatic ketones, and other typical BVMO substrates. BoBVMO and AmBVMO showed the highest activities (0.117 and 0.025 U/mg protein, respectively) toward thioanisole among the tested substrates. Furthermore, these BVMOs exhibited distinct activity and excellent stereoselectivity toward bulky and prochiral prazole thioethers, which is a unique feature of this family of BVMOs. No native enzyme has been reported for the asymmetric sulfoxidation of bulky prazole thioethers into chiral sulfoxides. The identification of BoBVMO and AmBVMO provides an important scaffold for discovering enzymes capable of asymmetrically oxidizing bulky thioether substrates by genome mining.IMPORTANCE Baeyer-Villiger monooxygenases (BVMOs) are valuable enzyme catalysts that are an alternative to the chemical Baeyer-Villiger oxidation reaction. Although BVMOs display broad substrate ranges, no native enzymes were reported to have activity toward the asymmetric oxidation of bulky prazole-like thioether substrates. Herein, we report the discovery of two type I BVMOs from Bradyrhizobium oligotrophicum (BoBVMO) and Aeromicrobium marinum (AmBVMO) which are able to catalyze the asymmetric sulfoxidation of bulky prazole thioethers (proton pump inhibitors [PPIs], a group of drugs whose main action is a pronounced and long-lasting reduction of gastric acid production). Efficient catalysis of omeprazole oxidation by BoBVMO was developed, indicating that this enzyme is a promising biocatalyst for the synthesis of bulky and pharmaceutically relevant chiral sulfoxide drugs. These results demonstrate that the newly identified enzymes are suitable templates for the discovery of more and better thioether-converting BVMOs.

Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis.

Streptomyces coelicolor CR1 (ScCR1) has been shown to be a promising biocatalyst for the synthesis of an atorvastatin precursor, ethyl-(S)-4-chloro-3-hydroxybutyrate [(S)-CHBE]. However, limitations of ScCR1 observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. First, the crystal structure of ScCR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of ScCR1 was increased from 38.8 U/mg to 168 U/mg (ScCR1I158V/P168S) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1I158V/P168S as a template, affording a triple mutant, ScCR1A60T/I158V/P168S, whose thermostability (T5015, defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (C5015, defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) were 6.2°C and 4.7-fold higher than those of the wild-type enzyme. Interestingly, the specific activity of the triple mutant was further increased to 260 U/mg. Protein modeling and docking analysis shed light on the origin of the improved activity and stability. In the asymmetric reduction of ethyl-4-chloro-3-oxobutyrate (COBE) on a 300-ml scale, 100 g/liter COBE could be completely converted by only 2 g/liter of lyophilized ScCR1A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of >99% and a space-time yield of 255 g liter-1 day-1 These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor.IMPORTANCE Application of the carbonyl reductase ScCR1 in asymmetrically synthesizing (S)-CHBE, a key precursor for the blockbuster drug Lipitor, from COBE has been hindered by its low catalytic activity and poor thermostability and substrate tolerance. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. The catalytic efficiency, thermostability, and substrate tolerance of ScCR1 were significantly improved by structure-guided engineering and directed evolution. The engineered ScCR1 may serve as a promising biocatalyst for the biosynthesis of (S)-CHBE, and the protein engineering strategy adopted in this work would serve as a useful approach for future engineering of other reductases toward potential application in organic synthesis.

Combinatorial evolution of phosphotriesterase toward a robust malathion degrader by hierarchical iteration mutagenesis.

Malathion is one of the most widely used organophosphorus pesticides in the United States and developing countries. Herein, we enhanced the degradation rate of malathion starting with a phosphotriesterase PoOPHM2 while also considering thermostability. In the first step, iterative saturation mutagenesis at residues lining the binding pocket (CASTing) was employed to optimize the enzyme active site for substrate binding and activity. Hot spots for enhancing activity were then discovered through epPCR-based random mutagenesis, and these beneficial mutations were then recombined by DNA shuffling. Finally, guided by in silico energy calculations (FoldX), thermostability of the variant was improved. The mutations extend from the core region to the enzyme surface during the evolutionary pathway. After screening <9,000 mutants, the best variant PoOPHM9 showed 25-fold higher activity than wild-type PoOPHM2 , with a thermostability (T50 (15) ) of 67.6°C. Thus, PoOPHM9 appears to be an efficient and robust candidate for malathion detoxification. Biotechnol. Bioeng. 2016;113: 2350-2357. © 2016 Wiley Periodicals, Inc.

An engineered microorganism can simultaneously detoxify cadmium, chlorpyrifos, and γ-hexachlorocyclohexane.

Many ecosystems are currently co-contaminated with heavy metals such as cadmium (Cd(2+) ) and pesticides such as chlorpyrifos (CP) and γ-hexachlorocyclohexane (γ-HCH). A feasible approach to remediate the combined pollution of heavy metals and pesticides is the use of γ-HCH degrading bacteria endowed with CP hydrolysis and heavy metal biosorption capabilities. In this work, a recombinant microorganism capable of simultaneously detoxifying Cd(2+) , CP, and γ-HCH was constructed by display of synthetic phytochelatins (EC20) and methyl parathion hydrolase (MPH) fusion protein on the cell surface of the γ-HCH degrading Sphingobium japonicum UT26 using the truncated ice nucleation protein (INPNC) as an anchoring motif. The surface localization of INPNC-EC20-MPH was verified by cell fractionation, Western blot analysis, immunofluorescence microscopy, and proteinase accessibility experiment. Expression of EC20 on the cell surface not only improved Cd(2+) binding but also alleviated the cellular toxicity of Cd(2+) . As expected, the rates of CP and γ-HCH degradation were reduced in the presence of Cd(2+) for cells without EC20 expression. However, expression of EC20 (higher Cd(2+) accumulation) significantly restored the levels of CP and γ-HCH degradation. These results demonstrated that surface display of EC20 enhanced not only Cd(2+) accumulation but also protected the recombinant strain against the toxic effects of Cd(2+) on CP and γ-HCH degradation.

Regulation of Intrinsic and Extrinsic Apoptotic Pathways in Osteosarcoma Cells Following Oleandrin Treatment.

Our previous study has reported the anti-tumor effect of oleandrin on osteosarcoma (OS) cells. In the current study, we mainly explored its potential regulation on intrinsic and extrinsic apoptotic pathway in OS cells. Cells apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected using fluorescence staining and flow cytometry. Caspase-3 activity was detected using a commercial kit. The levels of cytoplasmic cytochrome c, mitochondrial cytochrome c, bcl-2, bax, caspase-9, Fas, FasL, caspase-8 and caspase-3 were detected by Western blotting. z-VAD-fmk was applied to block both intrinsic and extrinsic apoptosis pathways, and cells apoptosis was also tested. Furthermore, we used z-LEHD-fmk and Fas blocking antibody to inhibit intrinsic and extrinsic pathways, separately, and the selectivity of oleandrin on these pathways was explored. Results showed that oleandrin induced the apoptosis of OS cells, which was accompanied by an increase in ROS and a decrease in MMP. Furthermore, cytochrome c level was reduced in mitochondria but elevated in the cytoplasm. Caspase-3 activity was enhanced by oleandrin in a concentration- and time-dependent manner. Oleandrin also down-regulated the expression of bcl-2, but up-regulated bax, caspase-9, Fas, FasL, caspase-8 and caspase-3. In addition, the suppression of both apoptotic pathways by z-VAD-fmk greatly reverted the oleandrin-induced apoptosis. Moreover, the suppression of one pathway by a corresponding inhibitor did not affect the regulation of oleandrin on another pathway. Taken together, we concluded that oleandrin induced apoptosis of OS cells via activating both intrinsic and extrinsic apoptotic pathways.

Unusually broad substrate profile of self-sufficient cytochrome P450 monooxygenase CYP116B4 from Labrenzia aggregata.

A new member of the CYP116B subfamily-P450LaMO-was discovered in Labrenzia aggregata by genomic data mining. It was successfully overexpressed in Escherichia coli, purified, and subsequently characterized spectroscopically, and its catalytic properties were assessed. Substrate profiling of the P450LaMO revealed that it was a versatile catalyst, exhibiting hydroxylation and epoxidation activities as well as O-dealkylation and asymmetric sulfoxidation activities. Diverse compounds, including alkylbenzenes, aromatic bicyclic molecules, and terpenoids, were shown to be hydroxylated by P450LaMO. Such diverse catalytic activities are uncommon for the bacterial P450s, and the P450LaMO-mediated stereoselective hydroxylation of inactivated C-H bonds-ubiquitous and relatively unreactive in organic molecules-is particularly unusual. The self-sufficient nature of P450LaMO, coupled with its broad substrate range, highlights it as an ideal template for directed evolution towards various applications.

Efficient production of (R)-(-)-mandelic acid using glutaraldehyde cross-linked Escherichia coli cells expressing Alcaligenes sp. nitrilase.

Recombinant Escherichia coli cells expressing Alcaligenes sp. nitrilase were simply immobilized by direct cross-linking using glutaraldehyde. About 85 % of the total nitrilase activity was recovered under the optimal cross-linking conditions. The thermal stabilities of the cross-linked cells measured at 30, 40 and 50 °C were 4.5-, 5.3-, and 5.1-fold those of the free cells, respectively. The concentration of (R)-(-)-mandelic acid reached 280 mM after merely 2 h transformation with the immobilized cells using 300 mM mandelonitrile as substrate, affording an extremely high productivity of 510.7 g L(-1) d(-1). In addition, operational stability of the immobilized cells was obviously superior to that of free cells, without significant activity loss after 15 cycles of batch reactions or 8 cycles of repeated fed-batch reactions. Therefore, the easy preparation and robust characteristics of the immobilized biocatalyst make it a very promising biocatalyst for high-performance and low-cost production of optically pure (R)-(-)-mandelic acid.

Development of a Thermodynamically Favorable Multi-enzyme Cascade Reaction for Efficient Sustainable Production of ω-Amino Fatty Acids and α,ω-Diamines.

Aliphatic ω-amino fatty acids (ω-AFAs) and α,ω-diamines (α,ω-DMs) are essential monomers for the production of nylons. Development of a sustainable biosynthesis route for ω-AFAs and α,ω-DMs is crucial in addressing the challenges posed by climate change. Herein, we constructed an unprecedented thermodynamically favorable multi-enzyme cascade (TherFavMEC) for the efficient sustainable biosynthesis of ω-AFAs and α,ω-DMs from cheap α,ω-dicarboxylic acids (α,ω-DAs). This TherFavMEC was developed by incorporating bioretrosynthesis analysis tools, reaction Gibbs free energy calculations, thermodynamic equilibrium shift strategies and cofactor (NADPH&ATP) regeneration systems. The molar yield of 6-aminohexanoic acid (6-ACA) from adipic acid (AA) was 92.3 %, while the molar yield from 6-ACA to 1,6-hexanediamine (1,6-HMD) was 96.1 %, which were significantly higher than those of previously reported routes. Furthermore, the biosynthesis of ω-AFAs and α,ω-DMs from 20.0 mM α,ω-DAs (C6-C9) was also performed, giving 11.2 mM 1,6-HMD (56.0 % yield), 14.8 mM 1,7-heptanediamine (74.0 % yield), 17.4 mM 1,8-octanediamine (87.0 % yield), and 19.7 mM 1,9-nonanediamine (98.5 % yield), respectively. The titers of 1,9-nonanediamine, 1,8-octanediamine, 1,7-heptanediamine and 1,6-HMD were improved by 328-fold, 1740-fold, 87-fold and 3.8-fold compared to previous work. Therefore, this work holds great potential for the bioproduction of ω-AFAs and α,ω-DMs.