Engineering of Halide Methyltransferase BxHMT through Dynamic Cross-Correlation Network Analysis.

Halide methyltransferases (HMTs) provide an effective way to regenerate S-adenosyl methionine (SAM) from S-adenosyl homocysteine and reactive electrophiles, such as methyl iodide (MeI) and methyl toluene sulfonate (MeOTs). As compared with MeI, the cost-effective unnatural substrate MeOTs can be accessed directly from cheap and abundant alcohols, but shows only limited reactivity in SAM production. In this study, we developed a dynamic cross-correlation network analysis (DCCNA) strategy for quickly identifying hot spots influencing the catalytic efficiency of the enzyme, and applied it to the evolution of HMT from Paraburkholderia xenovorans. Finally, the optimal mutant, M4 (V55T/C125S/L127T/L129P), exhibited remarkable improvement, with a specific activity of 4.08 U/mg towards MeOTs, representing an 82-fold increase as compared to the wild-type (WT) enzyme. Notably, M4 also demonstrated a positive impact on the catalytic ability with other methyl donors. The structural mechanism behind the enhanced enzyme activity was uncovered by molecular dynamics simulations. Our work not only contributes a promising biocatalyst for the regeneration of SAM, but also offers a strategy for efficient enzyme engineering.

Discovery of an Imine Reductase for Reductive Amination of Carbonyl Compounds with Sterically Challenging Amines.

The synthesis of structurally diverse amines is of fundamental significance in the pharmaceutical industry due to the ubiquitous presence of amine motifs in biologically active molecules. Biocatalytic reductive amination for amine production has attracted great interest owing to its synthetic advantages. Herein, we report the direct synthesis of a wide range of sterically demanding secondary amines, including several important active pharmaceutical ingredients and pharmaceutical intermediates, via reductive amination of carbonyl substrates and bulky amine nucleophiles employing imine reductases. Key to success for this route is the identification of an imine reductase from Penicillium camemberti with unusual substrate specificity and its further engineering, which empowered the accommodation of a broad range of sterically demanding amine nucleophiles encompassing linear alkyl and (hetero)aromatic (oxy)alkyl substituents and the formation of final amine products with up to >99% conversion. The practical utility of the biocatalytic route has been demonstrated by its application in the preparative synthesis of the anti-hyperparathyroidism drug cinacalcet.

Engineering a norcoclaurine synthase for one-step synthesis of (S)-1-aryl-tetrahydroisoquinolines.

Tetrahydroisoquinoline alkaloids (THIQAs) are ubiquitous compounds with important pharmaceutical and biological activity. Their key N-heterocyclic structural motifs are synthesised via Pictet-Spengler (P-S) reaction by norcoclaurine synthases (NCS) in plants. The synthesis of 1-aryl-tetrahydroisoquinoline alkaloids has attracted increasing attention due to their antitumor and antivirus activities. Herein, the L68T/M97V mutant of NCS from Thalictrum flavum with improved activity was developed by semi-rational design. This mutant not only showed higher catalytic performance (> 96% conversion) toward benzaldehyde and dopamine over the wild-type enzyme, but also catalysed the P-S reaction of the bulky substrate 4-biphenylaldehyde and dopamine with high conversion (> 99%) for the effective synthesis of 1-aryl-THIQA. In terms of stereoselectivity, all products synthesised by the L68T/M97V mutant showed high optical purity (92-99% enantiomeric excess).

Regiospecific C-H amination of (-)-limonene into (-)-perillamine by multi-enzymatic cascade reactions.

BACKGROUND: (-)-Limonene, one of cyclic monoterpenes, is an important renewable compound used widely as a key building block for the synthesis of new biologically active molecules and fine chemicals. (-)-Perillamine, as derived from (-)-limonene, is a highly useful synthon for constructing more complicated and functionally relevant chemicals. AIM: We aimed to report a more sustainable and more efficient method for the regiospecific C-H amination of (-)-limonene into (-)-perillamine. RESULTS: Here, we report an artificial penta-enzymatic cascade system for the transformation of the cheap and easily available (-)-limonene into (-)-perillamine for the first time. This system is composed of cytochrome P450 monooxygenase, alcohol dehydrogenase and w-transaminase for the main reactions, as well as formate dehydrogenase and NADH oxidase for cofactor recycling. After optimization of the multi-enzymatic cascade system, 10 mM (-)-limonene was smoothly converted into 5.4 mM (-)-perillamine in a one-pot two-step biotransformation, indicating the feasibility of multi-enzymatic C7-regiospecific amination of the inert C-H bond of (-)-limonene. This method represents a concise and efficient route for the biocatalytic synthesis of derivatives from similar natural products.

One-pot biocatalytic route from cycloalkanes to α,ω-dicarboxylic acids by designed Escherichia coli consortia.

Aliphatic α,ω-dicarboxylic acids (DCAs) are a class of useful chemicals that are currently produced by energy-intensive, multistage chemical oxidations that are hazardous to the environment. Therefore, the development of environmentally friendly, safe, neutral routes to DCAs is important. We report an in vivo artificially designed biocatalytic cascade process for biotransformation of cycloalkanes to DCAs. To reduce protein expression burden and redox constraints caused by multi-enzyme expression in a single microbe, the biocatalytic pathway is divided into three basic Escherichia coli cell modules. The modules possess either redox-neutral or redox-regeneration systems and are combined to form E. coli consortia for use in biotransformations. The designed consortia of E. coli containing the modules efficiently convert cycloalkanes or cycloalkanols to DCAs without addition of exogenous coenzymes. Thus, this developed biocatalytic process provides a promising alternative to the current industrial process for manufacturing DCAs.

Rational Engineering of Formate Dehydrogenase Substrate/Cofactor Affinity for Better Performance in NADPH Regeneration.

Formate dehydrogenases are critical tools for nicotinamide cofactor regeneration, but their limited catalytic efficiency (kcat/Km) is a major drawback. A formate dehydrogenase from Burkholderia stabilis 15516 (BstFDH) was the first native NADP+-dependent formate dehydrogenase reported and has the highest kcat/Km toward NADP+ (kcat/KmNADP+) compared with other FDHs that can utilize NADP+ as a hydrogen acceptor. However, the substrate and cofactor affinities of BstFDH are inferior to those of other FDHs, making its practical application difficult. Herein, we engineered recombinant BstFDH to enhance its HCOO- and NADP+ affinities. Based on sequence information analysis and homologous modeling results, I124, G146, S262, and A287 were found to affect the binding affinity for HCOO- and NADP+. By combining these mutations, we identified a BstFDH variant (G146M/A287G) that reduced KmNADP+ to 0.09 mM, with a concomitant decrease in KmHCOO-, and gave 1.6-fold higher kcat/KmNADP+ than the wild type (WT). Furthermore, BstFDH I124V/G146H/A287G, with the lowest KmHCOO- of 8.51 mM, showed a catalytic efficiency that was 2.3-fold higher than that of the wild type and a decreased KmNADP+ of 0.11 mM. These results are beneficial for improving the performance of NADP+-dependent formate dehydrogenase in the NADPH regeneration of various bioreductive reactions and provide a useful guide for engineering of the substrate and cofactor affinity of other enzymes.

Evolution of Glucose Dehydrogenase for Cofactor Regeneration in Bioredox Processes with Denaturing Agents.

Glucose dehydrogenase (GDH) is a general tool for driving nicotinamide (NAD(P)H) regeneration in synthetic biochemistry. An increasing number of synthetic bioreactions are carried out in media containing high amounts of organic cosolvents or hydrophobic substrates/products, which often denature native enzymes, including those for cofactor regeneration. In this work, we attempted to improve the chemical stability of Bacillus megaterium GDH (BmGDHM0 ) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDHM6 (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDHM6 was also more stable than BmGDHM0 when exposed to hydrophobic and enzyme-inactivating compounds such as acetophenone, ethyl 2-oxo-4-phenylbutyrate, and ethyl (R)-2-hydroxy-4-phenylbutyrate. Coupled with a Candida glabrata carbonyl reductase, BmGDHM6 was successfully used for the asymmetric reduction of deactivating ethyl 2-oxo-4-phenylbutyrate with total turnover number of 1800 for the nicotinamide cofactor, thus making it attractive for commercial application. Overall, the evolution of chemically robust GDH facilitates its wider use as a general tool for NAD(P)H regeneration in biocatalysis.

Stereocomplementary Synthesis of Pharmaceutically Relevant Chiral 2-Aryl-Substituted Pyrrolidines Using Imine Reductases.

Exploring a collection of naturally occurring imine reductases (IREDs) identified two stereocomplementary IREDs with reducing activity toward sterically hindered 2-aryl-substituted pyrrolines. Using (R)-selective ScIR and (S)-selective SvIR, various chiral 2-aryl-substituted pyrrolidines with excellent enantioselectivity (>99% ee) were stereocomplementarily synthesized in good yield (60-80%), demonstrating the feasibility of IREDs for generating pharmaceutically relevant chiral 2-aryl-substituted pyrrolidine intermediates.

Coevolution of the Activity and Thermostability of an ϵ-Keto Ester Reductase for Better Synthesis of an (R)-α-Lipoic Acid Precursor.

In this work, we have identified a significantly improved variant (S131Y/Q252I) of the natural ϵ-keto ester reductase CpAR2 from Candida parapsilosis for efficiently manufacturing (R)-8-chloro-6-hydroxyoctanoic acid [(R)-ECHO] through co-evolution of activity and thermostability. The activity of the variant CpAR2S131Y/Q252I towards the ϵ-keto ester ethyl 8-chloro-6-oxooctanoate was improved to 214 U mg-1 -from 120 U mg-1 in the case of the wild-type enzyme (CpAR2WT )-and the half-deactivating temperature (T50 , for 15 min incubation) was simultaneously increased by 2.3 °C in relation to that of CpAR2WT . Consequently, only 2 g L-1 of lyophilized E. coli cells harboring CpAR2S131Y/Q252I and a glucose dehydrogenase (GDH) were required in order to achieve productivity similar to that obtained in our previous work, under optimized reaction conditions (530 g L-1 d-1 ). This result demonstrated a more economical and efficient process for the production of the key (R)-α-lipoic acid intermediate ethyl 8-chloro-6-oxooctanoate.

An Ammonium-Formate-Driven Trienzymatic Cascade for ω-Transaminase-Catalyzed (R)-Selective Amination.

(R)-Amination mediated by (R)-specific ω-transaminases generally requires costly d-alanine in excess to obtain the desired chiral amines in high yield. Herein, a one-pot, trienzymatic cascade comprising an (R)-specific ω-transaminase, an amine dehydrogenase, and a formate dehydrogenase was developed for the economical and eco-friendly synthesis of (R)-chiral amines. Using inexpensive ammonium formate as the sole sacrificial agent, the established cascade system enabled efficient ω-transaminase-mediated (R)-amination of various ketones, with high conversions and excellent ee (>99%); water and CO2 were the only waste products.

One-Pot Enzyme Cascade for Controlled Synthesis of Furancarboxylic Acids from 5-Hydroxymethylfurfural by H2 O2 Internal Recycling.

Furancarboxylic acids are promising biobased building blocks in pharmaceutical and polymer industries. In this work, dual-enzyme cascade systems composed of galactose oxidase (GOase) and alcohol dehydrogenases (ADHs) are constructed for controlled synthesis of 5-formyl-2-furancarboxylic acid (FFCA) and 2,5-furandicarboxylic acid (FDCA) from 5-hydroxymethylfurfural (HMF), based on the catalytic promiscuity of ADHs. The byproduct H2 O2 , which is produced in GOase-catalyzed oxidation of HMF to 2,5-diformylfuran (DFF), is used for horseradish peroxidase (HRP)-mediated regeneration of the oxidized nicotinamide cofactors for subsequent oxidation of DFF promoted by an ADH, thus implementing H2 O2 internal recycling. The desired products FFCA and FDCA are obtained with yields of more than 95 %.

Efficient Synthesis of Methyl 3-Acetoxypropionate by a Newly Identified Baeyer-Villiger Monooxygenase.

Baeyer-Villiger monooxygenases (BVMOs) are an emerging class of promising biocatalysts for the oxidation of ketones to prepare corresponding esters or lactones. Although many BVMOs have been reported, the development of highly efficient enzymes for use in industrial applications is desirable. In this work, we identified a BVMO from Rhodococcus pyridinivorans (BVMORp) with a high affinity toward aliphatic methyl ketones (Km < 3.0 µM). The enzyme was highly soluble and relatively stable, with a half-life of 23 h at 30°C and pH 7.5. The most effective substrate discovered so far is 2-hexanone (kcat = 2.1 s-1; Km = 1.5 µM). Furthermore, BVMORp exhibited excellent regioselectivity toward most aliphatic ketones, preferentially forming typical (i.e., normal) products. Using the newly identified BVMORp as the catalyst, a high concentration (26.0 g/liter; 200 mM) of methyl levulinate was completely converted to methyl 3-acetoxypropionate after 4 h, with a space-time yield of 5.4 g liter-1 h-1 Thus, BVMORp is a promising biocatalyst for the synthesis of 3-hydroxypropionate from readily available biobased levulinate to replace the conventional fermentation.IMPORTANCE BVMOs are emerging as a green alternative to traditional oxidants in the BV oxidation of ketones. Although many BVMOs are discovered and used in organic synthesis, few are really applied in industry, especially in the case of aliphatic ketones. Herein, a highly soluble and relatively stable monooxygenase from Rhodococcus pyridinivorans (BVMORp) was identified with high activity and excellent regioselectivity toward most aliphatic ketones. BVMORp possesses unusually high substrate loading during the catalysis of the oxidation of biobased methyl levulinate to 3-hydroxypropionic acid derivatives. This study indicates that the synthesis of 3-hydroxypropionate from readily available biobased levulinate by BVMORp-catalyzed oxidation holds great promise to replace traditional fermentation.

Direct Access to Medium-Chain α,ω-Dicarboxylic Acids by Using a Baeyer-Villiger Monooxygenase of Abnormal Regioselectivity.

Baeyer-Villiger monooxygenases (BVMOs) are versatile biocatalysts in organic synthesis that can generate esters or lactones by inserting a single oxygen atom adjacent to a carbonyl moiety. The regioselectivity of BVMOs is essential in determining the ratio of two regioisomers for converting asymmetric ketones. Herein, we report a novel BVMO from Pseudomonas aeruginosa (PaBVMO); this has been exploited for the direct synthesis of medium-chain α,ω-dicarboxylic acids through a Baeyer-Villiger oxidation-hydrolysis cascade. PaBVMO displayed the highest abnormal regioselectivity toward a variety of long-chain aliphatic keto acids (C16 -C20 ) to date, affording dicarboxylic monoesters with a ratio of up to 95 %. Upon chemical hydrolysis, α,ω-dicarboxylic acids and fatty alcohols are readily obtained without further treatment; this significantly reduces the synthetic steps of α,ω-dicarboxylic acids from renewable oils and fats.

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.

Recent progress on deep eutectic solvents in biocatalysis.

Deep eutectic solvents (DESs) are eutectic mixtures of salts and hydrogen bond donors with melting points low enough to be used as solvents. DESs have proved to be a good alternative to traditional organic solvents and ionic liquids (ILs) in many biocatalytic processes. Apart from the benign characteristics similar to those of ILs (e.g., low volatility, low inflammability and low melting point), DESs have their unique merits of easy preparation and low cost owing to their renewable and available raw materials. To better apply such solvents in green and sustainable chemistry, this review firstly describes some basic properties, mainly the toxicity and biodegradability of DESs. Secondly, it presents several valuable applications of DES as solvent/co-solvent in biocatalytic reactions, such as lipase-catalyzed transesterification and ester hydrolysis reactions. The roles, serving as extractive reagent for an enzymatic product and pretreatment solvent of enzymatic biomass hydrolysis, are also discussed. Further understanding how DESs affect biocatalytic reaction will facilitate the design of novel solvents and contribute to the discovery of new reactions in these solvents.

Identification of an Imine Reductase for Asymmetric Reduction of Bulky Dihydroisoquinolines.

A new imine reductase from Stackebrandtia nassauensis (SnIR) was identified, which displayed over 25- to 1400-fold greater catalytic efficiency for 1-methyl-3,4-dihydroisoquinoline (1-Me DHIQ) compared to other imine reductases reported. Subsequently, an efficient SnIR-catalyzed process was developed by simply optimizing the amount of cosolvent, and up to 15 g L-1 1-Me DHIQ was converted completely without a feeding strategy. Furthermore, the reaction proceeded well for a panel of dihydroisoquinolines, affording the corresponding tetrahydroisoquinolines (mostly in S-configuration) in good yields (up to 81%) and with moderate to excellent enantioselectivities (up to 99% ee).

Highly efficient bioreduction of 2-hydroxyacetophenone to (S)- and (R)-1-phenyl-1,2-ethanediol by two substrate tolerance carbonyl reductases with cofactor regeneration.

Optically pure 1-phenyl-1,2-ethanediol is a very important chiral building block and intermediate in fine chemical and pharmaceutical industries. Reduction of 2-hydroxyacetophenone provides a straightforward approach to access these important compounds. In this study, two enantiocomplementary carbonyl reductases, BDHA (2,3-butanediol dehydrogenase from Bacillus subtilis) and GoSCR (polyol dehydrogenase from Gluconobacter oxydans) were discovered for the first time to convert 2-hydroxyacetophenone (2-HAP) to (R)-1-phenyl-1,2-ethanediol ((R)-PED) and (S)-1-phenyl-1,2-ethanediol ((S)-PED) with excellent stereochemical selectivity, respectively. The two enzymes were purified and characterized. In vitro bioreduction of 2-HAP catalyzed by BDHA and GoSCR coupled with glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration were demonstrated, affording both (R)-PED and (S)-PED in>99% ee and 99% conversion. Recombinant Escherichia coli whole cells co-expressing both GDH and BDHA or GoSCR genes were used to asymmetric reduction of 2-HAP to (R)-PED or (S)-PED. Under the optimized conditions, the bioreduction of 400mM (54g/L) substrate was proceeded smoothly without the external addition of cofactor, and the product (R)-PED and (S)-PED were obtained with 99% yield, >99% ee and 18.0g/L/h volumetric productivity. These results offer a practical biocatalytic method for the preparation of both (R)-PED and (S)-PED with high volumetric productivity.

Engineering of a novel carbonyl reductase with coenzyme regeneration in E. coli for efficient biosynthesis of enantiopure chiral alcohols.

The novel anti-Prelog stereospecific carbonyl reductase from Acetobacter sp. CCTCC M209061 was successfully expressed in E. coli combined with glucose dehydrogenase (GDH) to construct an efficient whole-cell biocatalyst with coenzyme NADH regeneration. The enzymatic activity of GAcCR (AcCR with a GST tag) reached 304.9U/g-dcw, even 9 folds higher than that of wild strain, and the activity of GDH for NADH regeneration recorded 46.0U/mg-protein in the recombinant E. coli. As a whole-cell biocatalyst, the recombinant E. coli BL21(DE3)pLysS (pETDuet-gaccr-gdh) possessed a broad substrate spectrum for kinds of carbonyl compounds with encouraging yield and stereoselectivity. Besides, the asymmetric reduction of ethyl 4-chloroacetoacetate (COBE) to optically pure ethyl 4-chloro-3-hydroxybutyrate (CHBE) catalyzed by the whole-cell biocatalyst was systematically investigated. Under the optimal reaction conditions, the optical purity of CHBE was over 99% e.e. for (S)-enantiomer, and the initial rate and product yield reached 8.04µmol/min and 99.4%, respectively. Moreover, the space-time yield was almost 20 folds higher than that catalyzed by the wild strain. Therefore, a new, high efficiency biocatalyst for asymmetric reductions was constructed successfully, and the enantioselective reduction of prochiral compounds using the biocatalyst was a promising approach for obtaining enantiopure chiral alcohols.

Cloning and Characterization of an Enantioselective l-Menthyl Benzoate Hydrolase from Acinetobacter sp. ECU2040.

A new esterase gene abmbh, encoding a benzoate hydrolase which can enantioselectively hydrolyze l-menthyl benzoate to l-menthol, was recently identified from the genomic library of a soil isolate Acinetobacter sp. ECU2040. The abmbh gene contains a 1080-bp open reading frame encoding a protein of 360 amino acids with a calculated molecular mass of 40.7 kDa. The corresponding enzyme AbMBH was functionally expressed in Escherichia coli BL21 (DE3), purified, and characterized. The AbMBH displayed the maximum activity towards p-nitrophenyl butyrate at 50 °C, and an optimum pH of 8.5. A K M of 2.6 mM and a k cat of 0.26 s(-1) were observed towards dl-menthyl benzoate. The AbMBH exhibited a moderate enantioselectivity (E = 27.5) towards dl-menthyl benzoate. It can also catalyze the enantioselective hydrolysis of a variety of racemic menthyl esters, including dl-menthyl acetate, dl-menthyl chloroacetate, and dl-menthyl butyrate.