Characterization of Phenylalanine Ammonia Lyases from Lettuce (Lactuca sativa L.) as Robust Biocatalysts for the Production of d- and l-Amino Acids.

Phenylalanine ammonia lyase (PAL) catalyzes the reversible conversion of l-phenylalanine into the corresponding trans-cinnamic acid, providing a route to optically pure α-amino acids. We explored the catalytic function of all five PALs encoded in the genome of lettuce (Lactuca sativa L.) that are previously known to be involved in wound browning. All LsPALs were active toward l-phenylalanine in the ammonia elimination reaction and displayed maximum activity at 55-60 °C and pH 9.0-9.5. However, four of them, LsPAL1-LsPAL4, showed significantly higher activity and thermal stability than LsPAL5, as well as a broader substrate spectrum including some challenging substrates with steric demanding or electron-donating substituents. The best one LsPAL3 was subjected to the kinetic resolution of a panel of 21 rac-phenylalanine derivatives, as well as the ammonia addition of 21 cinnamic acid derivatives. It showed excellent enantioselectivity in most cases and significantly better activity than previously described PALs for a number of challenging non-natural substrates, demonstrating its great potential in biocatalysis.

Thermostabilizing ketoreductase ChKRED20 by consensus mutagenesis at dimeric interfaces.

Protein stability is crucial in enzymatic catalysis. To improve the efficiency in the searching for thermostablizing mutations, we applied a sequence consensus approach focusing on dimeric interface residues of ketoreductase ChKRED20. The strategy returned a success rate of 43%, revealing 9 beneficial mutations from 21 candidates with improved kinetic or thermodynamic stability. Several combinatorial mutants were then constructed, and mutant M8K displayed the highest thermostability, with a melting temperature (Tm) of 89 °C and a half-inactivation temperature (T50) of 93.4 °C, both of over 35 °C increase compared to the wild-type. M8K could remain stable for at least 7 days at its optimal reaction temperature of 55 °C. Its inactivation half-life (t1/2) was 110 min at 90 °C, while the wild-type was 18.6 min at 60 °C. The results were interpreted in the context of structural and molecular dynamic simulation analysis, which revealed the addition of intramolecular interactions, decreased conformational flexibility and increased compactness, all in agreement with the observed effect.

Asymmetric Epoxidation and Sulfoxidation Catalyzed by a New Styrene Monooxygenase from Bradyrhizobium.

Asymmetric epoxidation catalyzed with styrene monooxygenase (SMO) is a powerful enzymatic process producing enantiopure styrene epoxide derivatives. To establish a more diversified reservoir of SMOs, a new SMO from Bradyrhizobium sp. ORS 375, named BrSMO, was mined from the database and characterized. BrSMO was constituted of an epoxygenase component of 415 amino acid residues and an NADH-dependent flavin reductase component of 175 residues. BrSMO catalyzed the epoxidation of styrene and 7 more styrene derivatives, yielding the corresponding (S)-epoxides with excellent enantiomeric excesses (95- > 99% ee), with the highest activity achieved for styrene. BrSMO also catalyzed the asymmetric sulfoxidation of 7 sulfides, producing the corresponding (R)-sulfoxides (20-90% ee) with good yields.

Structure-guided engineering of ChKRED20 from Chryseobacterium sp. CA49 for asymmetric reduction of aryl ketoesters.

ChKRED20 is a robust NADH-dependent ketoreductase identified from the genome of Chryseobacterium sp. CA49 that can use 2-propanol as the ultimate reducing agent. The wild-type can reduce over 100 g/l ketones for some pharmaceutical relevant substrates, exhibiting a remarkable potential for industrial application. In this work, to overcome the limitation of ChKRED20 to aryl ketoesters, we first refined the X-ray crystal structure of ChKRED20/NAD+ complex at a resolution of 1.6 Å, and then performed three rounds of iterative saturation mutagenesis at critical amino acid sites to reshape the active cavity of the enzyme. For methyl 2-oxo-2-phenylacetate and ethyl 3-oxo-3-phenylpropanoate, several gain-of-activity mutants were achieved, and for ethyl 2-oxo-4-phenylbutanoate, improved mutants were achieved with kcat/Km increasing to 196-fold of the wild-type. All three substrates were completely reduced at 100 g/l loading catalyzed with selected ChKRED20 mutants, and deliver the corresponding chiral alcohols with >90% isolated yield and 97 - >99%ee.

Stereoselective Bioreduction of Ethyl 3-Oxo-3-(2-Thienyl) Propanoate Using the Short-Chain Dehydrogenase/Reductase ChKRED12.

Ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate((S)-HEES)acts as a key chiral intermediate for the blockbuster antidepressant drug duloxetine, which canbe achieved viathe stereoselective bioreduction ofethyl 3-oxo-3-(2-thienyl) propanoate (KEES) that containsa 3-oxoacyl structure.The sequences of the short-chain dehydrogenase/reductases from Chryseobacterium sp. CA49 were analyzed, and the putative3-oxoacyl-acyl-carrier-protein reductase, ChKRED12, was able to stereoselectivelycatalyze theNADPH-dependent reduction to produce (S)-HEES.The reductase activity of ChKRED12 towardsothersubstrates with 3-oxoacyl structure were confirmed with excellent stereoselectivity (>99% enantiomeric excess) in most cases. When coupled with a cofactor recycling system using glucose dehydrogenase, the ChKRED12 was able to catalyze the complete conversion of 100 g/l KEES within 12h, yielding the enantiopure product with >99% ee, showing a remarkable potential to produce (S)-HEES.

Single mutations of ketoreductase ChKRED20 enhance the bioreductive production of (1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol.

(1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol ((S)-CFPL) is an intermediate for the drug ticagrelor, and is manufactured via chemical approaches. To develop a biocatalytic solution to (S)-CFPL, an inventory of ketoreductases from Chryseobacterium sp. CA49 were rescreened, and ChKRED20 was found to catalyze the reduction of the ketone precursor with excellent stereoselectivity (>99 % ee). After screening an error-prone PCR library of the wild-type ChKRED20, two mutants, each bearing a single amino acid substitution of H145L or L205M, were identified with significantly increased activity. Then, the two critical positions were each randomized by constructing saturation mutagenesis libraries, which delivered several mutants with further enhanced activity. Among them, the mutant L205A was the best performer with a specific activity of 178 µmol/min/mg, ten times of that of the wild-type. Its k cat/K m increased by 15 times and half-life at 50 °C increased by 70 %. The mutant catalyzed the complete conversion of 150 and 200 g/l substrate within 6 and 20 h, respectively, to yield enantiopure (S)-CFPL with an isolated yield of 95 %.

Rapid asymmetric reduction of ethyl 4-chloro-3-oxobutanoate using a thermostabilized mutant of ketoreductase ChKRED20.

Ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) is an important chiral intermediate for the synthesis of "blockbuster" drug statins. The carbonyl reductase ChKRED20 from Chryseobacterium sp. CA49 was found to catalyze the bio-reductive production of (S)-CHBE with excellent stereoselectivity (>99.5 % ee). Perceiving a capacity for improvement, we sought to increase the thermostability of ChKRED20 to allow a higher reaction temperature. After one round of error-prone PCR (epPCR) library screening followed by the combination of beneficial mutations, a triple-mutant MC135 was successfully achieved with substantially enhanced thermostablity. The activity of MC135 at 50 °C was similar to the wild type. However, at its temperature optima of 65 °C, the mutant displayed 63 % increase of activity compared to the wild type and remained >95 % activity after being incubated for 15 days, while the wild type had a half-life of 11.9 min at 65 °C. At a substrate/catalyst ratio of 100 (w/w), the mutant catalyzed the complete conversion of 300 g/l substrate within 1 h to yield enantiopure (S)-CHBE with an isolated yield of 95 %, corresponding to a space-time yield of 1824 mM/h.

Removal of the free cysteine residue reduces irreversible thermal inactivation of feruloyl esterase: evidence from circular dichroism and fluorescence spectra.

Feruloyl esterase A from Aspergillus niger (AnFaeA) contains three intramolecular disulfide bonds and one free cysteine at position 235. Saturated mutagenesis at Cys235 was carried out to produce five active mutants, all of which displayed unusual thermal inactivation patterns with the most residual activity achieved at 75°C, much higher than the parental AnFaeA. But their optimal reaction temperatures were lower than the parental AnFaeA. Extensive investigation into their free thiol and disulfide bond, circular dichroism spectra and fluorescence spectra revealed that the unfolding of the parental enzyme was irreversible on all the tested conditions, while that of the Cys235 mutants was reversible, and their ability to refold was highly dependent on the denaturing temperature. Mutants denatured at 75°C were able to efficiently reverse the unfolding to regain native structure during the cooling process. This study provided valid evidence that free cysteine substitutions can reduce irreversible thermal inactivation of proteins.

An enoate reductase Achr-OYE4 from Achromobacter sp. JA81: characterization and application in asymmetric bioreduction of C=C bonds.

A putative enoate reductase, Achr-OYE4, was mined from the genome of Achromobacter sp. JA81, expressed in Escherichia coli, and was characterized. Sequence analysis and spectral properties indicated that Achr-OYE4 is a typical flavin mononucleotide-dependent protein; it preferred NADH over NADPH as a cofactor. The heterologously expressed protein displayed good activity and excellent stereoselectivity toward some activated alkenes in the presence of NADH, NADPH, or their recycling systems. The glucose dehydrogenase-based recycling system yielded the best results in most cases, with a product yield of up to 99 % and enantiopurity of >99 % ee. Achr-OYE4 is an important addition to the asymmetric reduction reservoir as an "old yellow enzyme" from Achromobacter.

Design of mutants for enhanced thermostability of ß-glycosidase BglY from Thermus thermophilus.

Three design strategies, based on rational and semi-rational approaches, were employed to investigate the functional impact of thermostability-related amino acid substitutions in the ß-glycosidase BglY from Thermus thermophilus. Five beneficial mutations were identified, of which 1 mutation was located in the active cavity of the enzyme and contributed to the released substrate inhibition. Combining all 5 beneficial substitutions resulted in the mutant HF5 with a 4.7-fold increase in half-life, with thermal inactivation at 93 °C, and complete lack of substrate inhibition toward the substrate p-nitrophenyl-ß-D-glucopyranoside at lower reaction temperatures. The results of this study provide valuable information on amino acid substitutions related to thermostability and substrate inhibition of BglY.

Multiple amino acid substitutions significantly improve the thermostability of feruloyl esterase A from Aspergillus niger.

Feruloyl esterase A from Aspergillus niger (AnFaeA) is one of the most important feruloyl esterases of industrial relevance. Previous work aided by the PoPMuSiC algorithm has identified two beneficial mutants (D93G and S187F) with thermostabilization effect. In this work, twelve additional amino acid substitutions were identified to be beneficial to the thermostability of AnFaeA after screening a random mutagenesis library constructed in Pichia pastoris. Combination of these mutations resulted in a mutant with 80% residual activity after heat treatment at 90 °C for 15 min and a half-life increasing from 15 min to >4000 min at 55 °C. The thermostabilized mutant displayed significantly enhanced performance compared to the parental AnFaeA when applied to the treatment of steam-exploded corn stalk at 60 °C together with an xylanase, demonstrating its great potential for industrial application.

Asymmetric bioreduction of activated alkenes by a novel isolate of Achromobacter species producing enoate reductase.

The strain Achromobacter sp. JA81, which produced enoate reductase, was applied in the asymmetric reduction of activated alkenes. The strain could catalyze the bioreduction of alkenes to form enantiopure (R)-ß-aryl-ß-cyano-propanoic acids, a precursor of (R)-γ-amino butyric acids, including the pharmaceutically active enantiomer of the chiral drug (R)-baclofen with excellent enantioselectivity. It could catalyze as well the stereoselective bioreduction of other activated alkenes such as cyclic imides, ß-nitro acrylates, and nitro-alkenes with up to >99 % ee and >99 % conversion. The draft genome sequencing of JA81 revealed six putative old yellow enzyme homologies, and the transcription of one of them, Achr-OYE3, was detected using reverse transcription polymerase chain reaction. The recombinant Escherichia coli expressing Achr-OYE3 displayed enoate reductase activity toward (Z)-3-cyano-3-phenyl-propenoic acid (2a).

Three amino acid changes contribute markedly to the thermostability of ß-glucosidase BglC from Thermobifida fusca.

Thermostability of ß-glucosidase was enhanced by family shuffling, site saturation mutagenesis, and site-directed mutagenesis. Family shuffling was carried out based on ß-glucosidase BglC from Thermobifida fusca and ß-glucosidase BglB from Paebibacillus polymxyxa with the help of synthetic primers. High-throughput screening revealed mutants with higher thermostability than both parental enzymes. The most thermostable mutant VM2 containing three key amino acid changes in L444Y/G447S/A433V had a 144-fold increase in half-life of inactivation as compared to the parental enzyme BglC. The mutant VM2 showed 28% and 94% increase in k(cat) towards p-nitrophenyl-ß-D-glucopyranoside (pNPG) and cellobiose, respectively. The mutant with enhanced stability would facilitate the recycle of ß-glucosidase in the bioconversion of cellulosic biomass.

Introduction of glycine and proline residues onto protein surface increases the thermostability of endoglucanase CelA from Clostridium thermocellum.

A saturation mutagenesis library was constructed at the position 329 of the endoglucanase CelA from Clostridium thermocellum based on previous results (Yi and Wu, 2010), and one mutation, S329G, was identified to contribute to the enhanced thermostability. The result inspired a rational design approach focusing on the introduction of Gly or Pro residue onto the protein surface, which led to the identification of two additional beneficial mutations, H194G and S269P. Combination of these three mutations resulted in a mutant with a 10-fold increase in half-life of inactivation (60 min) at 86°C without compromising activity compared with the wild-type. Its reaction temperature for maximum activity increased from 75 to 85°C. The results provide valuable thermostability-related structural information on this thermophilic enzyme.

Improving the thermostability of Geobacillus stearothermophilus xylanase XT6 by directed evolution and site-directed mutagenesis.

Protein engineering of the thermostable xylanase XT6 from Geobacillus stearothermophilus was performed to obtain enzymes with improved thermal tolerance. Mutants producing such enzymes were obtained after several rounds of directed evolution using error-prone PCR and sequence family shuffling, in combination with a consensus-based semi-rational approach. The most thermostable mutant enzyme contained 13 amino acid substitutions and its half-life of inactivation was 52-fold of that of the wild-type. Its reaction temperature for maximum activity increased from 77 degrees C to 87 degrees C, and catalytic efficiency (k(cat)/K(m)) increased by 90%. The mutant is of potential interest for industrial applications.

Increased virulence using engineered protease-chitin binding domain hybrid expressed in the entomopathogenic fungus Beauveria bassiana.

Insect cuticles consist mainly of interlinked networks of proteins and the highly insoluble polysaccharide, chitin. Entomopathogenic fungi, such as Beauveria bassiana, invade insects by direct penetration of host cuticles via the action of diverse hydrolases including proteases and chitinases coupled to mechanical pressure. In order to better target cuticle protein-chitin structures and accelerate penetration speed, a hybrid protease (CDEP-BmChBD) was constructed by fusion of a chitin binding domain BmChBD from Bombyx mori chitinase to the C-terminal of CDEP-1, a subtilisin-like protease from B. bassiana. Compared to the wild-type, the hybrid protease was able to bind chitin and released greater amounts of peptides/proteins from insect cuticles. The insecticidal activity of B. bassiana was enhanced by including proteases, CDEP-1 or CDEP:BmChBD produced in Pichia pastoris, as an additive, however, the augment effect of CDEP:BmChBD was significantly higher than that of CDEP-1. Expression of the hybrid protease in B. bassiana also significantly increased fungal virulence compared to wild-type and strains overexpressing the native protease. These results demonstrate that rational design virulence factor is a potential strategy for strain improvement by genetic engineering.

Enhanced bacterial expression of several mammalian cytochrome P450s by codon optimization and chaperone coexpression.

To elucidate the effects of codon optimization and chaperone coexpression on the heterologous expression of mammalian cytochrome P450s (P450) in Escherichia coli, the expression of P450s 2B1, 2S1, 2U1, 2W1, and 27C1 were investigated. With codon optimization for N-terminus or the entire gene, the expression levels of P450 27C1, 2U1 and 2W1 increased 22-fold, 3.6-fold and 2.1-fold, respectively, while those for P450s 2B1 and 2S1 remained unchanged. With coexpression of E. coli molecular chaperones GroEL/ES, the expression level increased up to 14-fold for P450 27C1, and 3- to 5-fold for P450s 2B1, 2S1, and 2W1. Simultaneous application of these two techniques resulted in synergetic effects.

Expression of a Beauveria bassiana chitinase (Bbchit1) in Escherichia coli and Pichia pastoris.

Beauveria bassiana chitinase (Bbchit1) is an important cuticle degrading enzyme involved in pathogenesis of fungi against insect. To obtain enough active chitinase for performing in vitro functional analysis, Bbchit1 gene was expressed in Escherichia coli and Pichia pastoris, respectively. The high-level production of recombinant Bbchit1 was detected in E. coli expression system, however mainly located in inclusion bodies. Refolding of solubilized inclusion body proteins was achieved by dialysis. In P. pastoris expression system, Bbchit1 was secreted into the culture medium under the induction of methanol. Active Bbchit1 was purified to near 90% purity from culture medium by desalting chromatography and anion exchange chromatography. The yield of Bbchit1 produced by P. pastoris was estimated at 153 mg/L, significantly higher than that of the refolded Bbchit1 from E. coli inclusion bodies (50 mg/L). Additionally, the specific activity of Bbchit1 from P. pastoris was also higher than that from E. coli (3.9 U/mg versus 2.8 U/mg). These results indicated P. pastoris was a convenient expression system for the efficient production of Bbchit1.

Increased insect virulence in Beauveria bassiana strains overexpressing an engineered chitinase.

Entomopathogenic fungi are currently being used for the control of several insect pests as alternatives or supplements to chemical insecticides. Improvements in virulence and speed of kill can be achieved by understanding the mechanisms of fungal pathogenesis and genetically modifying targeted genes, thus improving the commercial efficacy of these biocontrol agents. Entomopathogenic fungi, such as Beauveria bassiana, penetrate the insect cuticle utilizing a plethora of hydrolytic enzymes, including chitinases, which are important virulence factors. Two chitinases (Bbchit1 and Bbchit2) have previously been characterized in B. bassiana, neither of which possesses chitin-binding domains. Here we report the construction and characterization of several B. bassiana hybrid chitinases where the chitinase Bbchit1 was fused to chitin-binding domains derived from plant, bacterial, or insect sources. A hybrid chitinase containing the chitin-binding domain (BmChBD) from the silkworm Bombyx mori chitinase fused to Bbchit1 showed the greatest ability to bind to chitin compared to other hybrid chitinases. This hybrid chitinase gene (Bbchit1-BmChBD) was then placed under the control of a fungal constitutive promoter (gpd-Bbchit1-BmChBD) and transformed into B. bassiana. Insect bioassays showed a 23% reduction in time to death in the transformant compared to the wild-type fungus. This transformant also showed greater virulence than another construct (gpd-Bbchit1) with the same constitutive promoter but lacking the chitin-binding domain. We utilized a strategy where genetic components of the host insect can be incorporated into the fungal pathogen in order to increase host cuticle penetration ability.