Directed evolution of a Baeyer–Villiger monooxygenase to enhance enantioselectivity

The Baeyer-Villiger monooxygenase (BVMO) BmoF1 from Pseudomonas fluorescens DSM 50106 was shown before to enantioselectively oxidize different 4-hydroxy-2-ketones to the corresponding hydroxyalkyl acetates, being the first example of a BVMO-catalyzed kinetic resolution of aliphatic acyclic ketones. However, the wild-type enzyme exhibited only moderate E values (E approximately 55). Thus, the enantioselectivity was enhanced by means of directed evolution and optimization of reaction conditions since it was found that higher E values (E approximately 70 for wild-type BmoF1) could already be obtained when performing biotransformations in shake flasks rather than small tubes. In a first step, random mutations were introduced by error-prone polymerase chain reaction, and BmoF1 mutants (>3,500 clones) were screened for improved activity and enantioselectivity using a microtiter-plate-based screening method. Mutations S136L and L252Q were found to increase conversion compared to wild type, while several mutations (H51L, F225Y, S305C, and E308V) were identified enhancing the enantioselectivity to a varying extent (E approximately 75-90). In a second step, beneficial mutations were recombined by consecutive cycles of QuikChange site-directed mutagenesis resulting in a double mutant (H51L/S136L) showing both improved conversion and enantioselectivity (E approximately 86).     

Optimization of chemoenzymatic Baeyer–Villiger oxidation of cyclohexanone to ε-caprolactone using response surface methodology

ε-Caprolactone (ε-CL) has attracted a great deal of attention and a high product concentration is of great significance for reducing production cost. The optimization of ε-CL synthesis through chemoenzymatic Baeyer–Villiger oxidation mediated by immobilized Trichosporon laibacchii lipase was studied using response surface methodology (RSM). The yield of ε-CL was 98.06% with about 1.2 M ε-CL concentration that has a substantial increase mainly due to both better stability of the cross-linked immobilized lipase used and the optimum reaction conditions in which the concentration of cyclohexanone was 1.22 M, the molar ratio of cyclohexanone:urea hydrogen peroxide (UHP) was 1:1.3, and the reaction temperature was 56.5°C. Based on our experimental results, it can be safely concluded that there are three reactions in this reaction system, not just two reactions, in which the third reaction is that the acetic acid formed reacts with UHP to form peracetic acid in situ catalyzed by the immobilized lipase. A quadratic polynomial model based on RSM experimental results was developed and the R² value of the equation is 0.9988, indicating that model can predict the experimental results with high precision. The experimental results also show that the molar ratio of cyclohexanone to UHP has very significant impact on the yield of ε-CL (p < .0006).     

Baeyer–Villiger oxidation with peracid generated in situ by CaLB-CLEA catalyzed perhydrolysis

Candida antarctica lipase B, immobilized as cross linked enzyme aggregates (CLEAs) was used to mediate the Baeyer–Villiger oxidation of cyclohexanone to ɛ-caprolactone, and the reaction was compared with the one using Novozym^® 435 as catalyst. The conversion was dependent on the initial concentration of cyclohexanone, and was about 90% after 48 h at concentrations of up to 0.25 M but was decreased at higher concentrations. Caprolactone concentrations up to 0.6 M had no effect on the reaction efficiency. Among the cyclic ketones tested, the highest degree of conversion was achieved for cyclopentanone (88%) and the lowest for cyclooctanone (about 2%). The effect of methyl substitution and position of substitution on the cycloketone was studied using methylcyclohexanone and it has shown to influence the conversion efficiency. Both hydrogen peroxide and the reaction by-product acetic acid had a deleterious effect on the stability of the biocatalyst.     

Control of chemoselectivity of microbial reaction with resin adsorbent: enhancement of Baeyer–Villiger oxidation over reduction

Amberlite XAD-7, a hydrophobic polymer, was used to change microbial reaction of ketones from reduction to Baeyer–Villiger (BV) oxidation. Thus, D. magnusii NBRC 4600 and G. reessii NBRC 1112 could catalyze the BV reaction of ketones in the presence of the polymer while reduction of the substrates proceeded, and BV oxidation was scarcely found in the absence of the polymer.     

Discovery of a thermostable Baeyer–Villiger monooxygenase by genome mining

Baeyer–Villiger monooxygenases represent useful biocatalytic tools, as they can catalyze reactions which are difficult to achieve using chemical means. However, only a limited number of these atypical monooxygenases are available in recombinant form. Using a recently described protein sequence motif, a putative Baeyer–Villiger monooxygenase (BVMO) was identified in the genome of the thermophilic actinomycete Thermobifida fusca. Heterologous expression of the respective protein in Escherichia coli and subsequent enzyme characterization showed that it indeed represents a BVMO. The NADPH-dependent and FAD-containing monooxygenase is active with a wide range of aromatic ketones, while aliphatic substrates are also converted. The best substrate discovered so far is phenylacetone (k_cat = 1.9 s^–1, K_M = 59 M). The enzyme exhibits moderate enantioselectivity with -methylphenylacetone (enantiomeric ratio of 7). In addition to Baeyer–Villiger reactions, the enzyme is able to perform sulfur oxidations. Different from all known BVMOs, this newly identified biocatalyst is relatively thermostable, displaying an activity half-life of 1 day at 52°C. This study demonstrates that, using effective annotation tools, genomes can efficiently be exploited as a source of novel BVMOs.     

Discovery of Baeyer–Villiger monooxygenases from photosynthetic eukaryotes

Baeyer–Villiger monooxygenases are attractive “green” catalysts able to produce chiral esters or lactones starting from ketones. They can act as natural equivalents of peroxyacids that are the catalysts classically used in the organic synthesis reactions, consisting in the cleavage of CC bonds with the concomitant insertion of an oxygen atom.In this study, two type I BVMOs have been identified for the first time in photosynthetic eukaryotic organisms, the red alga Cyanidioschyzon merolae (Cm) and the moss Physcomitrella patens (Pp). A biocatalytic characterization of these newly discovered enzymes, expressed in recombinant forms, was carried out. Both enzymes could be purified as holo enzymes containing a FAD cofactor. Their thermostability was investigated and revealed that the Cm-BVMO is the most thermostable type I BVMO with an apparent melting temperature of 56 °C. Substrate profiling revealed that both eukaryotic BVMOs accept a wide range of ketones which include aromatic, aliphatic, aryl aliphatic and bicyclic ketones. In particular, linear aliphatic ketones (C9 and C12), carrying the keto functionality in different positions, resulted to be the best substrates in steady state kinetic analyses. In order to restore the BVMO-typifying sequence motif in the Pp-BVMO, a mutant was prepared (Y160H). Intriguingly, this mutation resulted in higher activities on most tested substrates. The recombinant enzymes displayed k_cat values in the 0.1–0.2 s⁻¹ range, which is relatively low when compared with other known type I BVMOs. This may hint to a role in secondary metabolism in these photosynthetic organisms, though their exact function remains to be established.     

Scale-up of Baeyer–Villiger monooxygenase-catalyzed synthesis of enantiopure compounds

Several Baeyer–Villiger monooxygenases converting a wide spectrum of substrates have been discovered, cloned, and characterized throughout the last few years. Still, only a few of them are applicable for large-scale conversion predominantly due to their sensitivity towards high substrate and/or product concentrations. The recently cloned and characterized 4-hydroxyacetophenone monooxygenase from Pseudomonas putida JD1 shows excellent enantioselectivity towards 3-phenyl-2-butanone with E?>?100 but is inhibited by concentrations >10 mM of both substrate and product. This obstacle could be circumvented by in situ substrate feed and product removal using a hydrophobic Lewatit® adsorbent resin. Thus, the concentration of 3-phenyl-2-butanone could be increased from 1.4 to >26 mM without significant reduction in conversion.     

Enantiocomplementary access to carba-analogs of C-nucleoside derivatives by recombinant Baeyer–Villiger monooxygenases

A novel and stereoselective synthetic route towards carba-C-nucleosides was investigated applying an enantiodivergent biooxidation strategy by two different Baeyer–Villiger monooxygenases. Within only three chemo-enzymatic steps it was possible to introduce four chiral centers starting from commercially available non-chiral starting material.     

Extending the substrate scope of a Baeyer–Villiger monooxygenase by multiple-site mutagenesis

Baeyer–Villiger monooxygenase-catalysed reactions are attractive for industrial processes. Here we report on expanding the substrate scope of phenylacetone monooxygenase (PAMO). In order to introduce activity on alicyclic ketones in PAMO, we generated and screened a library of 1,500 mutants. Based on recently published structures of PAMO and its mutants, we selected previously uncharacterised positions as well as known hot-spots to be targeted by focused mutagenesis. We were able to mutate 11 positions in a single step by using the OmniChange method for the mutant library generation. Screening of the library using a phosphate-based activity detection method allowed identification of a quadruple mutant (P253F/G254A/R258M/L443F) active on cyclopentanone. The substrate scope of this mutant is extended to several aliphatic ketones while activity on aromatic compounds typical for PAMO was preserved. Moreover, the mutant is as thermostable as PAMO. Our results demonstrate the power of screening structure-inspired, focused mutant libraries for creating Baeyer–Villiger monooxygenases with new specificities.     

Development of a new hydrogen peroxide–based vaccine platform

Safe and effective vaccines are crucial for maintaining public health and reducing the global burden of infectious disease. Here we introduce a new vaccine platform that uses hydrogen peroxide (H(2)O(2)) to inactivate viruses for vaccine production. H(2)O(2) rapidly inactivates both RNA and DNA viruses with minimal damage to antigenic structure or immunogenicity and is a highly effective method when compared with conventional vaccine inactivation approaches such as formaldehyde or β-propiolactone. Mice immunized with H(2)O(2)-inactivated lymphocytic choriomeningitis virus (LCMV) generated cytolytic, multifunctional virus-specific CD8(+) T cells that conferred protection against chronic LCMV infection. Likewise, mice vaccinated with H(2)O(2)-inactivated vaccinia virus or H(2)O(2)-inactivated West Nile virus showed high virus-specific neutralizing antibody titers and were fully protected against lethal challenge. Together, these studies demonstrate that H(2)O(2)-based vaccines are highly immunogenic, provide protection against a range of viral pathogens in mice and represent a promising new approach to future vaccine development.     

Functional assembly of camphor converting two-component Baeyer–Villiger monooxygenases with a flavin reductase from E. coli

The major limitation in the synthetic application of two-component Baeyer–Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor.     

Regime analysis of a Baeyer–Villiger bioconversion in a three-phase (air–water–ionic liquid) stirred tank bioreactor

The aim of this work was to conduct a regime analysis on a three-phase (air–water–ionic liquid) stirred tank bioreactor of the Baeyer–Villiger bioconversion process, using [MeBuPyrr][BTA] ionic liquid as the dispersed phase. The regime analysis based on characteristic times of the different mechanisms involved (mixing, mass transfer, reaction) can yield a quantitative estimate of bioreactor performance. The characteristic time obtained for oxygen uptake rate (54 s⁻¹) was among the characteristic times determined for oxygen transfer (13–129 s⁻¹) under different operating conditions, suggesting that the oxygen transfer rate under certain operating conditions could be a limiting step in the bioconversion process. Further enhancement of oxygen transfer rates requires proper selection of the bioreactor operational conditions, and improved design of the ionic liquid used as oxygen transfer vector.     

Studies on Baeyer–Villiger oxidation of steroids: DHEA and pregnenolone d-lactonization pathways in Penicillium camemberti AM83

Penicillium camemberti AM83 strain is able to carry out effective Baeyer–Villiger type oxidation of DHEA, pregnenolone, androstenedione and progesterone to testololactone. Pregnenolone and DHEA underwent oxidation to testololactone via two routes: through 4-en-3-ketones (progesterone and/or androstenedione respectively) or through 3β-hydroxy-17a-oxa-d-homo-androst-5-en-17-one.Analysis of transformation progress of studied substrates as function of time indicates that the 17β-side chain cleavage and oxidation of 17-ketones to d-lactones are catalyzed by two different, substrate-induced, BVMOs. In the presence of a C-21 substrate (pregnenolone or progesterone) induction of the enzyme catalyzing cleavage at 17β-acetyl chain was observed, whereas DHEA and androstenedione induced activity of the BVMO responsible for the ring-D oxidation; 5-en-3β-alcohol was a more effective inducer that the respective 4-en-3-ketone.     

Microscale process evaluation of recombinant biocatalyst libraries: application to Baeyer–Villiger monooxygenase catalysed lactone synthesis

Microscale processing techniques are rapidly emerging as a cost- effective means for parallel experimentation and hence the evaluation of large libraries of recombinant biocatalysts. In this work, the potential of an automated microscale process is demonstrated in a linked sequence of operations comprising fermentation, enzyme induction and bioconversion using three whole-cell biocatalysts each expressing cyclohexanone monoxygenase (CHMO). The biocatalysts, Escherichia coli TOP 10 [pQR239], E. coli JM107 and Acinetobacter calcoaceticus NCIMB 9871, were first produced in 96-deep square well fermentations at various carbon source concentrations (10 and 20 g L⁻¹ glycerol). Following induction of CHMO activity biomass concentrations of up to 6 g_DCW L⁻¹ were obtained. Cells from each fermentation were subsequently used for the Baeyer–Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one, cyclohexanone and cyclopentanone. Each bioconversion was performed at two initial substrate concentrations (0.5 and 1.0 g L⁻¹) in order to simultaneously explore both substrate specificity and inhibition. The microscale process sequences yielded quantitative and reproducible data for each biocatalyst on maximum growth rate, biomass yield, initial rate of lactone formation, specific biocatalyst activity and bioconversion yield. E. coli TOP 10 [pQR239] was demonstrated to be an efficient biocatalyst showing substrate specificities and substrate inhibition effects in line with previous studies. Finally, in order to show that the data obtained with E. coli TOP 10 [pQR239] at microwell scale (1,000 μL) could be related to larger scales of operation, the process was performed in a 2-L stirred-tank bioreactor. Using conditions designed to enable microwell kinetic measurements under none oxygen-limited conditions, the fermentation and bioconversion data obtained at the two scales showed good quantitative agreement. This study therefore confirms the potential of automated microscale experimentation for the whole-process evaluation of recombinant biocatalyst libraries and the specification of pilot and process scale operating conditions.     

Production of cyclohexanone monooxygenase from Acinetobacter calcoaceticus for large scale Baeyer–Villiger monooxygenase reactions

Cyclohexanone monooxygenase was produced from Acinetobacter calcoaceticus grown on a medium containing both glutamate (30 g l^–1) and cyclohexanol (1 g l^–1). Productivity was increased to 650 U l^–1, an order of magnitude greater than previous production methods, thereby enhancing the potential commercial utility of this enzyme.     

Rational design,synthesis and anti-proliferative evaluation of novel benzosuberone tethered with hydrazide–hydrazones

Two different series of novel analogues of benzosuberones (5a–m and 9a–w) tethered with hydrazone–hydrazides (functional group alterations: Head group to Tail group and vice versa) have been synthesized by the reaction of appropriate aldehydes with substituted hydrazides in excellent yields (87–94%) and their structures were confirmed by ¹H NMR, ¹³C NMR, ESI-MS and HRMS. The newly synthesized compounds were evaluated for anti-proliferative activity against different human cancer cell lines (HeLa, MDA MB 231, MIAPACA and IMR32). Among the synthesized compounds, six compounds 5a, 5b, 5d, 5e, 5f and 9v exhibited potent anti-proliferative activity with GI₅₀ values less than 0.01 μM against MIAPACA, MDA-MB-231 and IMR32 human cancer cell lines.     

Rational design of a synthetic Entner–Doudoroff pathway for improved and controllable NADPH regeneration

NADPH is an essential cofactor for the biosynthesis of several high-value chemicals, including isoprenoids, fatty acid-based fuels, and biopolymers. Tunable control over all potentially rate-limiting steps, including the NADPH regeneration rate, is crucial to maximizing production titers. We have rationally engineered a synthetic version of the Entner–Doudoroff pathway from Zymomonas mobilis that increased the NADPH regeneration rate in Escherichia coli MG1655 by 25-fold. To do this, we combined systematic design rules, biophysical models, and computational optimization to design synthetic bacterial operons expressing the 5-enzyme pathway, while eliminating undesired genetic elements for maximum expression control. NADPH regeneration rates from genome-integrated pathways were estimated using a NADPH-binding fluorescent reporter and by the productivity of a NADPH-dependent terpenoid biosynthesis pathway. We designed and constructed improved pathway variants by employing the RBS Library Calculator to efficiently search the 5-dimensional enzyme expression space and by performing 40 cycles of MAGE for site-directed genome mutagenesis. 624 pathway variants were screened using a NADPH-dependent blue fluorescent protein, and 22 were further characterized to determine the relationship between enzyme expression levels and NADPH regeneration rates. The best variant exhibited 25-fold higher normalized mBFP levels when compared to wild-type strain. Combining the synthetic Entner–Doudoroff pathway with an optimized terpenoid pathway further increased the terpenoid titer by 97%.     

Characterization of a recombinant Escherichia coli TOP10 [pQR239] whole-cell biocatalyst for stereoselective Baeyer–Villiger oxidations

This paper describes the kinetic characterization of a recombinant whole-cell biocatalyst for the stereoselective Baeyer–Villiger type oxidation of bicyclo[3.2.0]hept-2-en-6-one to its corresponding regio-isomeric lactones (−)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (−)-(1R,5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one. Escherichia coli TOP10 [pQR239], expressing cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus (NCIMB 9871), was shown to be suitable for this biotransformation since it expressed CHMO at a high level, was simple to produce, contained no contaminating lactone hydrolase activity and allowed the intracellular recycle of NAD(P)H necessary for the biotransformation. A small-scale biotransformation reactor (20 ml) was developed to allow rapid collection of intrinsic kinetic data. In this system, the optimized whole-cell biocatalyst exhibited a significantly lower specific lactone production activity (55–60 μmol min⁻¹ g⁻¹ dry weight) than that of sonicated cells (500 μmol min⁻¹ g⁻¹ dry weight). It was shown that this shortfall was comprised of a difference in the pH optima of the two biocatalyst forms and mass transfer limitations of the reactant and/or product across the cell barrier. Both reactant and product inhibition were evident. The optimum ketone concentration was between 0.2 and 0.4 g l⁻¹ and at product concentrations above 4.5–5 g l⁻¹ the specific activity of the whole cells was zero. These results suggest that a reactant feeding strategy and in situ product removal should be considered in subsequent process design.     

Rational design of microRNA–siRNA chimeras for multifunctional target suppression

MicroRNAs (miRNAs) are involved in a variety of human diseases by simultaneously suppressing many gene targets. Thus, the therapeutic value of miRNAs has been intensely studied. However, there are potential limitations with miRNA-based therapeutics such as a relatively moderate impact on gene target regulation and cellular phenotypic control. To address these issues, we proposed to design new chimeric small RNAs (aiRNAs) by incorporating sequences from both miRNAs and siRNAs. These aiRNAs not only inherited functions from natural miRNAs, but also gained new functions of gene knockdown in an siRNA-like fashion. The improved efficacy of multifunctional aiRNAs was demonstrated in our study by design and testing of an aiRNA that inherited the functions of both miR-200a and an AKT1-targeting siRNA for simultaneous suppression of cancer cell motility and proliferation. The general principles of aiRNA design were further validated by engineering new aiRNAs mimicking another miRNA, miR-9. By regulating multiple cellular functions, aiRNAs could be used as an improved tool over miRNAs to target disease-related genes, thus alleviating our dependency on a limited number of miRNAs for the development of RNAi-based therapeutics.