Cascade enzymatic synthesis of a statin side chain precursor - the role of reaction engineering in process optimization.

Statins are an important class of drugs used to lower blood cholesterol levels and are often used to combat cardiovascular disease. In view of the importance of safe and reliable supply and production of statins in modern medicine and the global need for sustainable processes, various biocatalytic strategies for their synthesis have been investigated. In this work, a novel biocatalytic route to a statin side chain precursor was investigated in a one-pot cascade reaction starting from the protected alcohol N-(3-hydroxypropyl)-2-phenylacetamide, which is oxidized to the corresponding aldehyde in the first reaction step, and then reacts with two equivalents of acetaldehyde to form the final product N-(2-((2S,4S,6S)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)ethyl)-2-phenylacetamide (phenylacetamide-lactol). To study this complex reaction, an enzyme reaction engineering approach was used, i.e. the kinetics of all reactions occurring in the cascade (including side reactions) were determined. The obtained kinetic model together with the simulations gave an insight into the system and indicated the best reactor mode for the studied reaction, which was fed-batch with acetaldehyde feed to minimize its negative effect on the enzyme activity during the reaction. The mathematical model of the process was developed and used to simulate different scenarios and to find the reaction conditions (enzyme and coenzyme concentration, substrate feed concentration and flow rate) at which the highest yield of phenylacetamide-lactol (75%) can be obtained. In the end, our goal was to show that this novel cascade route is an interesting alternative for the synthesis of the statin side chain precursor and that is why we also calculated an initial estimate of the potential value addition.

A host-vector toolbox for improved secretory protein overproduction in Bacillus subtilis.

Target proteins in biotechnological applications are highly diverse. Therefore, versatile flexible expression systems for their functional overproduction are required. In order to find the right heterologous gene expression strategy, suitable host-vector systems, which combine different genetic circuits, are useful. In this study, we designed a novel Bacillus subtilis expression toolbox, which allows the overproduction and secretion of potentially toxic enzymes. This toolbox comprises a set of 60 expression vectors, which combine two promoter variants, four strong secretion signals, a translation-enhancing downstream box, and three plasmid backbones. This B. subtilis toolbox is based on a tailor-made, clean deletion mutant strain, which is protease and sporulation deficient and exhibits reduced autolysis and secondary metabolism. The appropriateness of this alternative expression platform was tested for the overproduction of two difficult-to-produce eukaryotic model proteins. These included the sulfhydryl oxidase Sox from Saccharomyces cerevisiae, which forms reactive hydrogen peroxide and undesired cross-linking of functional proteins, and the human interleukin-1ß, a pro-inflammatory cytokine. For the best performing Sox and interleukin, overproducing and secreting variants of these new B. subtilis toolbox fermentation strategies were developed and tested. This study demonstrates the suitability of the prokaryotic B. subtilis host-vector system for the extracellular production of two eukaryotic proteins with biotechnological relevance. KEY POINTS: • Construction of a versatile Bacillus subtilis gene expression toolbox. • Verification of the toolbox by the secretory overproduction of two difficult-to-express proteins. • Fermentation strategy for an acetoin-controlled overproduction of heterologous proteins.

Combining Photo-Organo Redox- and Enzyme Catalysis Facilitates Asymmetric C-H Bond Functionalization.

In this study, we combined photo-organo redox catalysis and biocatalysis to achieve asymmetric C-H bond functionalization of simple alkane starting materials. The photo-organo catalyst anthraquinone sulfate (SAS) was employed to oxyfunctionalise alkanes to aldehydes and ketones. We coupled this light-driven reaction with asymmetric enzymatic functionalisations to yield chiral hydroxynitriles, amines, acyloins and α-chiral ketones with up to 99 % ee. In addition, we demonstrate functional group interconversion to alcohols, esters and carboxylic acids. The transformations can be performed as concurrent tandem reactions. We identified the degradation of substrates and inhibition of the biocatalysts as limiting factors affecting compatibility, due to reactive oxygen species generated in the photocatalytic step. These incompatibilities were addressed by reaction engineering, such as applying a two-phase system or temporal and spatial separation of the catalysts. Using a selection of eleven starting alkanes, one photo-organo catalyst and 8 diverse biocatalysts, we synthesized 26 products and report for the model compounds benzoin and mandelonitrile > 97 % ee at gram scale.

Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining.

Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community.

Cloning, expression and characterization of a eukaryotic cycloalkanone monooxygenase from Cylindrocarpon radicicola ATCC 11011.

In this study, we have cloned and characterized a cycloalkanone monooxygenase (CAMO) from the ascomycete Cylindrocarpon radicicola ATCC 11011 (identical to Cylindrocarpon destructans DSM 837). The primary structure of this Baeyer-Villiger monooxygenase (BMVO) revealed 531 residues with around 45% sequence identity to known cyclohexanone monooxygenases. The enzyme was functionally overexpressed in Escherichia coli and investigated with respect to substrate spectrum and kinetic parameters. Substrate specificity studies revealed that a large variety of cycloaliphatic and bicycloaliphatic ketones are converted by this CAMO. A high catalytic efficiency against cyclobutanone was observed and seems to be a particular property of this BVMO. The thus produced butyrolactone derivatives are valuable building blocks for the synthesis of a variety of natural products and bioactive compounds. Furthermore, the enzyme revealed activity against open-chain ketones such as cyclobutyl, cyclopentyl and cyclohexyl methyl ketone which have not been reported to be accepted by typical cyclohexanone monooxygenases. These results suggest that the BVMO from C. radicicola indeed might be rather unique and since no BVMOs originating from eukaryotic organisms have been produced recombinantly so far, this study provides the first example for such an enzyme.

Increasing the synthesis/hydrolysis ratio of aminoacylase 1 by site-directed mutagenesis.

Aminoacylase-1 from pig kidney (pAcy1) catalyzes the highly stereoselective acylation of amino acids, a useful conversion for the preparation of optically pure N-acyl-l-amino acids. The kinetic of this thermodynamically controlled conversion is determined by maximal velocities for synthesis (V(mS)) and hydrolysis (V(mH)) of the N-acyl-l-amino acid. To investigate which parameter affects maximal velocities, we focused on the proton acceptor potential of the catalytic base, E146, and studied the influence of the active site architecture on its contribution to the pKa of residue E146. The modeled structure of pAcy1 identified residue D346 as having the strongest impact on the electrostatic features of the catalytic base. Substitutions of D346 generally decreased enzymatic activities but also altered both the pH-dependency of hydrolytic activity and the V(mS)/V(mH) ratio of pAcy1. A reduced theoretical pKa value and a lowered experimental pH optimum of hydrolytic rates for the D346A mutant were associated with a 9-fold increase in V(mS)/V(mH). This supports the importance of electrostatic contributions of D346 to the acid-base properties of E146 and demonstrates for the first time the possibility of engineering the V(mS)/V(mH) ratio of pAcy1.

Functional expression of porcine aminoacylase 1 in E. coli using a codon optimized synthetic gene and molecular chaperones.

Efficient recombinant expression of N-acyl-L-aminoacylase 1 from pig kidney (pAcy1) was achieved in the prokaryotic host Escherichia coli. An optimized nucleotide sequence (codon adaptation index 0.95 for E. coli), was cloned into vector pET-52(b) yielding an E. coli-expressible pAcy1 gene. Formation of inclusion bodies was alleviated by co-expression of molecular chaperones resulting in 2.7- and 4.2-fold increased recovery of active pAcy1 using trigger factor or GroEL-GroES, respectively. Facile purification was achieved via StrepTag affinity chromatography. Overall, more than 80 mg highly active pAcy1 (94 U/mg) was obtained per liter of cultivation broth. The protein was analyzed for structural and functional identity, and the performances of further described expression and purification systems for pAcy1 were compared.

A potential high-throughput method for the determination of lipase activity by potentiometric flow injection titrations.

Potentiometric FIA titrations were performed to determine enzyme activities of lipase type B from Candida antarctica, CAL-B. Two substrates, triacetin and tributyrin were hydrolyzed in phosphate buffer solutions, and the concentration change of the base component of the buffer was titrated in a carrier solution containing hydrochloric acid and potassium chloride. The system was calibrated with butyric acid and acetic acid, respectively. FIA titration peaks were evaluated with respect to peak height and peak area. Butyric acid and acetic acid could be titrated in the buffer solution from 3x10(-3) mol L(-1) to 0.1 mol L(-1). The detection limit of enzyme activity was determined to be 0.07 U mL(-1) (15 min reaction time) and the minimum activity was calculated to be 0.035 units corresponding to 35 nmol min(-1). The specific activities of lipase B for the hydrolysis of tributyrin and triacetin were determined as 16+/-2 U mg(-1) and 2+/-0.2 U mg(-1) (per mg commercial lipase preparation), respectively.

Microbiosensors for measurement of microbially available dissolved organic carbon: sensor characteristics and preliminary environmental application.

Microbial respiration-based microbiosensors used for quantification of available dissolved organic carbon (ADOC) instantaneously respired by microorganisms are described. The sensing membranes contained aerobic seawater microorganisms immobilized in a polyurethane hydrogel. Molecular investigations revealed that the bacterial strain used was most closely related to Staphylococcus warneri. This strain was characterized by low substrate selectivity, which was reflected in the response to various mono- and disaccharides, short-chain fatty acids, and amino acids, as determined using Biolog microplates. Specific emphasis was placed on critically assessing biosensor functioning that was affected by preconditioning of the selected bacterial strain, chemical and geometric properties of the sensing membrane (e.g., composition, permeability, and thickness), and the distribution, biomass, and physiological state of immobilized cells, as well as the exposure conditions (e.g., temperature and nutrient supply). The sensors revealed that there was a linear response up to a glucose concentration of 500 microM depending on the type, characteristics, and recent history of the sensors. The detection limit of the sensors was equivalent to about 6 to 10 microM glucose. The 90% response time ranged from 1 to 5 min. Generally, the response of the biosensors became weaker with time. The shelf lives of individual sensors were up to 2 weeks. Measurements based on optical ADOC microbiosensors revealed that in photoautotrophically dominated sandy coastal sediments, the pool sizes and turnover of ADOC were regulated by the photosynthetic activity of benthic microalgae and microbial aerobic respiration. A large increase in ADOC production was observed shortly after the microphytobenthic primary production reached the maximum value at midday, whereas ADOC was consumed by microbial respiration during the night.