Production of Hydroxy Acids:Selective Double Oxidation of Diols by Flavoprotein Alcohol Oxidase

Flavoprotein oxidases can catalyze oxidations of alcohols and amines by merely using molecular oxygen as the oxidant, making this class of enzymes appealing for biocatalysis. The FAD-containing (FAD=flavin adenine dinucleotide) alcohol oxidase from P. chrysosporium facilitated double and triple oxidations for a range of aliphatic diols. Interestingly, depending on the diol substrate, these reactions result in formation of either lactones or hydroxy acids. For example, diethylene glycol could be selectively and fully converted into 2-(2-hydroxyethoxy)acetic acid. Such a facile cofactor-independent biocatalytic route towards hydroxy acids opens up new avenues for the preparation of polyester building blocks

Introducing an Artificial Deazaflavin Cofactor in Escherichia coli and Saccharomyces cerevisiae

[Image: see text] Deazaflavin-dependent whole-cell conversions in well-studied and industrially relevant microorganisms such as Escherichia coli and Saccharomyces cerevisiae have high potential for the biocatalytic production of valuable compounds. The artificial deazaflavin FOP (FO-5′-phosphate) can functionally substitute the natural deazaflavin F(420) and can be synthesized in fewer steps, offering a solution to the limited availability of the latter due to its complex (bio)synthesis. Herein we set out to produce FOP in vivo as a scalable FOP production method and as a means for FOP-mediated whole-cell conversions. Heterologous expression of the riboflavin kinase from Schizosaccharomyces pombe enabled in vivo phosphorylation of FO, which was supplied by either organic synthesis ex vivo, or by a coexpressed FO synthase in vivo, producing FOP in E. coli as well as in S. cerevisiae. Through combined approaches of enzyme engineering as well as optimization of expression systems and growth media, we further improved the in vivo FOP production in both organisms. The improved FOP production yield in E. coli is comparable to the F(420) yield of native F(420)-producing organisms such as Mycobacterium smegmatis, but the former can be achieved in a significantly shorter time frame. Our E. coli expression system has an estimated production rate of 0.078 μmol L(–1) h(–1) and results in an intracellular FOP concentration of about 40 μM, which is high enough to support catalysis. In fact, we demonstrate the successful FOP-mediated whole-cell conversion of ketoisophorone using E. coli cells. In S. cerevisiae, in vivo FOP production by SpRFK using supplied FO was improved through media optimization and enzyme engineering. Through structure-guided enzyme engineering, a SpRFK variant with 7-fold increased catalytic efficiency compared to the wild type was discovered. By using this variant in optimized media conditions, FOP production yield in S. cerevisiae was 20-fold increased compared to the very low initial yield of 0.24 ± 0.04 nmol per g dry biomass. The results show that bacterial and eukaryotic hosts can be engineered to produce the functional deazaflavin cofactor mimic FOP

Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.</p

Mining the Genome of:Two New Type I Baeyer-Villiger Monooxygenases From Atacama Desert

Actinobacteria are an important source of commercial (bio)compounds for the biotechnological and pharmaceutical industry. They have also been successfully exploited in the search of novel biocatalysts. We set out to explore a recently identified actinomycete, Streptomyces leeuwenhoekii C34, isolated from a hyper-arid region, the Atacama desert, for Baeyer-Villiger monooxygenases (BVMOs). Such oxidative enzymes are known for their broad applicability as biocatalysts by being able to perform various chemical reactions with high chemo-, regio-, and/or enantioselectivity. By choosing this specific Actinobacterium, which comes from an extreme environment, the respective enzymes are also expected to display attractive features by tolerating harsh conditions. In this work, we identified two genes in the genome of S. leeuwenhoekii (sle_13190 and sle_62070) that were predicted to encode for Type I BVMOs, the respective flavoproteins share 49% sequence identity. The two genes were cloned, overexpressed in E. coli with phosphite dehydrogenase (PTDH) as fusion partner and successfully purified. Both flavin-containing proteins showed NADPH-dependent Baeyer-Villiger oxidation activity for various ketones and sulfoxidation activity with some sulfides. Gratifyingly, both enzymes were found to be rather robust by displaying a relatively high apparent melting temperature (45°C) and tolerating water-miscible cosolvents. Specifically, Sle_62070 was found to be highly active with cyclic ketones and displayed a high regioselectivity by producing only one lactone from 2-phenylcyclohexanone, and high enantioselectivity by producing only normal (-)-1S,5R and abnormal (-)-1R,5S lactones (ee > 99%) from bicyclo[3.2.0]hept-2-en-6-one. These two newly discovered BVMOs add two new potent biocatalysts to the known collection of BVMOs

Computational Design of Enantiocomplementary Epoxide Hydrolases for Asymmetric Synthesis of Aliphatic and Aromatic Diols

The use of enzymes in preparative biocatalysis often requires tailoring enzyme selectivity by protein engineering. Herein we explore the use of computational library design and molecular dynamics simulations to create variants of limonene epoxide hydrolase that produce enantiomeric diols from meso-epoxides. Three substrates of different sizes were targeted: cis-2,3-butene oxide, cyclopentene oxide, and cis-stilbene oxide. Most of the 28 designs tested were active and showed the predicted enantioselectivity. Excellent enantioselectivities were obtained for the bulky substrate cis-stilbene oxide, and enantiocomplementary mutants produced (S,S)- and (R,R)-stilbene diol with >97 % enantiomeric excess. An (R,R)-selective mutant was used to prepare (R,R)-stilbene diol with high enantiopurity (98 % conversion into diol, >99 % ee). Some variants displayed higher catalytic rates (kcat) than the original enzyme, but in most cases KM values increased as well. The results demonstrate the feasibility of computational design and screening to engineer enantioselective epoxide hydrolase variants with very limited laboratory screening

Multienzymatic Stereoselective Reduction of Tetrasubstituted Cyclic Enones to Halohydrins with Three Contiguous Stereogenic Centers

The asymmetric hydrogenation of conjugated tetrasubstituted alkenes with transition-metal catalysts is a challenging reaction, especially for substrates bearing a halide substituent. We describe a two-step multienzymatic reduction of a series of α-halo β-alkyl tetrasubstituted cyclic enones, affording halohydrins with three contiguous stereogenic centers, in good yield and with a high stereoselectivity. The reduction is catalyzed by a stereospecific ene-reductase (OYE2-3 or NemA) and a highly enantioselective alcohol dehydrogenase (ADH). The use of two enantiodivergent ADHs allows the control of the diastereoselectivity. The absolute stereochemical configurations of the products have been determined from the analysis of single-crystal structures (Flack's parameter). The enantiomeric excess (ee) has been determined by derivatization of the products with (R) Mosher's acid. Lastly, we extended our methodology also to a nonhalogenated substrate: the α-methyl ketoisophorone was reduced by two distinct enantiodivergent ene-reductases (flavin mononucleotide- and F 420-dependent), affording each enantiomer of the saturated ketone with ee > 98%

Facile Stereoselective Reduction of Prochiral Ketones by using an F ₄₂₀-dependent alcohol dehydrogenase

Effective procedures for the synthesis of optically pure alcohols are highly valuable. A commonly employed method involves the biocatalytic reduction of prochiral ketones. This is typically achieved by using nicotinamide cofactor-dependent reductases. In this work, we demonstrate that a rather unexplored class of enzymes can also be used for this. We used an F420-dependent alcohol dehydrogenase (ADF) from Methanoculleus thermophilicus that was found to reduce various ketones to enantiopure alcohols. The respective (S) alcohols were obtained in excellent enantiopurity (>99 % ee). Furthermore, we discovered that the deazaflavoenzyme can be used as a self-sufficient system by merely using a sacrificial cosubstrate (isopropanol) and a catalytic amount of cofactor F420 or the unnatural cofactor FOP to achieve full conversion. This study reveals that deazaflavoenzymes complement the biocatalytic toolbox for enantioselective ketone reductions

Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering

Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (ΔTm ≥ +3 °C). By combining mutations, a 10-fold mutant was generated with a Tm of 94 °C (+51 °C relative to wildtype), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wildtype, while displaying a Tm of 88 °C (+45 °C relative to wildtype). This work demonstrates the value of enzyme stabilization through computational library design

Gal-3 plays an important pro-inflammatory role in the induction phase of acute colitis by promoting activation of NLRP3 inflammasome and production of IL-β in macrophages

BACKGROUND AND AIMS: Galectin-3 [Gal-3] is an endogenous lectin with a broad spectrum of immunoregulatory effects: it plays an important role in autoimmune/inflammatory and malignant diseases, but the precise role of Gal-3 in pathogenesis of ulcerative colitis is still unknown. METHODS: We used a model of dextran sulphate sodium [DSS]-induced acute colitis. The role of Gal-3 in pathogenesis of this disease was tested by evaluating disease development in Gal-3 deficient mice and administration of Gal-3 inhibitor. Disease was monitored by clinical, histological, histochemical, and immunophenotypic investigations. Adoptive transfer was used to detect cellular events in pathogenesis. RESULTS: Genetic deletion or pharmacological inhibition of Gal-3 significantly attenuate DSS-induced colitis. Gal-3 deletion suppresses production of pro-inflammatory cytokines in colonic macrophages and favours their alternative activation, as well as significantly reducing activation of NOD-like receptor family, pyrin domain containing 3 [NLRP3] inflammasome in macrophages. Peritoneal macrophages isolated from untreated Gal-3(-/-) mice and treated in vitro with bacterial lipopolysaccharide or DSS produce lower amounts of tumour necrosis factor alpha [TNF-α] and interleukin beta [IL-1β] when compared with wild type [WT] cells. Genetic deletion of Gal-3 did not directly affect total neutrophils, inflammatory dendritic cells [DCs] or natural killer [NK] T cells. However, the total number of CD11c+ CD80+ DCs which produce pro-inflammatory cytokines, as well as TNF-α and IL-1β producing CD45+ CD11c- Ly6G+ neutrophils were significantly lower in colons of Gal-3(-/-) DSS-treated mice. Adoptive transfer of WT macrophages significantly enhanced the severity of disease in Gal-3(-/-) mice. CONCLUSIONS: Gal-3 expression promotes acute DSS-induced colitis and plays an important pro-inflammatory role in the induction phase of colitis by promoting the activation of NLRP3 inflammasome and production of IL-1β in macrophages

BENCHMARKING WORKSHOPS AS A TOOL TO RAISE BUSINESS EXCELLENCE

Annual competition for national award for business excellence appears to be a good opportunity for participating organizations to demonstrate their practices particularly those ones which enable them to excel. National quality award competition in Serbia (and Montenegro), namely "OSKAR KVALITETA" started in 1995 but was limited to competition cycle only. However, upon establishing Fund for Quality Culture and Excellence - FQCE in 2002, which took over OSKAR KVALITETA model, several changes took place. OSKAR KVALITETA turned to be annual competition in business excellence, but at the same time FQCE started to offer much wider portfolio of its services including levels of excellence programs, assessment and self-assessment training courses and benchmarking workshops. These benchmarking events have hosted by Award winners or other laureates in OSKAR KVALITETA competition who demonstrated excellence in regard of some particular criteria thus being in position to share their practice with other organizations. For six years experience in organizing benchmarking workshops FQCE scored 31 workshops covering major part of model issues. Increasing level of participation on the workshops and distinct positive trends of participants expressed satisfaction may serve as a reliable indicator that the workshops have been effective in actuating people to think and move in business excellence direction