Cell-free reduction of carboxylic acids with secreted carboxylic acid reductase.

Mycobacterium marinum CAR (MmCAR) is one of the most widely used CARs as the key enzyme for the synthesis of aldehydes, alcohols and further products from the respective carboxylic acids. Herein, we describe the first functionally secreted 131 kDa CAR and its isolated A-domain using Komagataella phaffii and a methanol-free constitutive expression strategy. Precipitated and lyophilized MmCAR (500 µg) was isolated from the culture supernatant and showed no decrease in activity for piperonylic acid (80% conversion), even when stored for up to 3 weeks at 4°C. Lyophilized MmCAR precipitate gave 48% yield of E/Z-nonanal-4-nitrobenzoyloxime from the reduction of nonanoic acid and in-situ derivatization with O-4-nitrobenzoyl-hydroxylamine. Furthermore, K. phaffii could successfully secrete the MmCAR adenylation domain. Its activity was confirmed by the amidation of benzoic acid with n-hexylamine. Neither enzyme variant was glycosylated by the yeast. In summary, functional CAR can be secreted by K. phaffii and used for cell free conversion of carboxylic acids to various products.

Organic Acid to Nitrile: A Chemoenzymatic Three-Step Route.

Various widely applied compounds contain cyano-groups, and this functional group serves as a chemical handle for a whole range of different reactions. We report a cyanide free chemoenzymatic cascade for nitrile synthesis. The reaction pathway starts with a reduction of carboxylic acid to aldehyde by carboxylate reductase enzymes (CARs) applied as living cell biocatalysts. The second - chemical - step includes in situ oxime formation with hydroxylamine. The final direct step from oxime to nitrile is catalyzed by aldoxime dehydratases (Oxds). With compatible combinations of a CAR and an Oxd, applied in one-pot two-step reactions, several aliphatic and aryl-aliphatic target nitriles were obtained in more than 80% conversion. Phenylacetonitrile, for example, was prepared in 78% isolated yield. This chemoenzymatic route does not require cyanide salts, toxic metals, or undesired oxidants in contrast to entirely chemical procedures.

Scanning aldoxime dehydratase sequence space and characterization of a new aldoxime dehydratase from Fusarium vanettenii.

The aim of this work was to map the sequence space of aldoxime dehydratases (Oxds) as enzymes with great potential for nitrile synthesis. Microbes contain an abundance of putative Oxds but fewer than ten Oxds were characterized in total and only two in fungi. In this work, we prepared and characterized a new Oxd (protein gb|EEU37245.1 named OxdFv) from Fusarium vanettenii 77-13-4. OxdFv is distant from the characterized Oxds with a maximum of 36% identity. Moreover, the canonical Oxd catalytic triad RSH is replaced by R141-E187-E303 in OxdFv. R141A and E187A mutants did not show significant activities, but mutant E303A showed a comparable activity as the wild-type enzyme. According to native mass spectrometry, OxdFv contained almost 1 mol of heme per 1 mol of protein, and was composed of approximately 88% monomer (41.8 kDa) and 12% dimer. A major advantage of this enzyme is its considerable activity under aerobic conditions (25.0 ± 4.3 U/mg for E,Z-phenylacetaldoxime at pH 9.0 and 55 °C). Addition of sodium dithionite (reducing agent) and Fe2+ was required for this activity. OxdFv favored (aryl)aliphatic aldoximes over aromatic aldoximes. Substrate docking in the homology model of OxdFv showed a similar substrate specificity. We conclude that OxdFv is the first characterized Oxd of the REE type.

Chemoenzymatic one-pot reaction from carboxylic acid to nitrile via oxime.

We report a new chemoenzymatic cascade starting with aldehyde synthesis by carboxylic acid reductase (CAR) followed by chemical in situ oxime formation. The final step to the nitrile is catalyzed by aldoxime dehydratase (Oxd). Full conversions of phenylacetic acid and hexanoic acid were achieved in a two-phase mode.

Characterization and immobilization of Pycnoporus cinnabarinus carboxylic acid reductase, PcCAR2.

Carboxylic acid reductases (CARs) are well-known for their eminent selective one-step synthesis of carboxylic acids to aldehydes. To date, however, few CARs have been identified and characterized, especially from fungal sources. In this study, the CAR from the white rot fungus Pycnoporus cinnabarinus (PcCAR2) was expressed in Escherichia coli. PcCAR2's biochemical properties were explored in vitro after purification, revealing a melting temperature of 53 °C, while the reaction temperature optimum was at 35 °C. In the tested buffers, the enzyme showed a pH optimum of 6.0 and notably, a similar activity up to pH 7.5. PcCAR2 was immobilized to explore its potential as a recyclable biocatalyst. PcCAR2 showed no critical loss of activity after six cycles, with an average conversion to benzaldehyde of more than 85% per cycle. Immobilization yield and efficiency were 82% and 76%, respectively, on Ni-sepharose. Overall, our findings contribute to the characterization of a thermotolerant fungal CAR, and established a more sustainable use of the valuable biocatalyst.

Characterization of Type IV Carboxylate Reductases (CARs) for Whole Cell-Mediated Preparation of 3-Hydroxytyrosol.

Fragrance and flavor industries could not imagine business without aldehydes. Processes for their commercial production raise environmental and ecological concerns. The chemical reduction of organic acids to aldehydes is challenging. To fulfill the demand of a mild and selective reduction of carboxylic acids to aldehydes, carboxylic acid reductases (CARs) are gaining importance. We identified two new subtype IV fungal CARs from Dichomitus squalens CAR (DsCAR) and Trametes versicolor CAR (Tv2CAR) in addition to literature known Trametes versicolor CAR (TvCAR). Expression levels were improved by the co-expression of GroEL-GroES with either the trigger factor or the DnaJ-DnaK-GrpE system. Investigation of the substrate scope of the three enzymes revealed overlapping substrate-specificities. Tv2CAR and DsCAR showed a preferred pH range of 7.0 to 8.0 in bicine buffer. TvCAR showed highest activity at pH 6.5 to 7.5 in MES buffer and slightly reduced activity at pH 6.0 or 8.0. TvCAR appeared to tolerate a wider pH range without significant loss of activity. Type IV fungal CARs optimal temperature was in the range of 25-35 °C. TvCAR showed a melting temperature (Tm) of 55 °C indicating higher stability compared to type III and the other type IV fungal CARs (Tm 51-52 °C). Finally, TvCAR was used as the key enzyme for the bioreduction of 3,4-dihydroxyphenylacetic acid to the antioxidant 3-hydroxytyrosol (3-HT) and gave 58 mM of 3-HT after 24 h, which correlates to a productivity of 0.37 g L-1 h-1.

Discovery of carboxylic acid reductase (CAR) from Thermothelomyces thermophila and its evaluation for vanillin synthesis.

A novel type III fungal CAR was identified from the organism Thermothelomyces thermophila. High expression levels were observed in E. coli using the pETDuet-1 plasmid system in combination with an autoinduction protocol. A broad substrate scope ranging from aromatic to aliphatic carboxylic acids was tested and TtCAR showed activity for all substrates. High specific activities for aromatic substrates and short chain aliphatic substrates were observed, comparable to those of NcCAR, the first type III fungal CAR. TtCAR's pH and temperature optima were at 6.5 and 30 °C, respectively. Up to 20% (v/v) cosolvents did not show a decrease in specific activity of TtCAR using (E)-cinnamic acid as a substrate. Its half-life at 40 °C was determined to be 8.25 h and its melting temperature (Tm) was 56 °C. In vitro reactions with TtCAR reduced 95.2% of 10 mM vanillic acid, which correlated to a titer of 1.4 g L-1 of vanillin. The space time yield of 0.029 g L-1 h-1 indicates that further improvements would be necessary for an industrially relevant application. This would be especially important when competing against de novo synthesis of bio vanillin by microbial strains producing >30 g L-1. In de novo and in vivo biosynthesis systems, by-products are fairly common. By contrast, we were pleased to observe less than 0.7% of vanillyl alcohol formed, making the cell-free acid reduction in the envisaged sequential two-step bioconversion from eugenol to vanillin very attractive.

Recombinant expression, purification and biochemical characterization of kievitone hydratase from Nectria haematococca.

Kievitone hydratase catalyzes the addition of water to the double bond of the prenyl moiety of plant isoflavonoid kievitone and, thereby, forms the tertiary alcohol hydroxy-kievitone. In nature, this conversion is associated with a defense mechanism of fungal pathogens against phytoalexins generated by host plants after infection. As of today, a gene sequence coding for kievitone hydratase activity has only been identified and characterized in Fusarium solani f. sp. phaseoli. Here, we report on the identification of a putative kievitone hydratase sequence in Nectria haematococca (NhKHS), the teleomorph state of F. solani, based on in silico sequence analyses. After heterologous expression of the enzyme in the methylotrophic yeast Pichia pastoris, we have confirmed its kievitone hydration activity and have assessed its biochemical properties and substrate specificity. Purified recombinant NhKHS is obviously a homodimeric glycoprotein. Due to its good activity for the readily available chalcone derivative xanthohumol (XN), this compound was selected as a model substrate for biochemical studies. The optimal pH and temperature for hydratase activity were 6.0 and 35°C, respectively, and apparent Vmax and Km values for hydration of XN were 7.16 µmol min-1 mg-1 and 0.98 ± 0.13 mM, respectively. Due to its catalytic properties and apparent substrate promiscuity, NhKHS is a promising enzyme for the biocatalytic production of tertiary alcohols.

Temporal brightness illusion changes color perception of "the dress".

"The dress" has provoked intensive commentary among psychophysicists, especially in relation to color vision. Researchers have shown that manipulating illuminance cues can influence the perceived colors of the dress. Here we investigate whether illusory shifts in brightness can shift color perception of the dress. Drifting achromatic gratings with fast off and fast on shading profiles are known to give an illusion of brightening or darkening, respectively. We superimposed rotating sawtooth gratings on a series of dress images that morphed from extreme white/gold through to blue/black. In a sample of 18 adults (11 with white/gold dress percept and seven with blue/black percept), a two-alternative, forced-choice constant stimulus task measured the morphed image point at which each observer was equally likely to categorize the dress as white/gold or blue/black (the point of subjective equality or PSE). Despite manifest individual differences in the PSE, the two sawtooth temporal profiles consistently changed the perceived colors of the dress. Perceptual dimming shifted color categorization toward blue/black whereas perceptual brightening shifted color categorization toward white/gold. We conclude that color categorization is influenced substantially by illusory shifts in brightness.