Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Horvat M

PROVIDER: S-EPMC6813634 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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

Horvat Melissa M Fritsche Susanne S Kourist Robert R Winkler Margit M

ChemCatChem 20190409 16

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 versic ...[more]

PMID: 31681448

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Project description:The rare actinomycete Actinoplanes missouriensis produces terminal sporangia containing a few hundred flagellated spores. After release from the sporangia, the spores swim rapidly in aquatic environments as zoospores. The zoospores stop swimming and begin to germinate in niches for vegetative growth. Here, we report the characterization and functional analysis of zoospore type IV pili in A. missouriensis The pilus gene (pil) cluster, consisting of three apparently ?FliA-dependent transcriptional units, is activated during sporangium formation similarly to the flagellar gene cluster, indicating that the zoospore has not only flagella but also pili. With a new method in which zoospores were fixed with glutaraldehyde to prevent pilus retraction, zoospore pili were observed relatively easily using transmission electron microscopy, showing 6?±?3 pili per zoospore (n?=?37 piliated zoospores) and a length of 0.62?±?0.35??m (n?=?206), via observation of fliC-deleted, nonflagellated zoospores. No pili were observed in the zoospores of a prepilin-encoding pilA deletion (?pilA) mutant. In addition, the deletion of pilT, which encodes an ATPase predicted to be involved in pilus retraction, substantially reduced the frequency of pilus retraction. Several adhesion experiments using wild-type and ?pilA zoospores indicated that the zoospore pili are required for the sufficient adhesion of zoospores to hydrophobic solid surfaces. Many zoospore-forming rare actinomycetes conserve the pil cluster, which indicates that the zoospore pili yield an evolutionary benefit in the adhesion of zoospores to hydrophobic materials as footholds for germination in their mycelial growth.IMPORTANCE Bacterial zoospores are interesting cells in that their physiological state changes dynamically: they are dormant in sporangia, show temporary mobility after awakening, and finally stop swimming to germinate in niches for vegetative growth. However, the cellular biology of a zoospore remains largely unknown. This study describes unprecedented zoospore type IV pili in the rare actinomycete Actinoplanes missouriensis Similar to the case for the usual bacterial type IV pili, zoospore pili appeared to be retractable. Our findings that the zoospore pili have a functional role in the adhesion of zoospores to hydrophobic solid surfaces and that the zoospores use both pili and flagella properly according to their different purposes provide an important insight into the cellular biology of the zoospore.

Dataset Information

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

Publications

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

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