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Chiral diaryl alcohols, such as (4-chlorophenyl)(pyridin-2-yl)methanol, are important intermediates for pharmaceutical synthesis. However, using alcohol dehydrogenases (ADHs) in the asymmetric reduction of diaryl ketones to produce the corresponding alcohols is challenging due to steric hindrance in the substrate binding pockets of the enzymes. In this study, the steric hindrance of the ADH from Geotrichum candidum NBRC 4597 ( G. candidum acetophenone reductase, Gc APRD) was eliminated by simultaneous site-directed mutagenesis of Phe56 (in the large pocket) and Trp288 (in the small pocket). As a result, two double mutants, Phe56Ile/Trp288Ala, and Phe56Ala/Trp288Ala, exhibited much higher specific activities towards 2-(4′-chlorobenzoyl)pyridine (4.5 μmol/min/mg and 3.4 μmol/min/mg, respectively) than the wild type (< 0.2 μmol/min/mg). In whole-cell-catalyzed asymmetric reductions of diaryl ketones, Phe56Ile/Trp288Ala significantly increased the isolated yields, which were over 90% for the reactions of most of the tested substrates. Regarding enantioselectivity, Phe56Ile/Trp288Ala and Phe56Ala/Trp288Ala, and Trp288Ala generally exhibited similar selectivity to produce ( R )-alcohols with up to 97% ee . Key points • Phe56 in Geotrichum reductase (GcAPRD) was mutated to eliminate steric hindrance. • Mutation at Phe56 increased enzymatic activity and expanded substrate specificity. • Phe56Ile/Trp288Ala showed high activity and (R)-selectivity towards diaryl ketones. Graphical Abstract

Camembert-type cheese ripening is driven mainly by fungal microflora including Geotrichum candidum and Penicillium camemberti. These species are major contributors to the texture and flavour of typical bloomy rind cheeses. Biochemical studies showed that G. candidum reduces bitterness, enhances sulphur flavors through amino acid catabolism and has an impact on rind texture, firmness and thickness, while P. camemberti is responsible for the white and bloomy aspect of the rind, and produces enzymes involved in proteolysis and lipolysis activities. However, very little is known about the genetic determinants that code for these activities and their expression profile over time during the ripening process. The metatranscriptome of an industrial Canadian Camembert-type cheese was studied at seven different sampling days over 77 days of ripening. A database called CamemBank01 was generated, containing a total of 1,060,019 sequence tags (reads) assembled in 7916 contigs. Sequence analysis revealed that 57% of the contigs could be affiliated to molds, 16% originated from yeasts, and 27% could not be identified. According to the functional annotation performed, the predominant processes during Camembert ripening include gene expression, energy-, carbohydrate-, organic acid-, lipid- and protein- metabolic processes, cell growth, and response to different stresses. Relative expression data showed that these functions occurred mostly in the first two weeks of the ripening period. These data provide further advances in our knowledge about the biological activities of the dominant ripening microflora of Camembert cheese and will help select biological markers to improve cheese quality assessment.

Enzyme engineering has been widely employed to tailor the substrate specificity and enantioselectivity of enzymes. In this study, we mutated Trp288, an unconserved residue in the small binding pocket of an acetophenone reductase from Geotrichum candidum NBRC 4597 ( Gc APRD). Trp288 mutants showed substrate specificity expansion towards bulky-bulky ketones and enantioselectivity alteration which was highly dependent on the substrate substituent length. In aliphatic ketone reduction, enantioselectivity inverted from ( S ) to ( R ) when one of the substituents to the carbonyl carbon was elongated from propyl to butyl or pentyl. The best ( R )-selective mutant, Trp288Val, achieved the reduction of 3-heptanone to its corresponding ( R )-alcohol with 97% ee . Our docking simulation suggested that when enantioselectivity inverted to ( R ), only pro- R binding poses were productive. Gly94 played an important role to stabilize the butyl or pentyl group for their productive pro- R poses. Interestingly, when the substituent was further elongated, the enantioselectivity inverted back to the ( S ) form.

Plant-associated endophytes are recognized as sources of novel bioactive molecules having diverse applications. In this study, an endophytic yeast-like fungal strain was isolated from the fruit of eggplant (Solanum melongena) and identified as Geotrichum candidum through phenotypic and genotypic characterizations. This endophytic G. candidum isolate PF005 was found to emit fruity scented volatiles. The compositional profiling of volatile organic compounds (VOCs) revealed the presence of 3-methyl-1-butanol, ethyl 3-methylbutanoate, 2-phenylethanol, isopentyl acetate, naphthalene, and isobutyl acetate in significant proportion when analyzed on a time-course basis. The VOCs from G. candidum exhibited significant mycelial growth inhibition (54%) of phytopathogen Rhizoctonia solani, besides having mild antifungal activity against a few other fungi. The source of carbon as a nutrient was found to be an important factor for the enhanced biosynthesis of antifungal VOCs. The antifungal activity against phytopathogen R. solani was improved up to 91% by feeding the G. candidum with selective precursors of alcohol and ester volatiles. Furthermore, the antifungal activity of VOCs was enhanced synergistically up to 92% upon the exogenous addition of naphthalene (1.0 mg/plate). This is the first report of G. candidum as an endophyte emitting antifungal VOCs, wherein 2-penylethanol, isopentyl acetate, and naphthalene were identified as important contributors to its antifungal activity. Possible utilization of G. candidum PF005 as a mycofumigant has been discussed based upon its antifungal activity and the qualified presumption of safety status.

The evolutionary history of the characters underlying the adaptation of microorganisms to food and biotechnological uses is poorly understood. We undertook comparative genomics to investigate evolutionary relationships of the dairy yeast Geotrichum candidum within Saccharomycotina. Surprisingly, a remarkable proportion of genes showed discordant phylogenies, clustering with the filamentous fungus subphylum (Pezizomycotina), rather than the yeast subphylum (Saccharomycotina), of the Ascomycota. These genes appear not to be the result of Horizontal Gene Transfer (HGT), but to have been specifically retained by G. candidum after the filamentous fungi–yeasts split concomitant with the yeasts’ genome contraction. We refer to these genes as SRAGs (Specifically Retained Ancestral Genes), having been lost by all or nearly all other yeasts and thus contributing to the phenotypic specificity of lineages. SRAG functions include lipases consistent with a role in cheese making and novel endoglucanases associated with degradation of plant material. Similar gene retention was observed in three other distantly related yeasts representative of this ecologically diverse subphylum. The phenomenon thus appears to be widespread in the Saccharomycotina and argues that, alongside neo-functionalization following gene duplication and HGT, specific gene retention must be recognized as an important mechanism for generation of biodiversity and adaptation in yeasts.

Background Geotrichum is a genus of fungi found in different habitats throughout the world. Although Geotrichum and its related species have been extensively reclassified and taxonomically revised, it is still the target for many researches. Methods and results In this study, phenotypic and molecular genetics comparisons were performed between Geotrichum candidum and Geotrichum silvicola . Mitis Salivarius Agar was used as the growing medium for the phenotypic comparison study, which was carried out at two temperatures (20–25 and 37 °C). For genotypic comparison, we compared the 18 S, ITS, and 28 S sequences of universal DNA barcode regions of both species. Important findings on the new culture media for fungal isolation were revealed by the results. The phenotypic variation between the two species’ colonies, including their shapes, sizes, textures and growth rates, were strikingly different. DNA sequences of both species showed that pairwise identities of the species were 99.9% for 18 S, 100% for ITS and 99.6% for 28 S regions. Conclusions Contrary to what is commonly seen, the results showed that 18 S, ITS and 28 S failed to discriminate the species. The first investigation into the performance of Mitis Salivarius Agar as a fungus culture medium is reported in this work, and proved its efficiency. Additionally, this is the first study to compare G. candidum with G. silvicola by means of both phenotypic and genotypic analysis.

Geotrichum candidum is a fungus-like yeast widely used as a starter culture for cheese ripening for its proteolytic and lipolytic activities and its contribution to the cheese flavours. The sequenced strain G. candidum CLIB 918 was isolated from cheese Pont-L'Evêque. This strain's ability to produce volatile compounds was compared to the ability of a known strong sulphur compound producer G. candidum strain (Gc203). The aminotransferase-coding genes BAT2 and ARO8 were identified to be involved in methionine catabolism. The production of volatile compounds indicated that the sequenced strain was a moderate producer compared to the strong producer strain. The major volatile compounds were produced from sulphur amino acid, branched-chain amino acid and fatty acid metabolisms. Metabolite content of the cells showed that the ability of the strain to produce volatile compounds was inversely proportional to its ability to store amino acids inside the cells. Reduced glutathione, hypotaurine and taurine intracellular concentrations and volatile fatty aldehyde production indicated the role of oxidative stress sensitivity in flavour production. The increase in expression of several genes in a Reblochon-type cheese at the end of ripening confirmed that oxygen and iron were key factors regulating cheese flavour production.

Higher alcohols are the byproducts of yeasts in alcohol fermentation and are harmful to human health at high concentrations. In this study, immobilized crude enzymes extracted from Geotrichum spp. strains S12 and S13 were separately employed to treat red wine, then GC and GC-MS analyses were used to determine the profiles of volatile compounds in untreated and treated wine samples. Immobilized enzymes from S13 (SA-S13E) were more active in decreasing higher alcohols than enzymes from S12. Conditions for preparing SA-S13E were optimized, and best results were obtained at a sodium alginate concentration of 35 g/L, calcium chloride of 20 g/L, and crude enzyme dosage of 3 mL. Treatment with SA-S13E significantly increased the ester content and sensory quality of wine. After being reused three times, SA-S13E still exhibited approximately 80% activity towards 1-propanol, isobutanol, and hexanol and had certain activity even after 3 months storage at −20 °C, indicating high stability in application and storage and thus showing potential in wine processing.

The gene encoding acetophenone reductase (APRD), a useful biocatalyst for producing optically pure alcohols, was cloned from the cDNA of Geotrichum candidum NBRC 4597. The gene contained an open reading frame that consisted of 1,029 nucleotides corresponding to 342 amino acid residues. The subunit molecular weight was calculated to be 36.7 kDa. The predicted amino acid sequence did not have significant similarity to those of the acetophenone reductase reported previously. The gene was inserted into the pET-21b(+) expression vector and expressed in Escherichia coli Rosetta™(DE3)pLysS by induction with 1 mM of isopropyl-β- d -thiogalactopyranoside. E. coli cell-free extract gave 21.9 U/mg APRD activity, which was 81 times that of the G. candidum cell-free extract. The enzyme was purified with a HisTrap FF crude column. The enzyme exhibited the highest activity at 60 °C, and optimum reducing and oxidizing activity were observed in a pH range around 7.0–8.0 and 8.5, respectively. The enzyme was most stable at 60 °C and pH 6.5–7.5. The Vmax and the apparent Km value of the reductase were 67.6 μmol/min per milligram of protein and 0.146 mM for acetophenone, respectively. From 4 % ( v / v ) 4-phenyl-2-butanone, ( S )-4-phenyl-2-butanol was obtained with a yield >80 % and an enantiomeric excess >99 % in a 20 h reaction recycling NADH with 15 % ( v / v ) 2-propanol.

The introduction of multilocus sequence typing (MLST) for strain characterization provided the first sequence-based approach for genotyping many fungi, leading to reproducible, reliable, and exchangeable data. A MLST scheme based on the analysis of six housekeeping genes was developed for genotyping Geotrichum candidum . The scheme was first developed using 18 isolates for which the complete sequences of the alanyl-tRNA synthetase ( ALA 1), pyruvate kinase ( CDC 19), acetyl-coA acetyltransferase ( ERG 10), glutaminyl-tRNA synthase ( GLN 4), phosphoglucoisomerase ( PGI 1), and phosphoglucomutase ( PGM 2) housekeeping genes were determined. Multiple sequence alignments of these genes were used to define a set of loci showing, as closely as possible, the same phylogenetic resolution level as complete gene sequences. This scheme was subsequently validated with 22 additional isolates from dairy and non-dairy sources. Overall, 58 polymorphic sites were indexed among 3,009 nucleotides analyzed. Depending on the loci, four to eight alleles were detected, generating 17 different sequence types, of which ten were represented by a single strain. MLST analysis suggested a predominantly clonal population for the 40  G . candidum isolates. Phylogenetic analysis of the concatenated sequences revealed a distantly related group of four isolates. Interestingly, this group diverged with respect to internal transcribed spacers 1 (ITS1), 5.8S, and ITS2 analysis. The reproducibility of the MLST approach was compared to random amplification of microsatellites by PCR (RAM-PCR), a gel profiling method previously proposed for G . candidum strain typing. Our results found MLST differentiation to be more efficient than RAM-PCR, and MLST also offered a non-ambiguous, unique language, permitting data exchange and evolutionary inference.