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Biocatalytic approaches have gained increasing attention as sustainable alternatives to metal-catalyzed asymmetric reductions of ketones to obtain enantiopure alcohols, important intermediates for pharmaceutical synthesis. For example, enzyme-catalyzed reduction of substituted benzophenone analogs to produce chiral diaryl methanols has attracted interest, as they are the key intermediates in the synthesis of antihistamines. However, benzophenone analogs are difficult to be reduced by enzymes due to steric hindrance. Moreover, the similarities between the two groups adjacent to the carbonyl group make achieving high enantioselectivity in reduction challenging. In this study, we examined the reduction of benzophenone and its analogs by Geotrichum candidum acetophenone reductase ( Gc APRD). However, the wild type did not exhibit activity toward benzophenone due to the substrate’s bulkiness. Then, two mutants of Gc APRD (Trp288Ala and Phe56Ile/Trp288Ala) were applied to catalyze the reduction of benzophenone, resulting in high reduction yield (≥ 80%). In addition, both mutants exhibited catalytic activity toward methyl- and halogen-substituted benzophenones, especially toward 3- and 4-substituted substrates. Regarding enantioselectivity, Trp288Ala generally reduced both 3- and 4-substituted substrates to ( R )-alcohols with up to 97% ee . In contrast, Phe56Ile/Trp288Ala reduced 3-substituted substrates to ( R )-alcohols with up to 89% ee but reduced 4-substituted substrates to ( S )-alcohols with up to 92% ee . At last, the reduction mechanism was investigated using molecular docking simulations. Key points • GcAPRD mutants exhibited catalytic performance toward benzophenone analogs. • GcAPRD Phe56Ile/Trp288Ala exhibited substituent-dependent enantioselectivity. • Introducing Phe56Ile into GcAPRD Trp288Ala resulted in a clear enantiopreference. Graphical Abstract

Two asexual arthroconidial yeast strains, TM3-44T and LYSM5T, were isolated, respectively, from estuarine water in a mangrove forest and soil in a terrestrial forest in Thailand. Analysis of the D1/D2 domains of the large-subunit rRNA gene sequences revealed that strain TM3-44T differed from the closest species in terms of pairwise sequence similarity, Dipodascus albidus, by 11.7% nucleotide substitutions, while strain LYSM5T was closest to Galactomyces geotrichum with only 2.9% nucleotide substitutions. The phylogenetic tree further demonstrated that strain TM3-44T was at a distant position from the closest species, D. albidus, and other related species in the Dipodascus clade, while strain LYSM5T clustered with G. geotrichum, it closest relative in the Galactomyces clade. The phenotypic characteristics of the two strains were typical of the genus Geotrichum. On the basis of the above findings, strain TM3-44T was assigned as a novel species of Geotrichum, for which the name Geotrichum siamensis sp. nov. is proposed. The type strain is TM3-44T (BCC 29903T=NBRC 104880T=CBS 10929T). Strain LYSM5T represented another novel species of Geotrichum, which was named Geotrichum phurueaensis sp. nov. The type strain is LYSM5T (BCC 34756T=NBRC 105674T=CBS 11418T).

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.

ObjectivePectinase is an industrially important enzyme which is employed in an array of commercial processes; cost of production, however, impedes its application. The main objective of this study was to design a two-layered strategy for the reduction of production cost, firstly by using a yeast co-culture in an immobilized form on an agricultural waste matrix, corncob (CB), secondly by utilizing orange peels (OP) as substrate.ResultsTwo yeast strains, Saccaromyces cerevisiae MK-157 and Geotrichum candidum AA15 were cultivated as mono-, as well as, co-culture after immobilization on CB and pectinase production was monitored. Initial experiments revealed that co-culture is beneficial to get sustainable product in subsequent 2nd and 3rd production cycles. The factors affecting pectinase production in consecutive three production cycles were studied by employing Plackett–Burman design and the significant factors were optimized through Box–Behnken design. Under optimized conditions, 17.89 IU mL−1 of pectinase was obtained. Scanning electron micrographs presented damaged immobilized yeast cells on CB after the 3rd production cycle.ConclusionThe pectinase production was improved substantially by using immobilized co-culture and hence the strategy was found effective at lab scale. Since, pectinase is applied in orange juice clarification, therefore, the study can be extended to move forward towards circular economy.

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.

Consumers demand the use of eco-friendly fungicides to treat fruit and vegetables and governmental authorities have unauthorized the application of chemical antifungals for the efficient control of sour rot. In the present research, the microwave irradiation (MW) method was used to encapsulate thymol into 2-hydroxylpropyl-beta-cyclodextrin (HP-β-CD) and the effect of these HP-β-CD on controlling sour rot in citrus fruit, caused by Geotrichum citri-aurantii, was evaluated. Amounts of 25 and 50 mM of HP-β-CD-thymol were used, and compared with propiconazole, to control the decay of inoculated lemon fruit. The treatments were performed in curative and preventive experiments. The incidence and severity of Geotrichum citri-aurantii in 25 and 50 mM HP-β-CD-thymol-treated fruit were reduced in both experiments. The preventive 50 mM HP-β-CD-thymol treatment showed the best effect, reducing the sour rot, respiration rate and fruit weight loss during storage at 20 °C. HP-β-CD-thymol increased polyphenol concentration and the activity of antioxidant enzymes, such as catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD) in lemon peel, and the highest effects were found with the 50-mM dose. In conclusion, the results show that the use of thymol encapsulated by MW into HP-β-CD could be an effective and sustainable tool, a substitute to the synthetic fungicides, for G. citri-auriantii control in citrus fruit.

This study investigated the potential anti-fungal mechanisms of sodium dehydroacetate (SD) against Geotrichum citri-aurantii. The results showed that the cell wall integrity of G. citri-aurantii was not affected, whereas the membrane permeability of G. citri-aurantii mycelia was visibly altered by SD. Dramatic morphological changes of the mycelia, such as loss of cytoplasm, plasmolysis, and dissolution of intracellular substances, were observed by scanning electron microscopy and transmission electron microscopy analyses, indicating that the mycelium is severely damaged by the SD treatment. Furthermore, SD apparently induced a decrease in the intracellular ATP content before 30 min of exposure. An increase in the activity of the Na+/K+-ATPase was also observed, indicating that Na+ ions might enter the cell and thus disturb the energy supply. Taken together, this study’s findings suggest that the anti-fungal activity of SD against G. citri-aurantii can be attributed to the disruption of cell membrane permeability and energy metabolism.

Endophytic fungi of healthy and brittle leaf diseased (BLD) date palm trees (Phoenix dactylifera L.) represent a promising source of bioactive compounds with biomedical, industrial, and pharmaceutical applications. The fungal endophytes Penicillium citrinum isolate TDPEF34, and Geotrichum candidum isolate TDPEF20 from healthy and BLD date palm trees, respectively, proved very effective in confrontation assays against three pathogenic bacteria, including two Gram-positive bacteria Bacillus thuringiensis (Bt) and Enterococcus faecalis (Ef), and one Gram-negative bacterium Salmonella enterica (St). They also inhibited the growth of three fungi Trichoderma sp. (Ti), Fusarium sporotrichioides (Fs), Trichoderma sp. (Ts). Additionally, their volatile organic compounds (VOCs) were shown to be in part responsible for the inhibition of Ti and Ts and could account for the full inhibition of Fs. Therefore, we have explored their potential as fungal cell factories for bioactive metabolites production. Four extracts of each endophyte were prepared using different solvent polarities, ethanol (EtOH), ethyl acetate (EtOAc), hexane (Hex), and methanol (MetOH). Both endophyte species showed varying degrees of inhibition of the bacterial and fungal pathogens according to the solvent used. These results suggest a good relationship between fungal bioactivities and their produced secondary metabolites. Targeting the discovery of potential anti-diabetic, anti-hemolysis, anti-inflammatory, anti-obesity, and cytotoxic activities, endophytic extracts showed promising results. The EtOAc extract of G. candidum displayed IC50 value comparable to the positive control diclofenac sodium in the anti-inflammatory assays. Antioxidant activity was evaluated using α,α-diphenyl-β-picrylhydrazyl (DPPH), β-carotene bleaching, reducing power (RP), and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonique) (ABTS) radical scavenging assays. The findings revealed strong anti-oxidant power with an IC50 of 177.55 µg/mL for G. candidum EtOAc extract using DPPH assay, probably due to high polyphenol and flavonoid content in both fungal extracts. Finally, LC-HRMS (Liquid Chromatography–High Resolution Mass Spectrometry) and GC-MS (Gas Chromatography–Mass Spectrometry) analysis of G. candidum and P. citrinum extracts revealed an impressive arsenal of compounds with previously reported biological activities, partly explaining the obtained results. Finally, LC-HRMS analysis indicated the presence of new fungal metabolites that have never been reported, which represent good candidates to follow for the discovery of new bioactive molecules.