Fungal glucosylceramide plays important role in cell division, hyphal formation and growth, spore germination and the modulation of virulence and has recently been considered as target for small molecule inhibitors. In this study, we characterized CgGCS, a protein encoding a glucosylceramide synthase (GCS) in Colletotrichum gloeosporioides. Disruption of CgGCS resulted in a severe reduction of mycelial growth and defects in conidiogenesis. Sphingolipid profile analysis revealed large decreases in glucosylceramide production in the mutant strains. Pathogenicity assays indicated that the ability of the ΔCgGCS mutants to invade both tomato and mango hosts was almost lost. In addition, the expression levels of many genes, especially those related to metabolism, were shown to be affected by the mutation of CgGCS via transcriptome analysis. Overall, our results demonstrate that C. gloeosporioides glucosylceramide is an important regulatory factor in fungal growth, conidiation, and pathogenesis in hosts.

pathogenicity, glucosylceramide, Colletotrichum gloeosporioides, GCS conidiogenesis, growth rate

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

R1 = LIP
R2 = xXSphd
[1*][2*]O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O

179.148 g/mol (C₆H₁₁R₂O₆, R = residue superclass(es), not counted in mol weight)

Fucosylated oligosaccharides are interesting molecules due to their bioactive properties. In particular, their application as active ingredient in milk powders is attractive for dairy industries. The objective of this study was to characterize the glycosyl hydrolase family 29 α-fucosidase produced by Aspergillus niger and test its ability to transfucosylate lactose with a view towards potential industrial applications such as the valorization of the lactose side stream produced by dairy industry. In order to reduce costs and toxicity the use of free fucose instead of environmentally questionable fucose derivatives was studied. In contrast to earlier studies, a recombinantly produced A. niger α-fucosidase was utilized. Using pNP-fucose as substrate, the optimal pH for hydrolytic activity was determined to be 3.8. The optimal temperature for a 30-min reaction was 60 °C, and considering temperature stability, the optimal temperature for a 24-h reaction was defined as 45 °C For the same hydrolysis reaction, the kinetic values were calculated to be 0.385 mM for the KM and 2.8 mmol/mg/h for the Vmax. Transfucosylation of lactose occurred at high substrate concentrations when reaction time was elongated to several days. The structure of the product trisaccharide was defined as 1-fucosyllactose, where fucose is α-linked to the anomeric carbon of the β-glucose moiety of lactose. Furthermore, the enzyme was able to hydrolyze its own transfucosylation product and 2'-fucosyllactose but only poorly 3-fucosyllactose. As a conclusion, α-fucosidase from A. niger can transfucosylate lactose using free fucose as substrate producing a novel non-reducing 1-fucosyllactose.

Aspergillus niger, 1-fucosyllactose, fucosyllactose, non-reducing sugar, transfucosylation, α-fucosidase

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
1,4	bDGalp	104.2	72.3	73.8	69.8	76.7	62.3
1	bDGlcp	98.5	73.7	75.5	79.5	76.2	61.2
	aLFucp	96.5	68.6	70.7	73.0	68.5	16.6


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
1,4	bDGalp	4.456	3.545	3.666	3.926	3.727	3.773
1	bDGlcp	4.658	3.423	3.674	3.701	3.598	3.826-3.971
	aLFucp	5.283	3.838	3.913	3.833	4.220	1.216


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
1,4	bDGalp	104.2/4.456	72.3/3.545	73.8/3.666	69.8/3.926	76.7/3.727	62.3/3.773
1	bDGlcp	98.5/4.658	73.7/3.423	75.5/3.674	79.5/3.701	76.2/3.598	61.2/3.826-3.971
	aLFucp	96.5/5.283	68.6/3.838	70.7/3.913	73.0/3.833	68.5/4.220	16.6/1.216

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

C[C@@H]1O[C@@H](O[C@@H]2O[C@H](CO)[C@@H](O[C@@H]3O[C@H](CO)[C@H](O)[C@H](O)[C@H]3O)[C@H](O)[C@H]2O)[C@@H](O)[C@H](O)[C@@H]1O

488.439 g/mol (C₁₈H₃₂O₁₅)

Sake, the Japanese rice wine, contains a variety of oligosaccharides and glucosides produced by fungal enzymes during the brewing process. This study investigates the effect of knocking out the Aspergillus oryzae α-glucosidase (agdA) gene on the transglycosylation products in brewed sake. In addition to α-ethyl glucoside and α-glyceryl glucoside, the amount of two compounds that have molecular mass values similar to that of ethyl maltose decreased by agdA gene knockout. Both compounds were synthesized, in vitro, from maltose and ethanol with purified agdA. Nuclear magnetic resonance analysis identified the two compounds as ethyl α-maltoside and ethyl α-isomaltoside, respectively, which are novel compounds in sake as well as in the natural environment. Quantitative analysis of 111 commercially available types of sake showed that these novel compounds were widely present at concentrations of several hundred mg/L, suggesting that both of them are ones of the common glycosides in sake.

glucoside, transglycosylation, Aspergillus oryzae, α-glucosidase, brewing, sake

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
1,4	aDGlcp	99.7	71.8	72.9	69.4	72.7	60.5
1	aDGlcp	97.8	71.1	73.7	77.0	70.2	60.6
	Et	64.0	14.2


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
1,4	aDGlcp	5.32	3.50	3.61	3.34	3.63	3.69-3.79
1	aDGlcp	4.85	3.51	3.88	3.56	3.73	3.69-3.80
	Et	3.50-3.71	1.15


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
1,4	aDGlcp	99.7/5.32	71.8/3.50	72.9/3.61	69.4/3.34	72.7/3.63	60.5/3.69-3.79
1	aDGlcp	97.8/4.85	71.1/3.51	73.7/3.88	77.0/3.56	70.2/3.73	60.6/3.69-3.80
	Et	64.0/3.50-3.71	14.2/1.15

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CCO[C@H]1O[C@H](CO)[C@@H](O[C@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O

370.351 g/mol (C₁₄H₂₆O₁₁)

Sake, the Japanese rice wine, contains a variety of oligosaccharides and glucosides produced by fungal enzymes during the brewing process. This study investigates the effect of knocking out the Aspergillus oryzae α-glucosidase (agdA) gene on the transglycosylation products in brewed sake. In addition to α-ethyl glucoside and α-glyceryl glucoside, the amount of two compounds that have molecular mass values similar to that of ethyl maltose decreased by agdA gene knockout. Both compounds were synthesized, in vitro, from maltose and ethanol with purified agdA. Nuclear magnetic resonance analysis identified the two compounds as ethyl α-maltoside and ethyl α-isomaltoside, respectively, which are novel compounds in sake as well as in the natural environment. Quantitative analysis of 111 commercially available types of sake showed that these novel compounds were widely present at concentrations of several hundred mg/L, suggesting that both of them are ones of the common glycosides in sake.

glucoside, transglycosylation, Aspergillus oryzae, α-glucosidase, brewing, sake

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
1,6	aDGlcp	98.1	71.7	73.3	69.8	72.1	60.7
1	aDGlcp	98.2	71.4	73.7	69.7	70.4	65.8
	Et	64.4	14.4


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
1,6	aDGlcp	4.95	3.55	3.72	3.43	3.71	3.77-3.85
1	aDGlcp	4.94	3.56	3.69	3.52	3.87	3.72-3.99
	Et	3.59-3.81	1.23


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
1,6	aDGlcp	98.1/4.95	71.7/3.55	73.3/3.72	69.8/3.43	72.1/3.71	60.7/3.77-3.85
1	aDGlcp	98.2/4.94	71.4/3.56	73.7/3.69	69.7/3.52	70.4/3.87	65.8/3.72-3.99
	Et	64.4/3.59-3.81	14.4/1.23

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CCO[C@H]1O[C@H](CO[C@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)[C@@H](O)[C@H](O)[C@H]1O

370.351 g/mol (C₁₄H₂₆O₁₁)