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A UDP-glycosyltransferase from Bacillus licheniformis was exploited for the glycosylation of phloretin. The in vitro glycosylation reaction confirmed the production of five phloretin glucosides, including three novel glucosides. Consequently, we demonstrated the application of the same glycosyltransferase for the efficient whole-cell biocatalysis of phloretin in engineered Escherichia coli .

A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d - and l -sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α- d -glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3- O -β- d -glucoside, resveratrol 4′- O -β- d -glucoside, resveratrol 3,5- O -β- d -diglucoside, and resveratrol 3,5,4′- O -β- d -triglucoside. The conversion rates and numbers of products formed were found to vary with the other NDP sugar donors. Resveratrol 3- O -β- d -2-deoxyglucoside and resveratrol 3,5- O -β- d -di-2-deoxyglucoside were found to be produced using TDP-2-deoxyglucose as a donor; however, the monoglycosides resveratrol 4′- O -β- d -galactoside, resveratrol 4′- O -β- d -viosaminoside, resveratrol 3- O -β- l -rhamnoside, and resveratrol 3- O -β- l -fucoside were produced from the respective sugar donors. Altogether, 10 diverse glycoside derivatives of the medically important resveratrol were generated, demonstrating the capacity of YjiC to produce structurally diverse resveratrol glycosides.

Extracellular Penicillium fellutanum exo-β- d -galactofuranosidase, with a mass of 70 kDa, was purified to apparent homogeneity. The enzyme was used to investigate the influence of phosphodiesters of the peptidophosphogalactomannans pP 2 GM ii and pP 25 GM ii (containing 2 and 25 phosphodiester residues, respectively, per mol of polymer) on the kinetic parameters of galactofuranosyl hydrolysis of these two polymers, of 1- O -methyl-β- d -galactofuranoside, and of two galactofuranooligosaccharides. The enzyme did not hydrolyze phosphorylated galactose residues of pP 2 GM ii or pP 25 GM ii . The k cat / K m value for pP 25 GM ii is 1.7 × 10 3 M −1 s −1 , that for 1- O -methyl-β- d -galactofuranoside is 1.1 × 10 4 M −1 s −1 , that for pP 2 GM ii is 1.7 × 10 4 M −1 s −1 , and those for 5- O -β- d -galactofuranooligosaccharides with degrees of polymerization of 3.4 and 5.5 are 1.7 × 10 5 and 4.1 × 10 5 M −1 s −1 , respectively. Variability in the k cat / K m values is due primarily to differences in K m values; the k −1 / k 1 ratio likely provides the most influence on K m . k cat increases as the degree of polymerization of galactofuranosyl residues increases. Most of the galactofuranosyl and phosphocholine residues were removed by day 8 in vivo from pP x GM ii added to day 3 cultures initiated in medium containing 2 mM phosphate but not from those initially containing 20 mM phosphate. The filtrates from day 9 cultures initiated in 2 mM inorganic phosphate in modified Raulin-Thom medium contained 0.2 mM inorganic phosphate and 2.2 U of galactofuranosidase ml −1 h −1 . No galactofuranosidase activity but 15 mM inorganic phosphate was found in filtrates from day 9 cultures initiated in 20 mM phosphate. In vivo the rate of galactofuranosyl hydrolysis of pP x GM ii and of related polymers is proportional to the k cat / K m value of each polymer. The kinetic data show that the k cat / K m value increases as the number of phosphodiesters of pP x GM ii decreases, also resulting in an increase in the activity of exo-β- d -galactofuranosidase.

Addition of 3 M NaCl to 72-h cultures of Penicillium fellutanum in 2 mM phosphate resulted in an increase in percentage of extracellular peptidophosphogalactomannan III (pP x GM iii ) and a decrease in that of pP x GM ii . The magnitude of 31 P nuclear magnetic resonance signals at 1.47 and 1.33 ppm of phospho-1- O -[ N -peptidyl-(2-aminoethanol)] phosphodiesters pP x GM ii and pP x GM iii decreased compared with controls. The data suggest that serine, glycine, and threonine residues from the 3-kDa peptide and from galactofuranosyl-6- O -phospho-1′- O -[ N -peptidyl-(2-aminoethanol)] residues were the precursors of the needed choline-derived osmolytes.

Choline is a major component of membranes and a structural component of some microbial cell wall polymers (12, 16, 20). In some bacteria and higher plants, choline is a precursor of the osmolyte glycine betaine (GB) (2, 3, 15). In Penicillium fellutanum cultured in a medium containing 3 M NaCl, 46 and 70 mM concentrations of the osmolytes choline-O-sulfate (COS) and GB, respectively, accumulated (14). Choline stimulates hyphal extension, inhibits initiation of branching (19, 22), and is an essential nutrient for growth of choline auxotrophs of Neurospora crassa (6) and Aspergillus nidulans (9, 10). COS is a known sulfate storage molecule in fungi (4, 5, 7), and it is also a known endogenous reserve source of choline that stimulates growth in choline-requiring auxotrophs of A. nidulans cultured in insufficient choline (11). However, Markham et al. (11) suggested that A. nidulans carrying mutations blocked in sulfate metabolism did not synthesize COS and that residual growth must have resulted from an unknown endogenous storage precursor of choline. We previously reported that, as phosphate in the nutrient medium becomes limiting, choline phosphodiesters of P. fellutanum extracellular glycopeptide (peptidophosphogalactomannan) provide phosphate and choline, and excess choline accumulates as cytoplasmic GB and COS (13, 14). This finding was exploited to determine the relationship between COS, GB, and choline in P. fellutanum under sulfate-limiting conditions. We assume that COS is a storage form of both sulfate and choline in filamentous fungi (11). This study was focused on determining if an alternative intracellular soluble precursor of choline or COS accumulates in P. fellutanum cultured in limiting sulfate or if the concentration of choline increases in the cytoplasm. Influence of phosphate concentration in the nutrient medium on accumulation of COS and GB. The sources of P. fellutanum, nutrients, and L-[methyl- 13 C]methionine, preparation of mycelial extracts, and 13 C nuclear magnetic resonance (NMR) spectroscopy analysis have been described recently (14). The 13 C-methyl signals of COS (56.77 ppm) and GB (56.23 ppm) in extracts of mycelium from 200 ml of 8-day cultures in lowphosphate standard growth (LPSG) (2 mM Pi) or standard growth (SG) (20 mM Pi) medium containing L-[methyl- 13 C]methionine were integrated, and their magnitudes were compared with that of the 0.22% TSP [(trimethylsilane)-1-propanesulfonate] (0.00 ppm) signal, all as described previously (13, 14). COS in extracts of day-5 mycelium from LPSG and SG medium was 9.3 and 6.3 mg (dry weight) per g, respectively (14). No significant GB was found in extracts of SG mycelium. The increases in COS and GB in mycelium from LPSG medium may result from decreases in the requirements for choline and ethanolamine. No detectable soluble choline occurred in mycelium from LPSG medium. This suggests that COS and GB are the primary choline precursors or storage products. Age-dependent accumulation of COS and GB. Mycelium