P450Jα : A New, Robust and α-Selective Fatty Acid Hydroxylase Displaying Unexpected 1-Alkene Formation.

Oxyfunctionalization of fatty acids (FAs) is a key step in the design of novel synthetic pathways for biobased/biodegradable polymers, surfactants and fuels. Here, we show the isolation and characterization of a robust FA α-hydroxylase (P450Jα ) which catalyses the selective conversion of a broad range of FAs (C6:0-C16:0) and oleic acid (C18:1) with H2 O2 as oxidant. Under optimized reaction conditions P450Jα yields α-hydroxy acids all with >95 % regioselectivity, high specific activity (up to 15.2 U mg-1 ) and efficient coupling of oxidant to product (up to 85 %). Lauric acid (C12:0) turned out to be an excellent substrate with respect to productivity (TON=394 min-1 ). On preparative scale, conversion of C12:0 reached 83 % (0.9 g L-1 ) when supplementing H2 O2 in fed-batch mode. Under similar conditions P450Jα allowed further the first biocatalytic α-hydroxylation of oleic acid (88 % conversion on 100 mL scale) at high selectivity and in good yields (1.1 g L-1 ; 79 % isolated yield). Unexpectedly, P450Jα displayed also 1-alkene formation from shorter chain FAs (≤C10:0) showing that oxidative decarboxylation is more widely distributed across this enzyme family than reported previously.

New flavanol O-glycosides in grape and wine.

The presence of monomeric and dimeric flavan-3-ol monohexosides was investigated in grapes and wines. Polyphenol extracts were prepared from grape seeds and skins (Syrah, Merlot, and Cabernet-Sauvignon) sampled at three different developmental stages. Different wines (Tannat, Alicante, Syrah, Merlot, and Grenache) were also studied. The different samples obtained were analyzed by UPLC-ESI-IT-MS. Specific molecular ions corresponding to flavan-3-ol hexosides were detected by using targeted molecular ions with specific m/z values: 451 for (epi)catechin hexosides, and 739 for procyanidin dimer hexosides. 4'-O-ß-glucosyl-(+)-catechin and 7-O-ß-glucosyl-(+)-catechin were obtained by using a glucosyl transferase from apple, UGT71A15, and their structures determined by NMR. These glucosylated monomers and the dimers were identified in all analyzed grape seed and several skin extracts at the different stages and in wines made from different varieties.

Comparison of broad-scope assays of nucleotide sugar-dependent glycosyltransferases.

Glycosyltransferases (GTs) are abundant in nature and diverse in their range of substrates. Application of GTs is, however, often complicated by their narrow substrate specificity. GTs with tailored specificities are highly demanded for targeted glycosylation reactions. Engineering of such GTs is, however, restricted by lack of practical and broad-scope assays currently available. Here we present an improvement of an inexpensive and simple assay that relies on the enzymatic detection of inorganic phosphate cleaved from nucleoside phosphate products released in GT reactions. This phosphatase-coupled assay (PCA) is compared with other GT assays: a pH shift assay and a commercially available immunoassay in Escherichia coli cell-free extract (CE). Furthermore, we probe PCA with three GTs with different specificities. Our results demonstrate that PCA is a versatile and apparently general GT assay with a detection limit as low as 1 mU. The detection limit of the pH shift assay is roughly 4 times higher. The immunoassay, by contrast, detected only nucleoside diphosphates (NDPs) but had the lowest detection limit. Compared with these assays, PCA showed superior robustness and, therefore, appears to be a suitable general screening assay for nucleotide sugar-dependent GTs.

Phosphoryl transfer from α-d-glucose 1-phosphate catalyzed by Escherichia coli sugar-phosphate phosphatases of two protein superfamily types.

The Cori ester α-d-glucose 1-phosphate (αGlc 1-P) is a high-energy intermediate of cellular carbohydrate metabolism. Its glycosidic phosphomonoester moiety primes αGlc 1-P for flexible exploitation in glucosyl and phosphoryl transfer reactions. Two structurally and mechanistically distinct sugar-phosphate phosphatases from Escherichia coli were characterized in this study for utilization of αGlc 1-P as a phosphoryl donor substrate. The agp gene encodes a periplasmic αGlc 1-P phosphatase (Agp) belonging to the histidine acid phosphatase family. Had13 is from the haloacid dehydrogenase-like phosphatase family. Cytoplasmic expression of Agp (in E. coli Origami B) gave a functional enzyme preparation (kcat for phosphoryl transfer from αGlc 1-P to water, 40 s(-1)) that was shown by mass spectrometry to exhibit no free cysteines and the native intramolecular disulfide bond between Cys(189) and Cys(195). Enzymatic phosphoryl transfer from αGlc 1-P to water in H2 (18)O solvent proceeded with complete (18)O label incorporation into the phosphate released, consistent with catalytic reaction through O-1-P, but not C-1-O, bond cleavage. Hydrolase activity of both enzymes was not restricted to a glycosidic phosphomonoester substrate, and d-glucose 6-phosphate was converted with a kcat similar to that of αGlc 1-P. By examining phosphoryl transfer from αGlc 1-P to an acceptor substrate other than water (d-fructose or d-glucose), we discovered that Agp exhibited pronounced synthetic activity, unlike Had13, which utilized αGlc 1-P mainly for phosphoryl transfer to water. By applying d-fructose in 10-fold molar excess over αGlc 1-P (20 mM), enzymatic conversion furnished d-fructose 1-phosphate as the main product in a 55% overall yield. Agp is a promising biocatalyst for use in transphosphorylation from αGlc 1-P.

Leloir Glycosyltransferases and Natural Product Glycosylation: Biocatalytic Synthesis of the C-Glucoside Nothofagin, a Major Antioxidant of Redbush Herbal Tea.

Nothofagin is a major antioxidant of redbush herbal tea and represents a class of bioactive flavonoid-like C-glycosidic natural products. We developed an efficient enzymatic synthesis of nothofagin based on a one-pot coupled glycosyltransferase-catalyzed transformation that involves perfectly selective 3'-C-ß-d-glucosylation of naturally abundant phloretin and applies sucrose as expedient glucosyl donor. C-Glucosyltransferase from Oryza sativa (rice) was used for phloretin C-glucosylation from uridine 5'-diphosphate (UDP)-glucose, which was supplied continuously in situ through conversion of sucrose and UDP catalyzed by sucrose synthase from Glycine max (soybean). In an evaluation of thermodynamic, kinetic, and stability parameters of the coupled enzymatic reactions, poor water solubility of the phloretin acceptor substrate was revealed as a major bottleneck of conversion efficiency. Using periodic feed of phloretin controlled by reaction progress, nothofagin concentrations (45 mM; 20 g l-1) were obtained that vastly exceed the phloretin solubility limit (5-10 mM). The intermediate UDP-glucose was produced from catalytic amounts of UDP (1.0 mM) and was thus recycled 45 times in the process. Benchmarked against comparable glycosyltransferase-catalyzed transformations (e.g., on quercetin), the synthesis of nothofagin has achieved intensification in glycosidic product formation by up to three orders of magnitude (µM→mM range). It thus makes a strong case for the application of Leloir glycosyltransferases in biocatalytic syntheses of glycosylated natural products as fine chemicals.

Nutritional requirements of the BY series of Saccharomyces cerevisiae strains for optimum growth.

Among the vast variety of Saccharomyces cerevisiae strains, the BY family is particularly important because the widely used deletion collections are based on this background. Here we demonstrate that some standard growth media recipes require substantial modifications to provide optimum growth conditions for auxotrophic BY strains and to avoid growth arrest before glucose is depleted. In addition to the essential supplements that are required to satisfy auxotrophic requirements, we found the four amino acids phenylalanine, glutamic acid, serine, and threonine to be indispensable for optimum growth, despite the fact that BY is 'prototrophic' for these amino acids. Interestingly, other widely used S. cerevisiae strains, such as strains of the CEN.PK family, are less sensitive to lack of the described supplements. Furthermore, we found that the concentration of inositol in yeast nitrogen base is too low to support fast proliferation of yeast cultures until glucose is exhausted. Depletion of inositol during exponential growth induces characteristic changes, namely a decrease in glucose uptake and maximum specific growth rate, increased cell size, reduced viability, and accumulation of lipid storage pools. Thus, several of the existing growth media recipes need to be revised to achieve optimum growth conditions for BY-derived strains.