Baccatin III, an intermediate of Taxol biosynthesis and a useful precursor for semisynthesis of the anti-cancer drug, is produced in yew (Taxus) species by a sequence of 15 enzymatic steps from primary metabolism. Ten genes encoding enzymes of this extended pathway have been described, thereby permitting a preliminary attempt to reconstruct early steps of taxane diterpenoid (taxoid) metabolism in Saccharomyces cerevisiae as a microbial production host. Eight of these taxoid biosynthetic genes were functionally expressed in yeast from episomal vectors containing one or more gene cassettes incorporating various epitope tags to permit protein surveillance and differentiation of those pathway enzymes of similar size. All eight recombinant proteins were readily detected by immunoblotting using specific monoclonal antibodies and each expressed protein was determined to be functional by in vitro enzyme assay, although activity levels differed considerably between enzyme types. Using three plasmids carrying different promoters and selection markers, genes encoding five sequential pathway steps leading from primary isoprenoid metabolism to the intermediate taxadien-5alpha- acetoxy-10beta-ol were installed in a single yeast host. Metabolite analysis showed that yeast isoprenoid precursors could be utilized in the reconstituted pathway because products accumulated from the first two engineered pathway steps (leading to the committed intermediate taxadiene); however, a pathway restriction was encountered at the first cytochrome P450 hydroxylation step. The means of overcoming this limitation are described in the context of further development of this novel approach for production of Taxol precursors and related taxoids in yeast.
Biosynthesis of the anticancer drug Taxol involves 19 enzymatic steps from the universal diterpenoid progenitor geranylgeranyl diphosphate derived by the plastidial methylerythritol phosphate pathway for isoprenoid precursor supply. To gain further insight about Taxol biosynthesis relevant to the improved production of this drug and to draw inferences about the organization, regulation, and origins of this complex natural product pathway, random sequencing of a cDNA library derived from Taxus cuspidata cells (induced for taxoid biosynthesis with methyl jasmonate) was undertaken. This effort revealed surprisingly high abundances for transcripts of several of the 12 defined genes of Taxol biosynthesis, yielded cDNAs encoding two previously uncharacterized cytochrome P450 taxoid hydroxylases, and provided candidate genes for all but one of the remaining seven steps of this extended sequence of reactions.paclitaxel ͉ Taxus cuspidata ͉ expressed sequence tags ͉ taxoid 10-hydroxylase
dHisto-blood group antigens (HBGAs) are important binding factors for norovirus infections. We show that two human milk oligosaccharides, 2=-fucosyllactose (2=FL) and 3-fucosyllactose (3FL), could block norovirus from binding to surrogate HBGA samples. We found that 2=FL and 3FL bound at the equivalent HBGA pockets on the norovirus capsid using X-ray crystallography. Our data revealed that 2=FL and 3FL structurally mimic HBGAs. These results suggest that 2=FL and 3FL might act as naturally occurring decoys in humans. Mothers' milk has long been seen as a great source of infant nutrition and protection against a large number of pathogens. Human milk oligosaccharides (HMOs), the third-mostabundant (10 to 15 g/liter) components of human milk, are thought to be in part accountable for these health benefits (1). HMOs are unconjugated complex glycans, and more than 200 isomers are known. HMOs consist of combinations of different monosaccharide building blocks, including fucose, glucose, galactose, N-acetylglucosamine, and the sialic acid derivative N-acetylneuraminic acid. HMOs have been demonstrated to protect against human noroviruses, rotavirus, and certain bacteria (reviewed in reference 2).Human noroviruses are also known to interact with histoblood group antigens (HBGAs), and the interaction is thought to be important for infection (3-6). HBGAs can be found as soluble antigens in saliva and are expressed on epithelial cells. HBGAs consist of monosaccharide building blocks similar to those of HMOs, and at least nine different HBGA types have been found to interact with human norovirus (7-12). HMOs are thought to act as a "receptor decoy" for certain pathogens, since HMOs and HBGAs mimic each other structurally. However, little is known about how HMOs block norovirus infections. One study found that human milk was able to block genogroup I genotype 1 (GI.1) and GII.4 norovirus strains from binding to saliva samples (13). A follow-up study suggested that certain HMOs might compete with the HBGA binding sites on the GI.1 and GII.4 norovirus capsid (14). Despite the fact that human noroviruses are the dominant cause of acute gastroenteritis, there are still no suitable antivirals or vaccines commercially available.In this study, we analyzed the ability of two HMOs, i.e., 2=-fucosyllactose (2=FL) and 3-fucosyllactose (3FL), to block GII.10 norovirus virus-like particles (VLPs) from binding to HBGAs (Fig. 1A). A slightly modified blocking enzyme-linked immunosorbent assay (ELISA) was developed using both porcine gastric mucin type III (PGM) and human saliva (A and B types) (3, 15). The PGM sample was confirmed to contain a mixture of A and H types using specific anti-HBGA monoclonal antibodies (data not shown).The GII.10 VLPs were expressed and purified as previously described (16). The untreated VLPs were first examined for binding to PGM and saliva samples using a direct ELISA. Maxisorp 96-well plates were coated with 100 l per well of 10 g/ml PGM for 4 h at room temperature. The saliva samples were processed in a si...
Aldolases are emerging as powerful and cost efficient tools for the industrial synthesis of chiral molecules. They catalyze enantioselective carbon-carbon bond formations, generating up to two chiral centers under mild reaction conditions. Despite their versatility, narrow substrate ranges and enzyme inactivation under synthesis conditions represented major obstacles for large-scale applications of aldolases. In this study we applied directed evolution to optimize Escherichia coli 2-deoxy-D-ribose 5-phosphate aldolase (DERA) as biocatalyst for the industrial synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside. This versatile chiral precursor for vastatin drugs like Lipitor (atorvastatin) is synthesized by DERA in a tandem-aldol reaction from chloroacetaldehyde and two acetaldehyde equivalents. However, E. coli DERA shows low affinity to chloroacetaldehyde and is rapidly inactivated at aldehyde concentrations useful for biocatalysis. Using high-throughput screenings for chloroacetaldehyde resistance and for higher productivity, several improved variants have been identified. By combination of the most beneficial mutations we obtained a tenfold improved variant compared to wild-type DERA with regard to (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside synthesis, under industrially relevant conditions.
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