Designer biocatalysts for direct incorporation of exogenous galactose into globotriose.

Galactose is ubiquitous. The synthesis of galactose-containing oligosaccharides using Leloir galactosyltransferase requires uridine diphosphate (UDP)-galactose as the precursor. Of all UDP-galactose synthesis pathways developed for in vitro synthesis, the salvage pathway represents the simplest route. In this study, for the first time, we designed and constructed an Escherichia coli strain to use salvage pathway for UDP-galactose synthesis, demonstrating effective and direct incorporation of exogenous galactose into globotriose (Gb3). Successful establishment of salvage pathway enabled a complete delineation of carbon and energy source. Consequently, the designed biocatalyst was able to achieve high yield synthesis from galactose (0.95 moles of Gb3/moles galactose consumed) and a high product titer (2 g/L) in shaker flask within 24 hr. Elimination of limitation in acceptor sugar via homologous overexpression of LacY, the transporter for lactose, further improved the synthesis, raising Gb3 titer to 6 g/L in 24 hr and 7.5 g/L in 48 hr. The design principles successfully demonstrated in this study could be broadly applied for synthesis of other galactose-containing oligosaccharides. This study also illustrates a valid strategy to overcome limitation in the transport of acceptor sugar. As lactose is one of the most important basal structures, the significant improvement in synthesis through its enhanced transport could be emulated in numerous other lactose-based oligosaccharides.

Photoinactivation of the Staphylococcus aureus Lactose-Specific EIICB Phosphotransferase Component with p-azidophenyl-ß-D-Galactoside and Phosphorylation of the Covalently Bound Substrate.

BACKGROUND: The phosphoenolpyruvate (PEP):lactose phosphotransferase system of Staphylococcus aureus transports and phosphorylates lactose and various phenylgalactosides. Their phosphorylation is catalyzed by the Cys476-phosphorylated EIIB domain of the lactose-specific permease enzyme IICB (EIICBLac). Phosphorylation causes the release of galactosides bound to the EIIC domain into the cytoplasm by a mechanism not yet understood. RESULTS: Irradiation of a reaction mixture containing the photoactivatable p-azidophenyl-ß-D-galactopyranoside and EIICBLac with UV light caused a loss of EIICBLac activity. Nevertheless, photoinactivated EIICBLac could still be phosphorylated with [32P]PEP. Proteolysis of photoinactivated [32P]P-EIICBLac with subtilisin provided an 11-kDa radioactive peptide. Only the sequence of its first three amino acids (-H-G-P-, position 245-247) could be determined. They are part of the substrate binding pocket in EIICs of the lactose/cellobiose PTS family. Surprisingly, while acid treatment caused hydrolysis of the phosphoryl group in active [32P]P∼EIICBLac, photoinactivated [32P]P-EIICBLac remained strongly phosphorylated. CONCLUSION: Phosphorylation of the -OH group at C6 of p-nitrenephenyl-ß-D-galactopyranoside covalently bound to EIICLac by the histidyl-phosphorylated [32P]P∼EIIBLac domain is a likely explanation for the observed acid resistance. Placing p-nitrenephenyl-ß-D-galactopyranoside into the active site of modelled EIICLac suggested that the nitrene binds to the -NH- group of Ser248, which would explain why no sequence data beyond Pro247could be obtained.

Ploidy Variation in Kluyveromyces marxianus Separates Dairy and Non-dairy Isolates.

Kluyveromyces marxianus is traditionally associated with fermented dairy products, but can also be isolated from diverse non-dairy environments. Because of thermotolerance, rapid growth and other traits, many different strains are being developed for food and industrial applications but there is, as yet, little understanding of the genetic diversity or population genetics of this species. K. marxianus shows a high level of phenotypic variation but the only phenotype that has been clearly linked to a genetic polymorphism is lactose utilisation, which is controlled by variation in the LAC12 gene. The genomes of several strains have been sequenced in recent years and, in this study, we sequenced a further nine strains from different origins. Analysis of the Single Nucleotide Polymorphisms (SNPs) in 14 strains was carried out to examine genome structure and genetic diversity. SNP diversity in K. marxianus is relatively high, with up to 3% DNA sequence divergence between alleles. It was found that the isolates include haploid, diploid, and triploid strains, as shown by both SNP analysis and flow cytometry. Diploids and triploids contain long genomic tracts showing loss of heterozygosity (LOH). All six isolates from dairy environments were diploid or triploid, whereas 6 out 7 isolates from non-dairy environment were haploid. This also correlated with the presence of functional LAC12 alleles only in dairy haplotypes. The diploids were hybrids between a non-dairy and a dairy haplotype, whereas triploids included three copies of a dairy haplotype.