Engineering two species of yeast as cell factories for 2'-fucosyllactose.

Oligosaccharides present in human breast milk have been linked to beneficial effects on infant health. Inclusion of these human milk oligosaccharides (HMOs) in infant formula can recapitulate these health benefits. As a result, there is substantial commercial interest in a cost-effective source of HMOs as infant formula ingredients. Here we demonstrate that the yeast species Saccharomyces cerevisiae and Yarrowia lipolytica both can be engineered to produce 2'-fucosyllactose (2'FL), which is the most abundant oligosaccharide in human breast milk, at high titer and productivity. Both yeast species were modified to enable uptake of lactose and synthesis of GDP-fucose - the two precursors of 2'FL - by installing a lactose transporter and enzymes that convert GDP-mannose to GDP-fucose. Production of 2'FL was then enabled by expression of α-1,2-fucosyltransferases from various organisms. By screening candidate transporters from a variety of sources, we identified transporters capable of exporting 2'FL from yeast, which is a key consideration for any biocatalyst for 2'FL production. In particular, we identified CDT2 from Neurospora crassa as a promising target for further engineering to improve 2'FL efflux. Finally, we demonstrated production of 2'FL in fermenters at rates and titers that indicate the potential of engineered S. cerevisiae and Y. lipolytica strains for commercial 2'FL production.

Melanization of flavonoids by fungal and bacterial laccases.

Laccase activity in plants results in the formation of a number of brown pigments, often referred to as tannins. Laccase-dependent pigment production is also catalogued in numerous fungal and bacterial species. The laccase of the haploid yeast Cryptococcus neoformans forms melanin-like pigmentation outside the cell wall in the presence of exogenous substrates. While this process is a contributing factor to its virulence in humans, the evolutionary intent for the laccase function remains a mystery. We show here that C. neoformans and Bacillus subtilis have the ability to create melanin-like pigments from a variety of flavonoid molecules across a range of conformations, preferring those with 3',4'-dihydroxylations. Since flavonoids are ubiquitous plant molecules and often-considered antimicrobial agents, we postulate that they are the intended natural targets of laccase activity and result in the formation of a defensive melanin-like coat. These results suggests a new mechanism by which flavonoid-melanin formation may occur, using not only A- and C-ring linkages, but also monomer links through the B-ring of the flavonoid structure. We also show that resveratrol and other non- and mono-hydroxylated polyphenol substrates have the ability to restrict pigment formation and may be potent inhibitors of laccase activity.

Utility of total lower extremity radiography investigation of nonweight bearing in the young child.

OBJECTIVES: The purpose of this work was to determine the utility of total lower extremity radiographs versus dedicated tibia radiographs in the evaluation of the young child presenting with nonweight bearing without localizing signs. METHODS: This was an institutional review board-approved retrospective review of 263 consecutive patients between the ages of 9 months and 4 years who were referred for total lower extremity radiography between September 29, 2001, and November 7, 2006. Among these, a total of 133 study subjects met inclusion criteria of presentation with nonweight bearing without localizing signs or history of previous trauma. The control population was selected from 1089 consecutive patients between the ages of 9 months and 4 years evaluated from January 5, 1999 and December 8, 2006, who had only tibia radiographs at presentation. From this group, a final control population of 128 patients was selected with similar presentation of nonweight bearing without localizing signs or history of previous trauma. Causes of nonweight bearing were recorded for both groups based on radiograph findings and additional studies performed during workup. RESULTS: At initial presentation, fractures were present in 13 study patients (9.8%) and in 23 control patients (17.9%). Total fractures (when including follow-up) were present in 14 study patients (10.5%) and in 26 control patients (20.3%). Fractures were located in the tibia alone in 100% of patients in the study group. Extratibial fracture (metatarsal) was present in 1 patient in the control group (0.7%). Among the study group, additional diagnoses included rickets (n = 1), cerebellar ataxia (n = 1), and discitis with epidural abscess (n = 1). CONCLUSIONS: Our study findings indicate that the diagnostic value of total lower extremity radiography is similar to dedicated tibia radiography in the workup of the nonweight-bearing young child without trauma history or localizing signs. Radiation and cost savings can be realized by reserving additional radiographs for patients with high clinical suspicion and normal findings on dedicated tibia radiography.