Search

Screening the genetic diversity of 45 Yarrowia lipolytica strains identified five candidates with unique metabolic capability and robustness in undetoxified switchgrass hydrolysates, including superior lipid production and efficient pentose sugar utilization. Here, we report the genome sequences of these strains to study their robustness and potential to produce fuels and chemicals.

Current glucose monitoring techniques for neonates rely heavily on blood glucose monitors which require intermittent blood collection through skin-penetrating pricks on the heel or fingers. This procedure is painful and often not clinically conducive, which presents a need for a noninvasive method for monitoring glucose in neonates. Our motivation for this study was to develop an in vitro method for measuring passive diffusion of glucose in premature neonatal skin using a porcine skin model. Such a model will allow us to initially test new devices for noninvasive glucose monitoring without having to do in vivo testing of newborns. The in vitro model is demonstrated by comparing uncompromised and tape-stripped skin in an in-line flow-through diffusion apparatus with glucose concentrations that mimic the hypo-, normo-, and hyper-glycemic conditions in the neonate (2.0, 5.0, and 20 mM, respectively). Transepidermal water loss (TEWL) of the tape-stripped skin was approximately 20 g m -2  h -1 , which closely mimics TEWL for neonatal skin at about 190 days post-conceptional age. The tape-stripped skin showed a >15-fold increase in glucose diffusion compared to the uncompromised skin. The very small concentrations of collected glucose were measured with a highly selective and highly sensitive fluorescent glucose biosensor based on the glucose binding protein (GBP). The demonstrated method of glucose determination is noninvasive and painless, which makes it especially desirable for glucose testing in neonates and children. This study is an important step towards an in vitro model for noninvasive real-time glucose monitoring that may be easily transferred to the clinic for glucose monitoring in neonates. Graphical Abstract Glucose diffusion through model skin was measured using an in-line flow-through diffusion apparatus with glucose solutions mimicking hypo-, normo- and hyperglycemia in the neonate. Phosphate buffered saline was added to the top chamber and the glucose that diffused through the model skin into the buffer was measured using a fluorescent glucose binding protein biosensor.

Yarrowia lipolytica is an oleaginous yeast species that has attracted attention as a model organism for synthesis of single cell oil. Among over 50 isolates of Y. lipolytica identified, only a few of the strains have been studied extensively. Furthermore, 12 other yeast species were recently assigned to the Yarrowia clade, and most are not well characterized in terms of cell growth and lipid accumulation, especially in industrially relevant conditions. In the present study, we investigated biomass and lipid production by 57 yeast isolates, representing all 13 species in the Yarrowia clade, on a non-detoxified dilute acid-pretreated switchgrass hydrolysate under highly aerobic conditions. The objective was to compare yeast physiology during growth in an abundant, low-cost biomass feedstock and to expand diversity of genetically tractable, oleaginous yeasts available for lipid research. Screening of 45 Y. lipolytica isolates demonstrated considerable variation within the species in terms of lipid accumulation (min = 0.1 g/L; max = 5.1 g/L; mean = 2.3 g/L); three strains (NRRL YB-420, YB-419, and YB-392) were especially promising for cellulosic biomass conversion with average improvements of 43, 57, and 64%, respectively, in final lipid titer as compared to control strain W29. Subsequently, evaluation of strains from 13 distinct species in the Yarrowia clade identified Candida phangngensis PT1-17 as the top lipid producer with a maximum titer of 9.8 g/L lipid, which was over twofold higher than the second-best species in the clade (Candida hollandica NRRL Y-48254). A small set of the most promising strains from the screenings was further characterized to evaluate inhibitor tolerance, lipid production kinetics, and fatty acid distribution. We expect that the results of this study will pave the way for new biotechnological applications involving previously overlooked and under-characterized strains within the Yarrowia clade.

In the important industrial yeast Saccharomyces cerevisiae, galactose metabolism requires energy production by respiration; therefore, this yeast cannot metabolize galactose under strict anaerobic conditions. While the respiratory dependence of galactose metabolism provides benefits in terms of cell growth and population stability, it is not advantageous for producing fuels and chemicals since a substantial fraction of consumed galactose is converted to carbon dioxide. In order to force S. cerevisiae to use galactose without respiration, a subunit (COX9) of a respiratory enzyme was deleted, but the resulting deletion mutant (Δcox9) was impaired in terms of galactose assimilation. Interestingly, after serial sub-cultures on galactose, the mutant evolved rapidly and was able to use galactose via fermentation only. The evolved strain (JQ-G1) produced ethanol from galactose with a 94% increase in yield and 6.9-fold improvement in specific productivity as compared to the wild-type strain. (13)C-metabolic flux analysis demonstrated a three-fold reduction in carbon flux through the TCA cycle of the evolved mutant with redirection of flux toward the fermentation pathway. Genome sequencing of the JQ-G1 strain revealed a loss of function mutation in a master negative regulator of the Leloir pathway (Gal80p). The mutation (Glu348*) in Gal80p was found to act synergistically with deletion of COX9 for efficient galactose fermentation, and thus the double deletion mutant Δcox9Δgal80 produced ethanol 2.4 times faster and with 35% higher yield than a single knockout mutant with deletion of GAL80 alone. When we introduced a functional COX9 cassette back into the JQ-G1 strain, the JQ-G1-COX9 strain showed a 33% reduction in specific galactose uptake rate and a 49% reduction in specific ethanol production rate as compared to JQ-G1. The wild-type strain was also subjected to serial sub-cultures on galactose but we failed to isolate a mutant capable of utilizing galactose without respiration. We concluded that the metabolic "death valley" (i.e. no galactose utilization by the Δcox9 mutant) is a necessary intermediate phenotype to facilitate galactose utilization without respiration in yeast. The results in this study demonstrate a promising approach for directing adaptive evolution toward fermentative metabolism and for generating evolved yeast strains with improved phenotypes under anaerobic conditions., (Copyright © 2016 Elsevier B.V. All rights reserved.)