Tailoring and optimizing fatty acid production by oleaginous yeasts through the systematic exploration of their physiological fitness.

BACKGROUND: The use of palm oil for our current needs is unsustainable. Replacing palm oil with oils produced by microbes through the conversion of sustainable feedstocks is a promising alternative. However, there are major technical challenges that must be overcome to enable this transition. Foremost among these challenges is the stark increase in lipid accumulation and production of higher content of specific fatty acids. Therefore, there is a need for more in-depth knowledge and systematic exploration of the oil productivity of the oleaginous yeasts. In this study, we cultivated Cutaneotrichosporon oleaginosus and Yarrowia lipolytica at various C/N ratios and temperatures in a defined medium with glycerol as carbon source and urea as nitrogen source. We ascertained the synergistic effect between various C/N ratios of a defined medium at different temperatures with Response Surface Methodology (RSM) and explored the variation in fatty acid composition through Principal Component Analysis. RESULTS: By applying RSM, we determined a temperature of 30 °C and a C/N ratio of 175 g/g to enable maximal oil production by C. oleaginosus and a temperature of 21 °C and a C/N ratio of 140 g/g for Y. lipolytica. We increased production by 71% and 66% respectively for each yeast compared to the average lipid accumulation in all tested conditions. Modulating temperature enabled us to steer the fatty acid compositions. Accordingly, switching from higher temperature to lower cultivation temperature shifted the production of oils from more saturated to unsaturated by 14% in C. oleaginosus and 31% in Y. lipolytica. Higher cultivation temperatures resulted in production of even longer saturated fatty acids, 3% in C. oleaginosus and 1.5% in Y. lipolytica. CONCLUSIONS: In this study, we provided the optimum C/N ratio and temperature for C. oleaginosus and Y. lipolytica by RSM. Additionally, we demonstrated that lipid accumulation of both oleaginous yeasts was significantly affected by the C/N ratio and temperature. Furthermore, we systematically analyzed the variation in fatty acids composition and proved that changing the C/N ratio and temperature steer the composition. We have further established these oleaginous yeasts as platforms for production of tailored fatty acids.

SALARECON connects the Atlantic salmon genome to growth and feed efficiency.

Atlantic salmon (Salmo salar) is the most valuable farmed fish globally and there is much interest in optimizing its genetics and rearing conditions for growth and feed efficiency. Marine feed ingredients must be replaced to meet global demand, with challenges for fish health and sustainability. Metabolic models can address this by connecting genomes to metabolism, which converts nutrients in the feed to energy and biomass, but such models are currently not available for major aquaculture species such as salmon. We present SALARECON, a model focusing on energy, amino acid, and nucleotide metabolism that links the Atlantic salmon genome to metabolic fluxes and growth. It performs well in standardized tests and captures expected metabolic (in)capabilities. We show that it can explain observed hypoxic growth in terms of metabolic fluxes and apply it to aquaculture by simulating growth with commercial feed ingredients. Predicted limiting amino acids and feed efficiencies agree with data, and the model suggests that marine feed efficiency can be achieved by supplementing a few amino acids to plant- and insect-based feeds. SALARECON is a high-quality model that makes it possible to simulate Atlantic salmon metabolism and growth. It can be used to explain Atlantic salmon physiology and address key challenges in aquaculture such as development of sustainable feeds.

Aspergillus niger citrate exporter revealed by comparison of two alternative citrate producing conditions.

Currently, there is no consensus regarding the mechanism underlying Aspergillus niger citrate biosynthesis and secretion. We hypothesise that depending on the experimental setup, extracellular citrate accumulation can have fundamentally different underlying transcriptomic landscapes. We show that varying the amount and type of supplement of an arginine auxotrophic A. niger strain results in transcriptional down-regulation of citrate metabolising enzymes in the condition in which more citrate is accumulated extracellularly. This contrasts with the transcriptional adaptations when increased citrate production is triggered by iron limitation. By combining gene expression data obtained from these two very distinct experimental setups with hidden Markov models and transporter homology approaches, we were able to compile a shortlist of the most likely citrate transporter candidates. Two candidates (An17g01710 and An09g06720m.01) were heterologously expressed in the yeast Saccharomyces cerevisiae, and one of the resultant mutants showed the ability to secrete citrate. Our findings provide steps in untangling the complex interplay of different mechanisms underlying A. niger citrate accumulation, and we demonstrate how a comparative transcriptomics approach complemented with further bioinformatics analyses can be used to pinpoint a fungal citrate exporter.

The effect of prebiotic fortified infant formulas on microbiota composition and dynamics in early life.

Gastrointestinal (GI) microbiota composition differs between breastfed and formula-fed infants. Today's infant formulas are often fortified with prebiotics to better mimic properties of human milk with respect to its effect on GI microbiota composition and function. We used Illumina HiSeq sequencing of PCR-amplified 16S rRNA gene fragments to investigate the composition of faecal microbiota in 2-12 week old infants receiving either breastmilk, infant formulas fortified with prebiotics, or mixed feeding. We compared these results with results from infants fed traditional formulas used in the Netherlands in 2002-2003, which contained no added prebiotics. We showed that today's formulas supplemented with either scGOS (0.24-0.50 g/100 ml) or scGOS and lcFOS (at a 9:1 ratio; total 0.6 g/100 ml) had a strong bifidogenic effect as compared to traditional formulas, and they also resulted in altered patterns of microbial colonisation within the developing infant gastrointestinal tract. We identified three microbial states (or developmental stages) in the first 12 weeks of life, with a gradual transition pattern towards a bifidobacteria dominated state. In infants receiving only fortified formulas, this transition towards the bifidobacteria dominated state was accelerated, whereas in infants receiving mixed feeding the transition was delayed, as compared to exclusively breastfed infants.

Pushing the limits of automatic computational protein design: design, expression, and characterization of a large synthetic protein based on a fungal laccase scaffold.

UNLABELLED: The de novo engineering of new proteins will allow the design of complex systems in synthetic biology. But the design of large proteins is very challenging due to the large combinatorial sequence space to be explored and the lack of a suitable selection system to guide the evolution and optimization. One way to approach this challenge is to use computational design methods based on the current crystallographic data and on molecular mechanics. We have used a laccase protein fold as a scaffold to design a new protein sequence that would adopt a 3D conformation in solution similar to a wild-type protein, the Trametes versicolor (TvL) fungal laccase. Laccases are multi-copper oxidases that find utility in a variety of industrial applications. The laccases with highest activity and redox potential are generally secreted fungal glycoproteins. Prokaryotic laccases have been identified with some desirable features, but they often exhibit low redox potentials. The designed sequence (DLac) shares a 50% sequence identity to the original TvL protein. The new DLac gene was overexpressed in E. coli and the majority of the protein was found in inclusion bodies. Both soluble protein and refolded insoluble protein were purified, and their identity was verified by mass spectrometry. Neither protein exhibited the characteristic T1 copper absorbance, neither bound copper by atomic absorption, and neither was active using a variety of laccase substrates over a range of pH values. Circular dichroism spectroscopy studies suggest that the DLac protein adopts a molten globule structure that is similar to the denatured and refolded native fungal TvL protein, which is significantly different from the natively secreted fungal protein. Taken together, these results indicate that the computationally designed DLac expressed in E. coli is unable to utilize the same folding pathway that is used in the expression of the parent TvL protein or the prokaryotic laccases. This sequence can be used going forward to help elucidate the sequence requirements needed for prokaryotic multi-copper oxidase expression. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11693-011-9080-9) contains supplementary material, which is available to authorized users.