Rhodotorula (Rhodosporidium) toruloides is an oleaginous basidiomycete yeast with great biotechnological potential. Capable of accumulating lipid up to 76 % of its dry biomass and well suited to the metabolism of lignocellulosic hydrolysate, it is a good candidate for production of advanced biofuels as well as a host of other potential roles in industry. However, molecular genetic tools for manipulation of this yeast are lacking and its high genomic GC content can make routine cloning difficult. Agrobacterium tumefaciens-mediated transformation of R. toruloides CBS 14 was demonstrated, and plasmid vectors were developed for transformation of R. toruloides, including elements for Saccharomyces cerevisiae in-yeast assembly. In-yeast assembly is robust to the manipulation of GC-rich DNA and of large plasmids. Using these vectors and an EGFP reporter, a screen to identify inducible promoters was performed, and promoters from the genes NAR1, ICL1, CTR3, and MET16 identified. These promoters have independent induction/repression conditions and different levels and rates of induction. Minimal inducible promoters were determined, which are as small as 200 bp. As well as showing tight regulation of the EGFP marker, the NAR1 promoter was able to drive conditional rescue of a leu2 mutant strain. In parallel, as a proof of principle for production of advanced biofuels, hydrocarbon biosynthesis pathways were expressed in R. toruloides and analysed by GC-MS. After co-expression of Synechococcus elongatus fatty acyl-ACP reductase and fatty aldehyde decarbonylase, and E. coli ferredoxin and ferredoxin reductase, production of the alkane heptadecane was observed. To increase the availability of free fatty acids (FFA) for production of hydrocarbons by other pathways, Thermomyces lanuginosus lipase 2 was expressed, resulting in a 1.3-fold increase in the concentration of FFAs.

Rhodotorula glutinis (Rhodosporidium toruloides) est une levure oléagineuse dont les fortes capacités d’accumulation lipidique à partir de glucose comme source carbonée ont fait de la souche un modèle d’étude. La capacité de cette levure à utiliser le glycérol ou le xylose en simple ou co-substrat avec le glucose est toutefois encore peu explorée. De l’analyse des travaux antérieurs il a été possible de dégager les verrous scientifiques qui nécessitent une amélioration des connaissances du comportement physiologique de cette levure pour la conversion des substrats cités. Des stratégies expérimentales adaptées à la quantification rationnelle des dynamiques de Rhodotorula glutinis en conditions de croissance et accumulation lipidique à partir de xylose et de glycérol en simple ou co-substrats avec le glucose ont été développées. Des résultats originaux ont été obtenus dont :- la mise en évidence des potentialités de co-consommation des substrats xylose et glucose ou glycérol et glucose sans accumulation de substrat ni production de métabolites en conditions contrôlées des flux de substrats. Il a été possible de déterminer la vitesse spécifique maximale de consommation du carbone de la souche qui diminue lorsque la part de xylose ou glycérol augmente dans l’apport de carbone total.- la quantification de la dynamique de croissance sur xylose et glycérol pur en terme de taux de croissance et de rendement : sur xylose µmax= 0.034h-1 et RS/X= 0.28 Cmolx.Cmolxylose-1; sur glycérol µmax=0.04h-1 RS/X=0.31Cmolx.Cmolglycérol-1.- la quantification des vitesses spécifiques et rendements de production de lipides à partir de xylose ou de glycérol en simple ou co-substrat avec du glucose : 20%xylose-80%glucose : qp=0.065CmolTAG.Cmolbiomasse.h-1, RS/P=0.3CmoleTAG.Cmolesubstrat-1 100%xylose : qp=0.035065CmolTAG.Cmolbiomasse.h-1, RS/P=0.31CmoleTAG.Cmolesubstrat-1, 25% glycérol-75%glucose : qp=0.07065CmolTAG.Cmolbiomasse.h-1, RS/P=0.25CmoleTAG.Cmolesubstrat-1 , 100% glycérol : qp=0.03065CmolTAG.Cmolbiomasse.h-1, RS/P= 0.29CmoleTAG.Cmolesubstrat-1.- L’impact de la nature des substrats sur le profil lipidique de Rhodotorula glutinis demeure léger : il apparait que le xylose entraîne une surproduction de C16:0 et C18:3et le glycérol favorise l’accumulation de C18:0 / Rhodotorula glutinis (Rhodosporidium toruloides) is an oleaginous yeast. The micro-organism has demonstrated high lipid accumulation when utilizing glucose as a substrate, and has become a model for oil production. Glycerol and xylose are interesting as substrates for production of oil from renewable resources, but the capacity of R. glutinis to utilize glycerol and xylose as substrates has not been characterized well. Fermentation strategies were designed to quantify growth and lipid accumulation dynamics of R. glutinis when utilizing glycerol and xylose - either as pure substrates, or as co-substrates with glucose. Several original results have been found, including: - Co-consumption of xylose or glycerol along with glucose was observed, without carbon substrate accumulation or byproduct formation, when the carbon feed rate was carefully controlled. The specific carbon consumption rate decreases when the proportion of the second substrate (glycerol or xylose) increases in the feed, relative to glucose. - Growth capacities were characterized on pure xylose and pure glycerol in terms of growth rate and carbon yields: on xylose μmax= 0.034h-1 and RS/X= 0.28 Cmolx.Cmolxylose-1; on glycerol μmax=0.04h-1 RS/X=0.31Cmolx.Cmolglycerol-1. - specific production rate of lipid production and substrate to product carbon conversion yields from xylose or glycerol as single or cosubstrate with glucose were determinated: 20%xylose-80%glucose : qp=0.065CmolTAG.Cmolbiomasse.h-1, RS/P=0.3CmoleTAG.Cmolesubstrat-1 100%xylose : qp=0.035065CmolTAG.Cmolbiomasse.h-1, RS/P=0.31CmoleTAG.Cmolesubstrat-1, 25% glycerol-75%glucose : qp=0.07065CmolTAG.Cmolbiomasse.h-1, RS/P=0.25CmoleTAG.Cmolesubstrat-1 , 100% glycerol : qp=0.03065CmolTAG.Cmolbiomasse.h-1, RS/P= 0.29CmoleTAG.Cmolesubstrat-1. - Substrate diversification slightly impacts Rhodotorula glutinis´s lipid profile: xylose leads to an overproduction of C16:0 and C18:3 and glycerol increases C18:0 accumulation