Guojie Jin

Triacylglycerols are among the most attractive alternative raw materials for biofuel development. Current oil plant-based technologies are limited in terms of triacylglycerol production capacity and rate. These limitations may be circumvented by biotransformation of carbohydrates into lipids; however, our understanding of microbial oleaginicity remains limited. Here we present the results of a multi-omic analysis of Rhodosporidium toruloides, a robust triacylglycerol-producing fungus. The assembly of genome and transcriptome sequencing data reveals a genome of 20.2 Mb containing 8,171 protein-coding genes, the majority of which have multiple introns. Genes including a novel fatty acid synthase are predicted to participate in metabolic pathways absent in non-oleaginous yeasts. Transcriptomic and proteomic data suggest that lipid accumulation under nitrogen-limited conditions correlates with the induction of lipogenesis, nitrogenous compound recycling, macromolecule metabolism and autophagy. The multi-omic map of R. toruloides therefore provides a valuable resource for efforts to rationally engineer lipid-production pathways. The ability of oleaginous fungi to produce lipids for biofuels remains untapped, in part due to a lack of genetic information required to engineer industrial strains. Zhuet al. present the genome of R. toruloides, and identify transcriptomic and proteomic changes associated with lipid production.

Microbial production can be advantageous over the extraction of phytoterpenoids from natural plant sources, but it remains challenging to rationally and rapidly access efficient pathway variants. Previous engineering attempts mainly focused on the mevalonic acid (MVA) or methyl-d-erythritol phosphate (MEP) pathways responsible for the generation of precursors for terpenoids biosynthesis, and potential interactions between diterpenoids synthases were unexplored. Miltiradiene, the product of the stepwise conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP) catalyzed by diterpene synthases SmCPS and SmKSL, has recently been identified as the precursor to tanshionones, a group of abietane-type norditerpenoids rich in the Chinese medicinal herb Salvia miltiorrhiza . Here, we present the modular pathway engineering (MOPE) strategy and its application for rapid assembling synthetic miltiradiene pathways in the yeast Saccharomyces cerevisiae . We predicted and analyzed the molecular interactions between SmCPS and SmKSL, and engineered their active sites into close proximity for enhanced metabolic flux channeling to miltiradiene biosynthesis by constructing protein fusions. We show that the fusion of SmCPS and SmKSL, as well as the fusion of BTS1 (GGPP synthase) and ERG20 (farnesyl diphosphate synthase), led to significantly improved miltiradiene production and reduced byproduct accumulation. The MOPE strategy facilitated a comprehensive evaluation of pathway variants involving multiple genes, and, as a result, our best pathway with the diploid strain YJ2X reached miltiradiene titer of 365 mg/L in a 15-L bioreactor culture. These results suggest that terpenoids synthases and the precursor supplying enzymes should be engineered systematically to enable an efficient microbial production of phytoterpenoids.

BACKGROUND: Biochemical conversion of lignocellulose hydrolysates remains challenging, largely because most microbial processes have markedly reduced efficiency in the presence of both hexoses and pentoses. Thus, identification of microorganisms capable of efficient and simultaneous utilization of both glucose and xylose is pivotal to improving this process. RESULTS: In this study, we found that the oleaginous yeast strain Trichosporon cutaneum AS 2.571 assimilated glucose and xylose simultaneously, and accumulated intracellular lipid up to 59 wt% with a lipid coefficient up to 0.17 g/g sugar, upon cultivation on a 2:1 glucose/xylose mixture in a 3-liter stirred-tank bioreactor. In addition, no classic pattern of diauxic growth behavior was seen; the microbial cell mass increased during the whole culture process without any lag periods. In shake-flask cultures with different initial glucose:xylose ratios, glucose and xylose were consumed simultaneously at rates roughly proportional to their individual concentrations in the medium, leading to complete utilization of both sugars at the same time. Simultaneous utilization of glucose and xylose was also seen during fermentation of corn-stover hydrolysate with a lipid content and coefficient of 39.2% and 0.15 g/g sugar, respectively. The lipid produced had a fatty-acid compositional profile similar to those of conventional vegetable oil, indicating that it could have potential as a raw material for biodiesel production. CONCLUSION: Efficient lipid production with simultaneous consumption of glucose and xylose was achieved in this study. This process provides an exciting opportunity to transform lignocellulosic materials into biofuel molecules, and should also encourage further study to elucidate this unique sugar-assimilation mechanism.