In Vivo Online Monitoring of Intracellular Lipid Accumulation in Ustilago maydis

Abstract

Single cell oils produced in microorganisms constitute appealing alternatives to plant oils. Oleaginous fungi accumulate triacylglycerols in lipid droplets (LD). Their biosynthesis is typically induced under nitrogen limitation. We exploit the fungal model Ustilago maydis for oil production. The stain 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY) can be used to track LD formation during cultivation but this expensive compound is only affordable at small-scale. Therefore, mutant screening for optimization of oil production and composition would benefit from an inexpensive online monitoring system. Accordingly, we aimed at developing an intrinsic reporter that is suitable to track oil formation even in larger cultures. From three tested candidates, the potential delta(24)-sterol C-methyltransferase Erg6 turned out to be the best reporter. Fluorescence microscopy confirmed its localization at the LD membrane. After optimization, Erg6 fused to mKate2, expressed from a promoter derived from glycolipid biosynthesis, showed a good correlation of fluorescence with oil accumulation. Time course experiments in micro-cultivators demonstrated that the fluorescence read-out can be used to track oil formation starting at the onset of nitrogen limitation to approximate the LD amount. In essence, our study introduces a biosensor for oil monitoring that can easily be transferred to other oleaginous yeasts. Summary: Microbial oils are promising, environmentally friendly alternatives to plant oils and have the potential of a huge market share once competitive production and isolation processes are accomplished. Online monitoring is key to efficient engineering of single cell oil producing microorganisms and bioprocess optimization in order to achieve competitive products. Here, we present an inexpensive, fluorescence-based reporter that can be used to track the approximate oil accumulation of microbial cultures in vivo. This omits the use of expensive dyes or offline methodology with a high workload. While we established the biosensor in the yeast form of the fungal microorganism U. maydis, the evolutionary conservation of the underlying protein Erg6 will allow for a straightforward transfer of the methodology to other oleaginous yeasts.