Neutral lipid production by the yeast Debaryomyces hansenii NCYC102 under different stress conditions

Oleaginous yeasts are very efficient in the accumulation of triacylglycerol, and are expected to be one of the most important feedstocks for the biofuel industry in the future. Lipid content can be enhanced through physiological stress or genetic manipulation. Debaryomyces hansenii NCYC102 was selected from three different yeast species (also including Yarrowia lipolytica NCYC476 and Cryptococcus curvatus NCYC2904) due to the highest neutral lipid content. The growth rate, osmolytes and neutral lipids were measured in cells grown under different concentrations (0, 0.8, 1.6 M) of NaCl. The maximum content of total osmolytes was found in 0.8 M NaCl YM medium. However, the highest level of glycerol was measured in 1.6 M NaCl grown cells. The main osmolytes identified by 1H NMR spectroscopy were glycerol, arabitol, glucose and trehalose. Debaryomyces. hansenii cells were grown in minimal medium with different carbon/nitrogen ratios using either glucose or glycerol as the sole carbon source along with ammonium sulphate as nitrogen source. Maximal neutral lipid production was observed in 48:0.5 glucose/ammonium sulphate ratio which achieved 1.4-fold increase compared with glycerol-based medium (8 glycerol: 0.25 ammonium sulphate). GC-MS analysis of the transesterified fatty acids showed that palmitic, oleic and stearic acids were the main fatty acids present, under normal and stress conditions (high salt and limited nitrogen source). Deletion of the GUT2 encoding for G3P dehydrogenase increased neutral lipid production up to a 1.4-fold compared to wild type strains. The mutant strains displayed slightly higher cell densities in medium with glucose when compared with wild type strains, while they failed to grow on glycerol as a sole carbon source. Collectively, these results indicate that D. hansenii is a good organism to produce biofuels as it has an intrinsic ability to accumulate neutral lipids and this can be further enhanced by genetic and metabolic engineering.