PNNL

Abstract

Background: Efficient and economically viable production of biofuels from lignocellulosic biomass is dependent on physical, chemical, and enzymatic pretreatment of plant material. This processing step(s) yield simple sugars as well as plant-derived and process-added organic acids, aldehydes, and phenolics that inhibit the growth of many microorganisms. Lipomyces starkeyi is an oleaginous yeast capable of robust growth and lipid accumulation on pretreated lignocellulosic substrates making it attractive as an industrial producer of biofuels. Here we examined gene expression during batch growth and lipid accumulation in a 20L bioreactor with either pure glucose and xylose or enzymatically digested corn stover hydrolysate as the carbon source. Results: We monitored sugar and ammonium utilization as well as biomass accumulation and found that growth of L. starkeyi is inhibited with enzymatically digested corn stover hydrolysate as the carbon source. Both acetic acid and furfural are present at concentrations toxic to L. starkeyi in enzymatically digested corn stover hydrolysate. We quantified gene expression at seven time-points for each carbon source during batch growth and found that gene expression is similar at physiologically equivalent points. Analysis of promoter regions revealed that gene expression during the transition to lipid accumulation is regulated by carbon and nitrogen catabolite repression regardless of carbon source and is associated with decreased expression of the translation machinery and suppression of the cell cycle. We identified 73 differentially expressed genes during growth phase in the bioreactor that may be involved in detoxification of corn stover hydrolysate. Conclusions: Growth of L. starkeyi is inhibited by compounds present in enzymatically digested corn stover hydrolysate. Here we monitored key metabolites to establish physiologically equivalent comparisons during a batch bioreactor run comparing enzymatically digested corn stover hydrolysate and purified sugars. L. starkeyi’s response to enzymatically digested corn stover hydrolysate is primarily at the beginning of the run during growth phase when inhibitory compounds are presumably being detoxified. Differentially expressed genes identified herein during growth phase will aid in the improvement of industrial strains capable of robust growth on less than ideal substrates.