Deletion of the KU70 homologue facilitates gene targeting in Lipomyces starkeyi strain NRRL Y-11558.

The objective of this study was to disrupt the non-homologous end-joining (NHEJ) pathway gene (Lsku70Δ) and evaluate the effects of selected gene deletions related to glycogen synthesis (LsGSY1) and lipid degradation (LsMFE1, LsPEX10, and LsTGL4) on lipid production in the oleaginous yeast Lipomyces starkeyi. Disruption of the NHEJ pathway to reduce the rate of non-homologous recombination is a common approach used to overcome low-efficiency targeted deletion or insertion in various organisms. Here, the homologue of the LsKU70 gene was identified and disrupted in L. starkeyi NRRL Y-11558. The LsGSY1, LsMFE1, LsPEX10, LsTGL4, and LsURA3 genes were then replaced with a resistance marker in the Lsku70Δ strain and several site-specific insertions were assessed for targeted over-expression of selected genes. The targeted disruption efficiency of five selected genes (LsGSY1, LsMFE1, LsPEX10, LsTGL4, and LsURA3) was increased from 0 to 10% in the parent to 50-100% of transformants screened in the Lsku70Δ strain with 0.8-1.4 kb homologous flanking sequences, while the efficiency of site-specific gene insertion with the ß-glucuronidase reporter gene was 100% in the locus near the 3'-end coding (LsKU70) and non-coding (LsGSY1, LsMFE1, and LsPEX10) regions. Disruption of LsKU70 in isolation and in conjunction with LsGSY1, LsMFE1, LsPEX10, or LsTGL4 did not affect lipid production in L. starkeyi. Furthermore, ß-glucuronidase reporter gene activity was similar in strains containing site-specific targeted insertions. Therefore, over-expression of genes related to lipid synthesis at targeted loci can be further examined for improvement of total lipid production in L. starkeyi.

Transcriptomic analysis of the oleaginous yeast Lipomyces starkeyi during lipid accumulation on enzymatically treated corn stover hydrolysate.

BACKGROUND: Efficient and economically viable production of biofuels from lignocellulosic biomass is dependent on mechanical and chemical pretreatment and enzymatic hydrolysis of plant material. These processing steps yield simple sugars as well as plant-derived and process-added organic acids, sugar-derived dehydration products, aldehydes, phenolics and other compounds that inhibit the growth of many microorganisms. Lipomyces starkeyi is an oleaginous yeast capable of robust growth on a variety of sugars 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 20-L bioreactor with either a blend of pure glucose and xylose or pretreated corn stover (PCS) that had been enzymatically hydrolyzed as the carbon sources. RESULTS: We monitored sugar and ammonium utilization as well as biomass accumulation and found that growth of L. starkeyi is inhibited with PCS hydrolysate as the carbon source. Both acetic acid and furfural are present at concentrations toxic to L. starkeyi in PCS 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 PCS hydrolysate. Here, we monitored key metabolites to establish physiologically equivalent comparisons during a batch bioreactor run comparing PCS hydrolysate and purified sugars. L. starkeyi's response to PCS hydrolysate is primarily at the beginning of the run during growth phase when inhibitory compounds are presumably at their highest concentration and inducing the general detoxification response by L. starkeyi. Differentially expressed genes identified herein during growth phase will aid in the improvement of industrial strains capable of robust growth on substrates containing various growth inhibitory compounds.