Engineering xylose metabolism in Thraustochytrid T18

Thraustochytrids spp are oleaginous marine protists with significant potential for biofuel production at industrial levels; however, the cost of feedstocks has been a major challenge in making this process economical. On a quest for cheaper and renewable feedstocks, we investigated the ability of Thraustochytrid strain T18 to grow in the presence of xylose and demonstrated its ability to produce xylitol. However, genome sequencing and in vivo enzymatic assays revealed the presence of a xylose isomerase which indicates there are two xylose metabolism pathways in Thraustochytrid T18: a xylose reductase/xylitol dehydrogenase pathway as well as an isomerase pathway. Characterization of the two native pathways suggested that xylitol production is a bottleneck to T18 xylose metabolism. Through various strain improvement strategies, including over-expression of the endogenous xylose isomerase and heterologous xylulose kinases, we enhanced xylose usage while reducing xylitol production by \u3e50% and 80%, respectively, compared to wild-type. Highest levels of xylose metabolism were obtained through selection of strains possessing multiple copies of the transgenes. The xylose usage of the best xylose metabolizing isolate was further validated through fermentation. These newly engineered strains pave the way to using T18 for biofuel production using hemicellulosic feedstock

Engineering xylose metabolism in thraustochytrid T18

Abstract Background Thraustochytrids are heterotrophic, oleaginous, marine protists with a significant potential for biofuel production. High-value co-products can off-set production costs; however, the cost of raw materials, and in particular carbon, is a major challenge to developing an economical viable production process. The use of hemicellulosic carbon derived from agricultural waste, which is rich in xylose and glucose, has been proposed as a sustainable and low-cost approach. Thraustochytrid strain T18 is a commercialized environmental isolate that readily consumes glucose, attaining impressive biomass, and oil production levels. However, neither thraustochytrid growth capabilities in the presence of xylose nor a xylose metabolic pathway has been described. The aims of this study were to identify and characterize the xylose metabolism pathway of T18 and, through genetic engineering, develop a strain capable of growth on hemicellulosic sugars. Results Characterization of T18 performance in glucose/xylose media revealed diauxic growth and copious extracellular xylitol production. Furthermore, T18 did not grow in media containing xylose as the only carbon source. We identified, cloned, and functionally characterized a xylose isomerase. Transcriptomics indicated that this xylose isomerase gene is upregulated when xylose is consumed by the cells. Over-expression of the native xylose isomerase in T18, creating strain XI 16, increased xylose consumption from 5.2 to 7.6 g/L and reduced extracellular xylitol from almost 100% to 68%. Xylose utilization efficiency of this strain was further enhanced by over-expressing a heterologous xylulose kinase to reduce extracellular xylitol to 20%. Moreover, the ability to grow in media containing xylose as a sole sugar was dependent on the copy number of both xylose isomerase and xylulose kinase present. In fed-batch fermentations, the best xylose metabolizing isolate, XI-XK 7, used 137 g of xylose versus 39 g by wild type and produced more biomass and fatty acid. Conclusions The presence of a typically prokaryotic xylose isomerase and xylitol production through a typically eukaryotic xylose reductase pathway in T18 is the first report of an organism naturally encoding enzymes from two native xylose metabolic pathways. Our newly engineered strains pave the way for the growth of T18 on waste hemicellulosic feedstocks for biofuel production