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.

Keywords: Biomass production; Lipid production for biofuel; Metabolic engineering; Thraustochytrid; Xylose isomerase; Xylose metabolism.

Fig. 1

Two common xylose metabolism pathways in microorganisms. Xylose can be directly converted to xylulose by a xylose isomerase (XI) or converted to xylitol by a xylose reductase (XR). Xylitol is then converted to xylulose by a xylitol dehydrogenase (XD). Both the XR and XD reactions require co-factors. Xylulose is then converted to xylulose-5-phosphate by an ATP-dependent xylulose kinase (XK)

Fig. 2

Sugar utilization and xylose isomerase transcription by wild type grown in xylose with and without glucose. a The amount of xylose (squares) remaining in the culture supernatant when grown in the absence of glucose. b Amount of glucose (circles), xylose, and xylitol (triangles) present in the culture supernatant when grown in the presence of both sugars. Error bars represent standard deviation from triplicates. c Cultures were batched in glucose and xylose then re-fed glucose after the first xylose uptake period as indicated by the asterisk. Error bars represent standard deviation from three fermentations

Fig. 3

qPCR analysis of wild-type (WT) and XI transformants strains. The gene copy number of a the endogenous and His-tagged transgenic xi genes and b the transgenic ble gene were measured. Experiments were done in triplicate. Error bars represent the higher and lower relative quantity limits

Fig. 4

Wild-type (WT) and XI transformants growth in minimal-media containing glucose with and without xylose. Glucose consumption (a) and biomass accumulation (b) in media containing glucose alone. Consumption of glucose (c) and xylose (d), xylitol production by day 7 (e), and biomass accumulation (f) in media containing glucose and xylose. The experiments were done in triplicate. Error bars represent standard deviations. Symbols: diamonds, wild type; squares, XI 4; triangles, XI 6; hexagons, XI 8; and circles, XI 16

Fig. 5

qPCR analysis of wild-type (WT) and XI-XK transformants strains. Gene copy numbers were measured for a aph7, b the native and His-tagged xylose isomerase, and c ble. Experiments were done in triplicate. Error bars represent the higher and lower relative quantity limits

Fig. 6

Wild-type and XI-XK transformants growth in minimal media containing glucose with and without xylose. Glucose consumption (a) and biomass accumulation (b) in media containing glucose alone. Consumption of glucose (c) and xylose (d), xylitol production (e), and biomass accumulation (f) in media containing glucose and xylose. The experiments were done in triplicate. Error bars represent standard deviations. Symbols: diamonds, WT; circles, XI 16; triangles, XB; hexagons, XI-XK 1; stars, XI-XK 3; squares, XI-XK 7

Fig. 7

Growth of wild-type and transformants in rich media containing xylose as the main carbon source. a Xylose consumed, b xylitol produced, and c biomass made. Experiments were done in triplicate. The error bars represent standard deviation. Symbols: diamonds, WT; circles, XI 16; triangles, XB; hexagons, XI-XK 1; stars, XI-XK 3; squares, XI-XK 7

Fig. 8

Fed-batch fermentations of wild-type and transformant XI-XK 7. a Glucose consumption, b xylose consumption, and c xylitol production throughout the fermentation. d Total carbon consumed at 96 h indicating the proportion of glucose (black portion) and xylose (white portion) used, e total biomass (black bar) and fatty acid (grey bar) produced, and f lipid profiles of transformant XI-XK 7 versus wild type (WT). Symbols: diamonds, WT; filled and unfilled squares, duplicate XI-XK 7 fermentations