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

Protein Glycosylation in Helicobacter pylori: Beyond the Flagellins?

Glycosylation of flagellins by pseudaminic acid is required for virulence in Helicobacter pylori. We demonstrate that, in H. pylori, glycosylation extends to proteins other than flagellins and to sugars other than pseudaminic acid. Several candidate glycoproteins distinct from the flagellins were detected via ProQ-emerald staining and DIG- or biotin- hydrazide labeling of the soluble and outer membrane fractions of wild-type H. pylori, suggesting that protein glycosylation is not limited to the flagellins. DIG-hydrazide labeling of proteins from pseudaminic acid biosynthesis pathway mutants showed that the glycosylation of some glycoproteins is not dependent on the pseudaminic acid glycosylation pathway, indicating the existence of a novel glycosylation pathway. Fractions enriched in glycoprotein candidates by ion exchange chromatography were used to extract the sugars by acid hydrolysis. High performance anion exchange chromatography with pulsed amperometric detection revealed characteristic monosaccharide peaks in these extracts. The monosaccharides were then identified by LC-ESI-MS/MS. The spectra are consistent with sugars such as 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Ac7Ac) previously described on flagellins, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Am7Ac), bacillosamine derivatives and a potential legionaminic acid derivative (Leg5AmNMe7Ac) which were not previously identified in H. pylori. These data open the way to the study of the mechanism and role of protein glycosylation on protein function and virulence in H. pylori

The Role of Protein Glycosylation in the Virulence of the Gastric Pathogens Helicobacter pylori and Campylobacter jejuni

H. pylori and C. jejuni are Gram-negative gastro-intestinal pathogens whose virulence is highly affected by protein glycosylation. The former causes gastric ulcers and cancer, while the latter causes enteritis and neurological disorders. Due to emerging drug-resistant strains, new treatments are needed. In both bacteria, the flagellins are essential virulence factors glycosylated by pseudaminic acid (PA). We have identified and disrupted genes required for PA synthesis in both bacteria, and shown that this affects flagellin production. Further analysis and glycoprotein staining revealed that in H. pylori, the PA pathway is necessary for the glycosylation of proteins other than flagellins and for the synthesis of additional virulence factors, including LPS and urease. Enzymatic deglycosylation analysis uncovered a second set of H. pylori proteins glycosylated with an unknown sugar synthesized by a PA-independent pathway. We have identified one as a membrane-associated isoform of an immunodominant antigen that protects H. pylori from oxidative stress. Numerous C. jejuni glycoproteins also possess a heptasaccharide containing diacetamidobacillosamine (DAB). Our DAB biosynthesis mutant was deficient in at least one glycoprotein and was avirulent in a chicken model, underscoring the role of this pathway in virulence. We have shown that both PA and DAB pathways are present in C. jejuni cell extracts and are investigating through activity-based assays and glycoprotein blotting how different growth conditions related to pathogenesis affect the relative activities of both pathways. Understanding how each pathway affects virulence will reveal the best targets for the development of glycosylation inhibitors to treat these major infections

Investigating the Glycosylation of GroEL and Its Role in the Virulence of Campylobacter jejuni

Campylobacter jejuni is a food-borne human pathogen and a major cause of gastroenteritis, reactive arthritis and neurological disorders. Due to emerging drug resistant strains, new treatments and/or vaccines need to be developed. Protein glycosylation, or the post-translational modification of proteins by sugar molecules, plays an important role in this bacterium\u27s virulence including affecting cell-host interactions, immunogenicity and pathogenicity. C jejuni harbours numerous proteins glycosylated by a heptasaccharide containing diacetamidobacillosamine. Our laboratory investigates the enzymes required to make this sugar as well as the glycoproteins on which this glycan is found. Analysis of C jejuni proteins by Western blot suggests that the molecular chaperone GroEL is post-translationally modified. A stress-induced protein as well as a major immunogenic antigen in C jejuni infections, GroEL prevents misfolding and aggregation of partially denatured proteins through an ATP-dependent process and may play an essential role in intestinal tract colonization and bacterial survival at high temperatures. GroEL and other chaperones have been shown to be glycosylated in other bacteria including another Campylobacter species. It has also been proposed that C jejuni GroEL is glycosylated based on its reactivity with a lectin. The focus of this work is to determine if GroEL is really glycosylated, identify the glycan present on GroEL and determine the function of the glycosylation modification on GroEL. We are using ATP affinity followed by anion exchange chromatography to purify GroEL and will determine its exact molecular weight and the glycan attachment site by mass spectrometry (MS). Initial enzymatic deglycosylation using five different enzymes suggests that the glycan present on the protein is not recognized by the enzymes available. The glycan present will be identified by ion-exchange chromatography, MS and NMR. The effect of the sugar on the activity of GroEL will be ascertained. We will also determine the role of the glycosylated form of GroEL and its glycan in the virulence of C jejuni by looking at the ability of mutants with defects in either the expression of the glycoprotein or its glycosylation to adhere and invade intestinal epithelial cells. This information will permit us to infer what enzymes are required to make these sugars, allowing the enzymes to later be targeted to inhibit protein glycosylation should glycosylation be shown to be important for virulence

The Helicobacter pylori flaA1 and wbpB Genes Control Lipopolysaccharide and Flagellum Synthesis and Function

flaA1 and wbpB are conserved genes with unknown biological function in Helicobacter pylori. Since both genes are predicted to be involved in lipopolysaccharide (LPS) biosynthesis, flagellum assembly, or protein glycosylation, they could play an important role in the pathogenesis of H. pylori. To determine their biological role, both genes were disrupted in strain NCTC 11637. Both mutants exhibited altered LPS, with loss of most O-antigen and core modification, and increased sensitivity to sodium dodecyl sulfate compared to wild-type bacteria. These defects could be complemented in a gene-specific manner. Also, flaA1 could complement these defects in the wbpB mutant, suggesting a potential redundancy of the reductase activity encoded by both genes. Both mutants were nonmotile, although the wbpB mutant still produced flagella. The defect in the flagellum functionality of this mutant was not due to a defect in flagellin glycosylation since flagellins from wild-type strain NCTC 11637 were shown not to be glycosylated. The flaA1 mutant produced flagellins but no flagellum. Overall, the similar phenotypes observed for both mutants and the complementation of the wbpB mutant by flaA1 suggest that both genes belong to the same biosynthesis pathway. The data also suggest that flaA1 and wbpB are at the interface between several pathways that govern the expression of different virulence factors. We propose that FlaA1 and WbpB synthesize sugar derivatives dedicated to the glycosylation of proteins which are involved in LPS and flagellum production and that glycosylation regulates the activity of these proteins

Genetics and Biochemistry of Protein Glycosylation in Campylobacter jejuni

BACKGROUND: C. jejuni produces numerous glycoproteins, including flagellins, which are important for virulence. The flagellins harbour pseudaminic acid (PA) whereas other glycoproteins harbour diacetamidobacillosamine (DAB). We are investigating the genetics and biochemistry of protein glycosylation in C. jejuni to identify the enzymes involved, and determine their activity and roles in virulence. We focused on two homologous pathways comprising each a putative dehydratase, aminotransferase and acetyltransferase, namely {Cj1293, Cj1294 and Cj1298} and {Cj1120c, Cj1121c and Cj1123c}. METHODS: All enzymes were overexpressed and purified before monitoring their activity by capillary electrophoresis. The cj1121c and cj1294 genes were disrupted by a chloramphenicol resistance cassette and virulence-related phenotypes were investigated. RESULTS: We determined that Cj1293 is a UDP-GlcNAc C6 dehydratase. It leads to the formation of 4-keto-arabino and 4-keto-gluco intermediates. We showed that Cj1294 and Cj1121c are aminotransferases that use the arabino and gluco intermediates generated by Cj1293 as substrates, respectively. Both activities are present in C. jejuni extracts, with preponderance of the Cj1121c activity. We showed that Cj1123c is a N-acetyltransferase that uses the Cj1121c reaction product as a substrate. The cj1294 and cj1121c mutants are non-motile but the cj1121c mutant produces normal flagellins and flagella, whereas the cj1294 mutant does not. cj1121c is important for interactions with Caco2 cells whereas cj1294 is not. Finally, cj1121c is essential for colonization of chicken intestine and for the glycosylation of proteins other than flagellins. DISCUSSION: Our data indicate that Cj1293 and Cj1294 are involved in flagellin glycosylation via PA biosynthesis, and that Cj1121c and Cj1123c are involved in general protein glycosylation via DAB biosynthesis. The data demonstrate a cross-talk between both pathways, with a preponderant role of Cj1121c on virulence, hence identifying Cj1121c as a target for inhibitor development

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