New inducible promoter for gene expression and synthetic biology in Yarrowia lipolytica.

BACKGROUND: The oleaginous yeast Yarrowia lipolytica is increasingly used as alternative cell factory for the production of recombinant proteins. At present, several promoters with different strengths have been developed based either on the constitutive pTEF promoter or on oleic acid inducible promoters such as pPOX2 and pLIP2. Although these promoters are highly efficient, there is still a lack of versatile inducible promoters for gene expression in Y. lipolytica. RESULTS: We have isolated and characterized the promoter of the EYK1 gene coding for an erythrulose kinase. pEYK1 induction was found to be impaired in media supplemented with glucose and glycerol, while the presence of erythritol and erythrulose strongly increased the promoter induction level. Promoter characterization and mutagenesis allowed the identification of the upstream activating sequence UAS1EYK1. New hybrid promoters containing tandem repeats of either UAS1XPR2 or UAS1EYK1 were developed showing higher expression levels than the native pEYK1 promoter. Furthermore, promoter strength was improved in a strain carrying a deletion in the EYK1 gene, allowing thus the utilization of erythritol and erythrulose as free inducer. CONCLUSIONS: Novel tunable and regulated promoters with applications in the field of heterologous protein production, metabolic engineering, and synthetic biology have been developed, thus filling the gap of the absence of versatile inducible promoter in the yeast Y. lipolytica.

Induction of lignocellulose-degrading enzymes in Neurospora crassa by cellodextrins.

Neurospora crassa colonizes burnt grasslands in the wild and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source such as sucrose to cellulose, N. crassa dramatically upregulates expression and secretion of a wide variety of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Here, we show that an N. crassa mutant carrying deletions of two genes encoding extracellular ß-glucosidase enzymes and one intracellular ß-glucosidase lacks ß-glucosidase activity, but efficiently induces cellulase gene expression in the presence of cellobiose, cellotriose, or cellotetraose as a sole carbon source. These data indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression in N. crassa. Furthermore, the inclusion of a deletion of the catabolite repressor gene, cre-1, in the triple ß-glucosidase mutant resulted in a strain that produces higher concentrations of secreted active cellulases on cellobiose. Thus, the ability to induce cellulase gene expression using a common and soluble carbon source simplifies enzyme production and characterization, which could be applied to other cellulolytic filamentous fungi.

Erythrose inhibits the progression to invasiveness and reverts drug resistance of cancer stem cells of glioblastoma.

Glioblastoma (GBM) is the most frequent brain cancer and more lethal than other cancers. Characteristics of this cancer are its high drug resistance, high recurrence rate and invasiveness. Invasiveness in GBM is related to overexpression of matrix metalloproteinases (MMPs) which are mediated by wnt/ß-catenin and induced by the activation of signaling pathways extracellularly activated by the cytokine neuroleukin (NLK) in cancer stem cells (CSC). Therefore, in this work we evaluated the effect of the tetrose saccharide, erythrose (Ery), a NLK inhibitor of invasiveness and drug sensitization in glioblastoma stem cells (GSC). GSC were obtained from parental U373 cell line by a CSC phenotype enrichment protocol based on microenvironmental stress conditions such as hypoxia, hipoglycemia, drug exposition and serum starvation. Enriched fraction of GSC overexpressed the typical markers of brain CSC: low CD133+ and high CD44; in addition, epithelial to mesenchyme transition (EMT) markers and MMPs were increased several times in GSC vs. U373 correlating with higher invasiveness, elongated and tubular mitochondrion and temozolomide (TMZ) resistance. IC50 of Ery was found at nM concentration and at 24 h induced a severe diminution of EMT markers, MMPs and invasiveness in GSC. Furthermore, the phosphorylation pattern of NLK after Ery exposition also was affected. In addition, when Ery was administered to GSC at subIC50, it was capable of reverting TMZ resistance at concentrations innocuous to non-tumor cancer cells. Moreover, Ery added daily induced the death of all GSC. Those findings indicated that the phytodrug Ery could be used as adjuvant therapy in GBM.

Cellotriose and cellotetraose as inducers of the genes encoding cellobiohydrolases in the basidiomycete Phanerochaete chrysosporium.

The wood decay basidiomycete Phanerochaete chrysosporium produces a variety of cellobiohydrolases belonging to glycoside hydrolase (GH) families 6 and 7 in the presence of cellulose. However, no inducer of the production of these enzymes has yet been identified. Here, we quantitatively compared the transcript levels of the genes encoding GH family 6 cellobiohydrolase (cel6A) and GH family 7 cellobiohydrolase isozymes (cel7A to cel7F/G) in cultures containing glucose, cellulose, and cellooligosaccharides by real-time quantitative PCR, in order to evaluate the transcription-inducing effect of soluble sugars. Upregulation of transcript levels in the presence of cellulose compared to glucose was observed for cel7B, cel7C, cel7D, cel7F/G, and cel6A at all time points during cultivation. In particular, the transcription of cel7C and cel7D was strongly induced by cellotriose or cellotetraose. The highest level of cel7C transcripts was observed in the presence of cellotetraose, whereas the highest level of cel7D transcripts was found in the presence of cellotriose, amounting to 2.7 x 10(6) and 1.7 x 10(6) copies per 10(5) actin gene transcripts, respectively. These numbers of cel7C and cel7D transcripts were higher than those in the presence of cellulose. In contrast, cellobiose had a weaker transcription-inducing effect than either cellotriose or cellotetraose for cel7C and had little effect in the case of cel7D. These results indicate that cellotriose and cellotetraose, but not cellobiose, are possible natural cellobiohydrolase gene transcription inducers derived from cellulose.

Kinetic characterization and comparison of various protein crosslinking reagents for matrix modification.

We have characterized the relative efficacies of a number of protein crosslinking agents that have the potential for use in the crosslinking of proteinaceous matrices both in vitro and in vivo. The crosslinkers tested were; L: -threose (LT), Genipin (GP), Methylglyoxal (MG), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), proanthrocyanidin (PA) and glutaraldehyde (GA). The relative effectiveness of the crosslinkers with regard to their saturating concentrations was: GA > PA > EDC > MG = GP >> LT. Most of the crosslinkers displayed a pH dependence and were more effective at more alkaline pH. At optimal pH and saturating conditions, the relative reaction rates of the crosslinkers were: PA = GA > EDC > GP > MG >> LT.

Activation of Innate Immune Responses by a CpG Oligonucleotide Sequence Composed Entirely of Threose Nucleic Acid.

Recent advances in synthetic biology have led to the development of nucleic acid polymers with backbone structures distinct from those found in nature, termed xeno-nucleic acids (XNAs). Several unique properties of XNAs make them attractive as nucleic acid therapeutics, most notably their high resistance to serum nucleases and ability to form Watson-Crick base pairing with DNA and RNA. The ability of XNAs to induce immune responses has not been investigated. Threose nucleic acid (TNA), a type of XNA, is recalcitrant to nuclease digestion and capable of undergoing Darwinian evolution to produce high affinity aptamers; thus, TNA is an attractive candidate for diverse applications, including nucleic acid therapeutics. In this study, we evaluated a TNA oligonucleotide derived from a cytosine-phosphate-guanine oligonucleotide sequence known to activate toll-like receptor 9-dependent immune signaling in B cell lines. We observed a slight induction of relevant mRNA signals, robust B cell line activation, and negligible effects on cellular proliferation.

A new biomimetic assay reveals the temporal role of matrix stiffening in cancer cell invasion.

Tumor initiation and growth is associated with significant changes in the surrounding tissue. During carcinoma progression, a global stiffening of the extracellular matrix is observed and is interpreted as a signature of aggressive invasive tumors. However, it is still unknown whether this increase in matrix rigidity promotes invasion and whether this effect is constant along the course of invasion. Here we have developed a biomimetic in vitro assay that enabled us to address the question of the importance of tissue rigidity in the chronology of tumor invasion. Using low concentrations of the sugar threose, we can effectively stiffen reconstituted collagen I matrices and control the stiffening in time with no direct effect on residing cells. Our findings demonstrate that, depending on the timing of its stiffening, the extracellular matrix could either inhibit or promote cancer cell invasion and subsequent metastasis: while matrix stiffening after the onset of invasion promotes cancer cell migration and tumor spreading, stiff matrices encapsulate the tumor at an early stage and prevent cancer cell invasion. Our study suggests that adding a temporal dimension in in vitro models to analyze biological processes in four dimensions is necessary to fully capture their complexity.

Effect of non-nutritive sugars to decrease the survivorship of spotted wing drosophila, Drosophila suzukii.

In this study, we investigated the effects of non-nutritive sugars and sugar alcohols on the survivorship of spotted wing drosophila, Drosophila suzukii, and found erythritol and erythrose as potentially insecticidal to the fly. In a dose-dependent study, erythritol and erythrose significantly reduced fly longevity, with 100% mortality with 1, 0.5, 0.1 & 0.05M doses after feeding for 7days. When sucrose and erythritol solutions were provided separately to flies for 7days, there was no effect on survivorship regardless of erythritol concentrations. However, with a serial combination of sucrose and erythritol solutions, fly survivorship was significantly decreased for the same period. Also, the higher dose of erythritol regardless of the sucrose dose combined showed greater mortality. In a no-choice assay, D. suzukii ingested more erythritol than sucrose or water, indicating the fly continuously fed on erythritol for 72h. Also under no-choice conditions, erythritol and sucrose-fed flies gained more weight than water-fed flies. However, in two-choice assays, the amount of erythritol ingested was less than sucrose or water. Total sugar and glycogen levels among erythritol and erythrose-fed flies were significantly less than mannitol, sorbitol, xylitol, and sucrose-fed flies after 48h. This indicates that these two non-nutritive sugars can't be used a substrate for enzymes involved in sugar metabolism. Although the metabolism of erythritol and erythrose is unknown in insects, the mortality of D. suzukii flies ingesting these sugars might be caused by two potential physiological changes. The fly is starved by feeding of non-metabolizable erythritol and erythrose, or experiences abnormally high osmotic pressure in the hemolymph with erythritol molecules diffused from the midgut. Non-nutritive sugars might be used as an insecticide alone or combined with conventional or biological insecticides to enhance efficacy. If other sugar sources are present, a palatable sugar might be mixed with erythritol to elicit feeding.

Inhibitors of advanced glycation end product-associated protein cross-linking.

The reaction of lens proteins with sugars over time results in the formation of protein-bound advanced glycation end products (AGEs). The most damaging element of AGE formation may be the synthesis of protein-protein cross-links in long-lived proteins, such as collagen or lens crystallins. A quantitative cross-linking assay, involving the sugar-dependent incorporation of [U-(14)C]lysine into protein, was employed to determine the efficacy of a variety of potential cross-linking inhibitors. Reaction mixtures contained 5.0 mM L-threose, 2.5 microCi [(14)C]lysine (1.0 mCi/mmole), 5.0 mg/ml bovine lens proteins, 0-10 mM inhibitor and 1.0 mM DTPA in 100 mM phosphate buffer, pH 7.0. Of 17 potential inhibitors tested, 11 showed 50% inhibition or less at 10 mM. The dicarbonyl-reactive compounds 2-aminoguanidine, semicarbazide and o-phenylenediamine inhibited 50% at 2.0 mM, whereas 10 mM dimethylguanidine had no effect. Several amino acids failed to compete effectively with [(14)C]lysine in the cross-linking assay; however, cysteine inhibited 50% at 1.0 mM. This was likely due to the sulfhydryl group of cysteine, because 3-mercaptopropionic acid and reduced glutathione exhibited similar activity. Sodium metabisulfite had the highest activity, inhibiting 50% at only 0.1-0.2 mM. Protein dimer formation, as determined by SDS-PAGE, was inhibited in a quantitatively similar manner. The dicarbonyl-reactive inhibitors and the sulfur-containing compounds produced similar inhibition curves for [(14)C]lysine incorporation over a 3 week assay with 250 mM glucose. A much lesser effect was observed on either the incorporation of [(14)C]glucose, or on fluorophore formation (360/420 nm), suggesting that non-cross-link fluorophores were also formed. The inhibitor data were consistent with cross-linking by a dicarbonyl intermediate. This was supported by the fact that the inhibitors were uniformly less effective when the 5.0 mM threose was replaced by either 3.0 mM 3-deoxythreosone or 3.0 mM threosone.

The influence of urea and guanidine chloride on the binding of the binding of the bacterial substrate and inhibitors to hen lysozyme at physiological temperature (40 degrees) (*).

The influence of urea and of guanidine chloride on the binding of the bacterial substrate and of inhibitors such as N-acetylglucosamine or chitotetraose to hen lysozyme were studied at 20 degrees and at 40 degrees C (physiological temperature). The action of urea did not prevent a certain degree of organization of the enzyme compatible with its usual behaviour in the presence of some inhibitors and with its crystallization ; guanidine chloride, already at low concentrations, seemed to have a more severe effect on lysozyme.

[Inhibitory effects of two oligosaccharides on murine melanoma experimental metastasis].

OBJECTIVE: To observe the effects of a chemically synthesized tetrose and a natural yeast mannan on mouse melanoma experimental liver metastasis. METHODS: After treated with 4 mg tetrose (tetrose group) or 4 mg mannan (mannan group) for 30 minutes at 37 degrees C, 0.5 ml 1 x 10(6)B16-MBK melanoma cells were injected intraspleen. 55 days later, melanoma metastasis nodes in the surfaces of the liver and other organs as well as mouse survival time were observed. RESULTS: Of 6 mice in control (B16 cell+PBS), 4 died naturally within 55 days, 2 were dissected in the 55th day. All of the 6 mice had metastases in the livers, the total number of the melanoma nodes on each liver surface ranged from 2 to 30, with the largest one fused to the whole liver. One mouse had a neoplasm in the remnant site of injection, 3 had metastases in lungs, while of the 6 mice in the tetrose group, one died on the 50th day on injection. In mannan group, all of the 6 mice survived and no metastasis was seen except the largest diameter of < 1 mm of 2 liver nodes in one mouse. Neither tetrose nor mannan group had metastasis of the liver, and the weights of liver in the two groups were significantly lower than in the control. CONCLUSION: Both tetrose and mannan had the effects in blocking melanoma experimental liver metastasis, inhibiting transmigration of the liver, and prolonging the survival time of the mouse.

Synthesis and evaluation of amino-threoses in D- and L-series: are five membered ring amino-sugars more potent glycosidase inhibitors than the six membered ones?

Cyclic D- and L-4-aminothreose were synthesised from ethyl D- and L-tartrate, respectively. D-aminothreose was a potent inhibitor of alpha-glucosidase and of alpha-mannosidase. From the glycosidase inhibition potencies of the four 4-amino-4-deoxy-tetroses, the contribution of binding of each functionality of the 5 and 6 membered ring amino-sugars towards the various glycosidases is discussed.

Deoxythreosyl phosphonate nucleosides as selective anti-HIV agents.

Out of a series of eight new phosphonate nucleosides with an l-threose and an l-2-deoxythreose sugar moiety, two new compounds were identified (PMDTA and PMDTT) that showed potent anti-HIV-1 (HIV-2) activity [EC50 = 2.53 microM (PMDTA) and 6.59 microM (PMDTT)], while no cytoxicity was observed at the highest concentration tested [CC50 > 316 microM (PMDTA) and > 343 microM (PMDTT)]. The kinetics of incorporation of PMDTA into DNA (using the diphosphate of PMDTA as substrate and HIV-1 reverse transcriptase as catalyst) was similar to the kinetics observed for dATP, while the diphosphate of PMDTA was a very poor substrate for DNA polymerase alpha. The incorporated PMDTA fits very well in the active site pocket of HIV-1 reverse transcriptase.