De-novo Biosynthesis Research Articles

Optimization of hydrogenobyrinic acid biosynthesis in Escherichia coli using multi-level metabolic engineering strategies

BackgroundHydrogenobyrinic acid is a key intermediate of the de-novo aerobic biosynthesis pathway of vitamin B12. The introduction of a heterologous de novo vitamin B12 biosynthesis pathway in Escherichia coli offers an alternative approach for its production. Although E. coli avoids major limitations that currently faced by industrial producers of vitamin B12, such as long growth cycles, the insufficient supply of hydrogenobyrinic acid restricts industrial vitamin B12 production.ResultsBy designing combinatorial ribosomal binding site libraries of the hemABCD genes in vivo, we found that their optimal relative translational initiation rates are 10:1:1:5. The transcriptional coordination of the uroporphyrinogen III biosynthetic module was realized by promoter engineering of the hemABCD operon. Knockdown of competitive heme and siroheme biosynthesis pathways by RBS engineering enhanced the hydrogenobyrinic acid titer to 20.54 and 15.85 mg L−1, respectively. Combined fine-tuning of the heme and siroheme biosynthetic pathways enhanced the hydrogenobyrinic acid titer to 22.57 mg L−1, representing a remarkable increase of 1356.13% compared with the original strain FH215-HBA.ConclusionsThrough multi-level metabolic engineering strategies, we achieved the metabolic balance of the uroporphyrinogen III biosynthesis pathway, eliminated toxicity due to by-product accumulation, and finally achieved a high HBA titer of 22.57 mg L−1 in E. coli. This lays the foundation for high-yield production of vitamin B12 in E. coli and will hopefully accelerate its industrial production.

Guanine Limitation Results in CodY-Dependent and -Independent Alteration of Staphylococcus aureus Physiology and Gene Expression.

In Staphylococcus aureus, the global transcriptional regulator CodY modulates the expression of hundreds of genes in response to the availability of GTP and the branched-chain amino acids isoleucine, leucine, and valine (ILV). CodY DNA-binding activity is high when GTP and ILV are abundant. When GTP and ILV are limited, CodY's affinity for DNA drops, altering expression of CodY-regulated targets. In this work, we investigated the impact of guanine nucleotides (GNs) on S. aureus physiology and CodY activity by constructing a guaA null mutant (ΔguaA strain). De novo biosynthesis of guanine monophosphate is abolished due to the guaA mutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked out guaA, activating the Agr two-component system and increasing secreted protease activity. Notably, in a rich, complex medium, we detected an increase in alternative sigma factor B activity in the ΔguaA mutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, ΔguaA cells failed to form robust biofilms when limited for guanine or guanosine. Transcriptome sequencing (RNA-Seq) analysis of the S. aureus transcriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alterations of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting that S. aureus exhibits a more invasive lifestyle when limited for guanosine. Further, gene products upregulated under GN limitation, including those necessary for lipoic acid biosynthesis and sugar transport, may prove to be useful drug targets for treating Gram-positive infections.IMPORTANCEStaphylococcus aureus infections impose a serious economic burden on health care facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing fitness and virulence of S. aureus in response to environmental cues is critical for developing efficient diagnostics and treatments. De novo purine biosynthesis is essential for both fitness and virulence in S. aureus since inhibiting production cripples S. aureus's ability to cause infection. Here, we corroborate these findings and show that blocking guanine nucleotide synthesis severely affects S. aureus fitness by altering metabolic and virulence gene expression. Characterizing pathways and gene products upregulated in response to guanine limitation can aid in the development of novel adjuvant strategies to combat S. aureus infections.

De Novo Biosynthesis of Apigenin, Luteolin, and Eriodictyol in the Actinomycete Streptomyces albus and Production Improvement by Feeding and Spore Conditioning.

Nutraceutical compounds as plant flavonoids play an important role in prevention and modulation of diverse heath conditions, as they exert interesting antifungal, antibacterial, antioxidant, and antitumor effects. They also possess anti-inflammatory activities in arthritis, cardiovascular disease or neurological diseases, as well as modulatory effects on the CYP450 activity on diverse drugs. Most flavonoids are bioactive molecules of plant origin, but their industrial production is sometimes hindered due to reasons as low concentration in the plant tissues, presence in only some species or as a complex mixture or inactive glycosides in plant vacuolae. In this work, we describe the de novo biosynthesis of two important flavones, apigenin and luteolin, and one known flavanone, eriodictyol. Their plant biosynthetic pathways have been reconstructed for heterologous expression in Streptomyces albus, an actinomycete bacterium manageable at industrial production level. Also, production levels for apigenin have been improved by feeding with naringenin precursor, and timing for settlement of secondary metabolism has been advanced by spore conditioning. In the cases of eriodictyol and luteolin, their production in this important type of biotechnology-prone bacteria, the actinomycetes, had not been described in the literature yet.

Statin use and survival in patients with metastatic castration-resistant prostate cancer treated with abiraterone acetate or enzalutamide.

e16503 Background: Statins have been associated with favorable effects in various solid malignancies. In prostate cancer, statins may synergize with novel anti-androgen agents enzalutamide and abiraterone via pharmacokinetic interactions and by decreasing substrate availability for androgen de-novo biosynthesis. Methods: Medical records of patients with metastatic castration-resistant prostate cancer (mCRPC) between September, 2011 and August, 2016 were reviewed at multiple participating centers. Results: 187 men received abiraterone, while 129 received enzalutamide. Patients treated with enzalutamide had a more advanced disease, with a median overall survival (OS) of 7.8 vs. 18 months in the abiraterone cohort. In the abiraterone cohort, statin use was a statistically significant prognostic factor for longer OS (HR = 0.40, 95% CI = 0.27 to 0.59, p < 0.001) and statin users vs. non users had greater 30% PSA declines rates at 12 wks(77.9 vs. 52.9%, p < .01) In the enzalutamide cohort, statin use was not prognostic for survival or PSA declines. Conclusions: Our study suggests a prognostic effect of statins plus abiraterone but not enzalutamide for mCRPC. The mechanism of this interaction warrants elucidation but may include enhancing the anti-tumor activity of abiraterone as well as cardioprotective effects. [Table: see text]

Caffeic Acid Expands Anti-Tumor Effect of Metformin in Human Metastatic Cervical Carcinoma HTB-34 Cells: Implications of AMPK Activation and Impairment of Fatty Acids De Novo Biosynthesis.

The efficacy of cancer treatments is often limited and associated with substantial toxicity. Appropriate combination of drug targeting specific mechanisms may regulate metabolism of tumor cells to reduce cancer cell growth and to improve survival. Therefore, we investigated the effects of anti-diabetic drug Metformin (Met) and a natural compound caffeic acid (trans-3,4-dihydroxycinnamic acid, CA) alone and in combination to treat an aggressive metastatic human cervical HTB-34 (ATCC CRL­1550) cancer cell line. CA at concentration of 100 µM, unlike Met at 10 mM, activated 5'-adenosine monophosphate-activated protein kinase (AMPK). What is more, CA contributed to the fueling of mitochondrial tricarboxylic acids (TCA) cycle with pyruvate by increasing Pyruvate Dehydrogenase Complex (PDH) activity, while Met promoted glucose catabolism to lactate. Met downregulated expression of enzymes of fatty acid de novo synthesis, such as ATP Citrate Lyase (ACLY), Fatty Acid Synthase (FAS), Fatty Acyl-CoA Elongase 6 (ELOVL6), and Stearoyl-CoA Desaturase-1 (SCD1) in cancer cells. In conclusion, CA mediated reprogramming of glucose processing through TCA cycle via oxidative decarboxylation. The increased oxidative stress, as a result of CA treatment, sensitized cancer cells and, acting on cell biosynthesis and bioenergetics, made HTB-34 cells more susceptible to Met and successfully inhibited neoplastic cells. The combination of Metformin and caffeic acid to suppress cervical carcinoma cells by two independent mechanisms may provide a promising approach to cancer treatment.

Salmonella Persistence in Tomatoes Requires a Distinct Set of Metabolic Functions Identified by Transposon Insertion Sequencing.

Human enteric pathogens, such as Salmonella spp. and verotoxigenic Escherichia coli, are increasingly recognized as causes of gastroenteritis outbreaks associated with the consumption of fruits and vegetables. Persistence in plants represents an important part of the life cycle of these pathogens. The identification of the full complement of Salmonella genes involved in the colonization of the model plant (tomato) was carried out using transposon insertion sequencing analysis. With this approach, 230,000 transposon insertions were screened in tomato pericarps to identify loci with reduction in fitness, followed by validation of the screen results using competition assays of the isogenic mutants against the wild type. A comparison with studies in animals revealed a distinct plant-associated set of genes, which only partially overlaps with the genes required to elicit disease in animals. De novo biosynthesis of amino acids was critical to persistence within tomatoes, while amino acid scavenging was prevalent in animal infections. Fitness reduction of the Salmonella amino acid synthesis mutants was generally more severe in the tomato rin mutant, which hyperaccumulates certain amino acids, suggesting that these nutrients remain unavailable to Salmonella spp. within plants. Salmonella lipopolysaccharide (LPS) was required for persistence in both animals and plants, exemplifying some shared pathogenesis-related mechanisms in animal and plant hosts. Similarly to phytopathogens, Salmonella spp. required biosynthesis of amino acids, LPS, and nucleotides to colonize tomatoes. Overall, however, it appears that while Salmonella shares some strategies with phytopathogens and taps into its animal virulence-related functions, colonization of tomatoes represents a distinct strategy, highlighting this pathogen's flexible metabolism.IMPORTANCE Outbreaks of gastroenteritis caused by human pathogens have been increasingly associated with foods of plant origin, with tomatoes being one of the common culprits. Recent studies also suggest that these human pathogens can use plants as alternate hosts as a part of their life cycle. While dual (animal/plant) lifestyles of other members of the Enterobacteriaceae family are well known, the strategies with which Salmonella colonizes plants are only partially understood. Therefore, we undertook a high-throughput characterization of the functions required for Salmonella persistence within tomatoes. The results of this study were compared with what is known about genes required for Salmonella virulence in animals and interactions of plant pathogens with their hosts to determine whether Salmonella repurposes its virulence repertoire inside plants or whether it behaves more as a phytopathogen during plant colonization. Even though Salmonella utilized some of its virulence-related genes in tomatoes, plant colonization required a distinct set of functions.

Inducible De Novo Biosynthesis of Isoflavonoids in Soybean Leaves by Spodoptera litura Derived Elicitors: Tracer Techniques Aided by High Resolution LCMS.

Isoflavonoids are a characteristic family of natural products in legumes known to mediate a range of plant-biotic interactions. For example, in soybean (Glycine max: Fabaceae) multiple isoflavones are induced and accumulate in leaves following attack by Spodoptera litura (Lepidoptera: Noctuidae) larvae. To quantitatively examine patterns of activated de novo biosynthesis, soybean (Var. Enrei) leaves were treated with a combination of plant defense elicitors present in S. litura gut content extracts and L-α-[13C9, 15N]phenylalanine as a traceable isoflavonoid precursor. Combined treatments promoted significant increases in 13C-labeled isoflavone aglycones (daidzein, formononetin, and genistein), 13C-labeled isoflavone 7-O-glucosides (daidzin, ononin, and genistin), and 13C-labeled isoflavone 7-O-(6″-O-malonyl-β-glucosides) (malonyldaidzin, malonylononin, and malonylgenistin). In contrast levels of 13C-labeled flavones and flavonol (4',7-dihydroxyflavone, kaempferol, and apigenin) were not significantly altered. Curiously, application of fatty acid-amino acid conjugate (FAC) elicitors present in S. litura gut contents, namely N-linolenoyl-L-glutamine and N-linoleoyl-L-glutamine, both promoted the induced accumulation of isoflavone 7-O-glucosides and isoflavone 7-O-(6″-O-malonyl-β-glucosides), but not isoflavone aglycones in the leaves. These results demonstrate that at least two separate reactions are involved in elicitor-induced soybean leaf responses to the S. litura gut contents: one is the de novo biosynthesis of isoflavone conjugates induced by FACs, and the other is the hydrolysis of the isoflavone conjugates to yield isoflavone aglycones. Gut content extracts alone displayed no hydrolytic activity. The quantitative analysis of isoflavone de novo biosynthesis, with respect to both aglycones and conjugates, affords a useful bioassay system for the discovery of additional plant defense elicitor(s) in S. litura gut contents that specifically promote hydrolysis of isoflavone conjugates.

Functional characterization of the first filamentous fungal tRNA-isopentenyltransferase and its role in the virulence of Claviceps purpurea.

In plants, cytokinins (CKs) are synthesized denovo or by the degradation of modified tRNAs. Recently, the first fungal denovo pathway was identified within the plant pathogen Claviceps purpurea. As the deletion of the denovo pathway did not lead to a complete loss of CKs, this work focuses on the tRNA-modifying protein tRNA-isopentenyltransferase (CptRNA-IPT). The contribution of this enzyme to the CK pool of Claviceps and the role of CKs in the host-pathogen interaction are emphasized. The effects of the deletion of cptRNA-ipt and the double deletion of cptRNA-ipt and the key gene of denovo biosynthesis cpipt-log on growth, CK biosynthesis and virulence were analyzed. In addition, the sites of action of CptRNA-IPT were visualized using reporter gene fusions. In addition to CK-independent functions, CptRNA-IPT was essential for the biosynthesis of cis-zeatin (cZ) and contributed to the formation of isopentenyladenine (iP) and trans-zeatin (tZ). Although ΔcptRNA-ipt was reduced in virulence, the 'CK-free' double deletion mutant was nearly apathogenic. The results prove a redundancy of the CK biosynthesis pathway in C.purpurea for iP and tZ formation. Moreover, we show, for the first time, that CKs are required for the successful establishment of a host-fungus interaction.

Denovo fatty acid biosynthesis and elongation in very long-chain acyl-CoA dehydrogenase-deficient mice supplemented with odd or even medium-chain fatty acids.

An even medium-chain triglyceride (MCT)-based diet is the mainstay of treatment in very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD). Previous studies with magnetic resonance spectroscopy have shown an impact of MCT on the average fatty acid chain length in abdominal fat. We therefore assume that medium-chain fatty acids (MCFAs) are elongated and accumulate in tissue as long-chain fatty acids. In this study, we explored the hepatic effects of long-term supplementation with MCT or triheptanoin, an odd-chain C7-based triglyceride, in wild-type and VLCAD-deficient (VLCAD(-/-) ) mice after 1year of supplementation as compared with a control diet. The denovo biosynthesis and elongation of fatty acids, and peroxisomal β-oxidation, were quantified by RT-PCR. This was followed by a comprehensive analysis of hepatic and cardiac fatty acid profiles by GC-MS. Long-term application of even and odd MCFAs strongly induced denovo biosynthesis and elongation of fatty acids in both wild-type and VLCAD(-/-) mice, leading to an alteration of the hepatic fatty acid profiles. We detected denovo-synthesized and elongated fatty acids, such as heptadecenoic acid (C17:1n9), eicosanoic acid (C20:1n9), erucic acid (C22:1n9), and mead acid (C20:3n9), that were otherwise completely absent in mice under control conditions. In parallel, the content of monounsaturated fatty acids was massively increased. Furthermore, we observed strong upregulation of peroxisomal β-oxidation in VLCAD(-/-) mice, especially when they were fed an MCT diet. Our data raise the question of whether long-term MCFA supplementation represents the most efficient treatment in the long term. Studies on the hepatic toxicity of triheptanoin are still ongoing.

Improved method for quantitative analysis of methylated phosphatidylethanolamine species and its application for analysis of diabetic-mouse liver samples.

N-monomethyl phosphatidylethanolamine (MMPE) and N,N-dimethyl phosphatidylethanolamine (DMPE) species are intermediates of phosphatidylcholine (PC) de-novo biosynthesis through methylation of phosphatidylethanolamine (PE). This synthesis pathway for PC is especially important in the liver when choline is deficient in the diet. Despite some efforts focused on the analysis of MMPE and DMPE species, a cost-effective and high-throughput method for determination of individual MMPE and DMPE species, including their regioisomeric structures, is still missing. Therefore we adopted and improved the "mass-tag" strategy for determining these PE-like species by methylating PE, MMPE, and DMPE molecules with deuterated methyl iodide to generate PC molecules with nine, six, and three deuterium atoms, respectively. On the basis of the principles of multidimensional mass-spectrometry-based shotgun lipidomics we could directly identify and quantify these methylated PE species, including their fatty-acyl chains and regiospecific positions. The method provided remarkable sensitivity, with a limit of detection at 0.5 fmol μL(-1), high specificity, and a broad linear-dynamics range of >2500 folds. By applying this method to liver samples from streptozotocin (STZ)-induced diabetic mice and controls, we found that the levels of PC species tended to decrease and the amounts of PE species tended to increase in the liver of STZ-induced diabetic mice compared with controls, but no significant changes in MMPE and DMPE species were determined. However, remodeling of fatty-acyl chains in the determined lipids was observed in the liver of STZ-induced diabetic mice, with reduction in 16:1 and increases in 18:2, 18:1, and 18:0 acyl chains. These results indicated the improved method to be a powerful tool to reveal the function of the PC de-novo biosynthesis pathway through methylation of PE species in biological systems.

Abstract 167: A Cell-Autonomous Role for Endothelial GTP Cyclohydrolase 1 and Tetrahydrobiopterin in Blood Pressure Regulation

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) function and nitric oxide (NO) generation. Augmentation of BH4 levels can prevent eNOS uncoupling and improve endothelial dysfunction in vascular disease states. However, the physiological requirement for de-novo endothelial cell BH4 biosynthesis in eNOS function remains unclear. We generated a novel mouse model with endothelial cell-specific deletion of GCH1, encoding GTP cyclohydrolase 1, an essential enzyme for BH4 biosynthesis, to test the cell-autonomous requirement for endothelial BH4 biosynthesis in vivo. Mice with a floxed GCH1 allele ( GCH1 fl/fl ) were crossed with Tie2cre mice to delete GCH1 in endothelial cells. GCH1 fl/fl Tie2cre mice demonstrated virtually absent NO bioactivity and significantly greater O 2 • - production. GCH1 fl/fl Tie2cre aortas and mesenteric arteries had enhanced vasoconstriction to phenylephrine and impaired endothelium-dependent vasodilatations to acetylcholine and SLIGRL. Endothelium-dependent vasodilatations in GCH1 fl/fl Tie2cre aortas were in part mediated by NOS-derived hydrogen peroxide (H 2 O 2 ), which mediated vasodilatation through soluble guanylate cyclase. Ex vivo supplementation of aortic rings with the BH4 analogue sepiapterin restored normal endothelial function and abolished eNOS-derived H 2 O 2 production in GCH1 fl/fl Tie2cre aortas. GCH1 fl/fl Tie2cre mice had higher systemic blood pressure than wild-type littermates, which was normalised by NOS inhibitor, L-NAME. Taken together, these studies reveal an endothelial cell-autonomous requirement for GCH1 and BH4 in regulation of vascular tone and blood pressure, and identify endothelial cell BH4 as a pivotal regulator of NO vs. H 2 O 2 as alternative eNOS-derived endothelial derived relaxing factors.

Arginine Decarboxylase and Agmatinase: An Alternative Pathway for De Novo Biosynthesis of Polyamines for Development of Mammalian Conceptuses1

Ornithine decarboxylase (ODC1) is considered the rate-controlling enzyme for the classical de novo biosynthesis of polyamines (putrescine, spermidine, and spermine) in mammals. However, metabolism of arginine to agmatine via arginine decarboxylase (ADC) and conversion of agmatine to polyamines via agmatinase (AGMAT) is an alternative pathway long recognized in lower organisms, but only recently suggested for neurons and liver cells of mammals. We now provide evidence for a functional ADC/AGMAT pathway for the synthesis of polyamines in mammalian reproductive tissue for embryonic survival and development. We first investigated cellular functions of polyamines by in vivo knockdown of translation of mRNA for ODC1 in ovine conceptus trophectoderm using morpholino antisense oligonucleotides (MAOs) and found that one-half of the conceptuses were morphologically and functionally either normal or abnormal. Furthermore, we found that increases in ADC/AGMAT mRNA levels and in the translation of AGMAT mRNA among conceptuses in MAO-ODC1 knockdown compensated for the loss of ODC1, supporting polyamine synthesis from arginine and accounting for the normal and abnormal phenotypes of conceptuses. We conclude that the majority of polyamine synthesis is by the conventional ODC1-dependent pathway (arginine-ornithine-putrescine) and that deficiencies in ODC1 result in increased activity of the rescue ADC/AGMAT-dependent pathway (arginine-agmatine-putrescine) for production of polyamines. The presence of an alternative ADC/AGMAT pathway for converting arginine into putrescine is functionally important for supporting survival and development of mammalian conceptuses.

Pathway Assembly and Optimization in E. coli for de Novo Biosynthesis of Resveratrol

Pathway Assembly and Optimization in E. coli for de Novo Biosynthesis of Resveratrol

Metabolic profiling reveals therapeutic biomarkers of processed Aconitum Carmichaeli Debx in treating hydrocortisone induced Kidney-Yang deficiency syndrome rats

Ethnopharmacological relevanceKidney-Yang Deficiency syndrome (KYDS) is a diagnostic pattern in traditional Chinese medicine (TCM) and clinical data showed that the unbalance in adrenal cortical hormone is the key issue in KYDS patients. The processed Ranunculaceae Aconitum carmichaeli debx (Bai-Fu-Pian in Chinese, BFP) is one of the most commonly used Chinese herbs for treating KYDS. The present study was conducted to explore the therapeutic biomarkers of the BFP in treating hydrocortisone administration induced KYDS rats. Materials and methodsThirty male Sprague-Dawley rats were randomly divided into five groups with six in each group. KYDS in rats was induced by i.p. injection of hydrocortisone at the dose of 10mg/kg per day for 15 days as described previously. The rats with KYDS were administered orally, starting from the day of hydrocortisone administration stopped, with BFP extract at the dose of 0.32g/kg, 0.64g/kg and 1.28g/kg per day respectively for 15 days. The blood samples were collected for the liquid chromatography quadruple time-of-flight mass spectrometry (LC-Q-TOF-MS) test, as well as radioimmunoassay to determine the concentrations of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and adrenocorticotrophic hormone (ACTH). The metabolic responses to BFP administration were investigated by using the principal components analysis (PCA) and orthogonal partial least squares analysis (OPLS). Bioinformatics analyses were performed by using the Ingenuity Pathway Analysis (IPA). Variance analysis and linear regression analysis were used in this study. ResultsThe signs and concentrations of cAMP, cGMP and ACTH in the model rats were similar to those previously described about KYDS rats and BFP treatment can reverse the changes. Seventeen significantly changed metabolites among different groups were identified. Thirteen metabolites were identified in the KYDS rats comparing to healthy rats with nine up-regulated and four down-regulated. After BFP treatment at three dosages, five up-regulated metabolites including phosphate, betaine, (4-hydroxyphenyl) acetaldehyde, 5-hydroxyindol-3-acetic acid and 5'-phosphoribosyl-N-formylglycinamide were dose-dependently reversed. The network analysis with IPA showed that four canonical pathways including superpathway of methionine degradation, purine nucleotides De Novo biosynthesis II, tyrosine synthesis and serotonin receptor signaling involved the therapeutic mechanism of BFP in treating the KYDS rats. ConclusionsFive therapeutic biomarkers (phosphate, betaine, (4-hydroxyphenyl) acetaldehyde, 5-hydroxyindol-3-acetic acid and 5'-phosphoribosyl-N-formylglycinamide) and two corresponding canonical pathways (amino acid metabolism and purine nucleotide metabolism) were identified to be involved in the therapeutic mechanism of BFP treating the KYDS.

BI-HEX®-GlymaxX® cells enable efficient production of next generation biomolecules with enhanced ADCC activity

Background Despite the succes story of therapeutic monoclonal antibodies (mAbs), a medical need remains to improve their efficacy. One possibility to achieve this is to modulate important effector functions such as the antibody dependent cellular cytotoxicity (ADCC). The advantage of highly active biotherapeutic molecules is apart from the enhanced efficacy the reduction of side effects due to lower administered doses. Furthermore, these therapeutic antibodies may enable treatment of current nonresponders, e.g. patients with low antigen bearing tumors. Enhancement of the effector functions of antibodies can be achieved either by directly mutating the antibody’s amino acid sequence or by modifying its glycosylation pattern, e.g. by using a novel host cell line able to attach a desired glycostructure to the product. The latter approach has the advantage of not impacting the antibody structure itself, thereby avoiding negative effects on the PK/PD of the molecule. During the last decade it has been shown that antibodies with a reduced level of glycan fucosylation are much more potent in mediating ADCC, a mode of action particularly relevant for cancer therapeutics. Therefore, defucosylated antibodies are of major interest for biotherapeutics developers. To produce such antibodies, Boehringer Ingelheim has inlicensed the GlymaxX system from ProBioGen, Germany. This technology utilises the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) which, when stably integrated into host cell lines, inhibits fucose de-novo biosynthesis. The enzyme deflects the fucosylation pathway by turning an intermediate (GDP-4-Keto-6-Deoxymannose) into GDP-Rhamnose, a sugar that cannot be metabolised by CHO cells. As a consequence, recombinant antibodies generated by such host cells exhibit reduced glycan fucosylation and 20100 fold higher ADCC activity. Here, we show the establishment of a new host cell line, termed BI-HEXGlymaxX which is capable of producing highly active therapeutic antibodies. We furthermore present data on the cell line properties concerning cell culture performance (e.g. titer, growth, transfection efficiency), process robustness and product quality reproducibility.

De Novo Biosynthesis of Tetrahydrobiopterin is Critical in Protecting against Ionizing Radiation

De Novo Biosynthesis of Tetrahydrobiopterin is Critical in Protecting against Ionizing Radiation

Sphingosine 1-Phosphate (S1P) Lyase Deficiency Increases Sphingolipid Formation via Recycling at the Expense of de Novo Biosynthesis in Neurons

Sphingosine 1-phosphate lyase (S1P lyase) irreversibly cleaves sphingosine 1-phosphate (S1P) in the final step of sphingolipid catabolism. As sphingoid bases and their 1-phosphate are not only metabolic intermediates but also highly bioactive lipids that modulate a wide range of physiological processes, it would be predicted that their elevation might induce adjustments in other facets of sphingolipid metabolism and/or alter cell behavior. Indeed, we have previously reported that S1P lyase deficiency causes neurodegeneration and other adverse symptoms. We next asked the question whether and how S1P lyase deficiency affects the metabolism of (glyco)sphingolipids and cholesterol, two lipid classes that might be involved in the neurodegenerative processes observed in S1P lyase-deficient mice. As predicted, there was a considerable increase in free and phosphorylated sphingoid bases upon elimination of S1P lyase, but to our surprise, rather than increasing, the mass of (glyco)sphingolipids persisted at wild type levels. This was discovered to be due to reduced de novo sphingoid base biosynthesis and a corresponding increase in the recycling of the backbones via the salvage pathway. There was also a considerable increase in cholesterol esters, although free cholesterol persisted at wild type levels, which might be secondary to the shifts in sphingolipid metabolism. All in all, these findings show that accumulation of free and phosphorylated sphingoid bases by loss of S1P lyase causes an interesting readjustment of the balance between de novo biosynthesis and recycling to maintain (glyco)sphingolipid homeostasis. These changes, and their impact on the metabolism of other cellular lipids, should be explored as possible contributors to the neurodegeneration in S1P lyase deficiency.

Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD + -Auxotrophic Mutant

NAD (NAD(+)) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD(+) level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD(+) auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. coli mutant YJE001, which had a deletion of the nadC gene responsible for NAD(+) de novo biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD(+). We then constructed the NAD(+)-auxotrophic mutant YJE003 by disrupting the essential gene nadE, which is responsible for the last step of NAD(+) biosynthesis in cells harboring the ntt4 gene. The minimal NAD(+) level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD(+), while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD(+) was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD(+) concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044× and 9.6× those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed.

De Novo Biosynthesis of Fatty Acids Plays Critical Roles in the Response of the Photosynthetic Machinery to Low Temperature in Arabidopsis

The Arabidopsis thaliana kas3 mutant was isolated based on the hypersensitivity of PSII to low temperature using a Chl fluorescence imaging technique. Chl content was lower in kas3 seedlings cultured at 23 degrees C than in the wild type, but PSII activity was only mildly affected. However, after the chilling treatment at 4 degrees C for 7 d, PSII activity was severely impaired in kas3. PSII was more sensitive to light at 4 degrees C in the presence of lincomycin, suggesting that the kas3 mutation accelerates at least the PSII photodamage. The kas3 mutation causes an amino acid alteration in 3-ketoacyl-ACP synthase III (KasIII), leading to the partial loss of the de novo synthesis pathway for fatty acids in plastids. Consequently, the total fatty acid level was reduced to 75% of the wild-type level in kas3 at 23 degrees C and was further reduced to 60% at 4 degrees C. The composition of fatty acids was also slightly affected in kas3 at both 4 and 23 degrees C. Consistent with the results of the electron transport analysis, the chilling treatment also destabilized PsaA and cytochrome (Cyt) f and D1 in kas3. An analysis of double mutants with pgr1 conditionally defective in Cyt b(6)f activity and with var2 defective in FtsH protease suggested that the kas3 mutation has pleiotropic effects on chloroplast function, probably impacting both the Cyt b(6)f activity and translation in chloroplasts at 23 degrees C. The full activity of KasIII is required for the biogenesis of the intact electron transport machinery in thylakoid membranes and is especially important for the process of responding to low temperature.

Novel function of the human presqualene diphosphate phosphatase as a type II phosphatidate phosphatase in phosphatidylcholine and triacylglyceride biosynthesis pathways

Phosphatidate phosphatases, PAPs, are key enzymes in lipid biosynthesis and signaling. Type I PAP enzymes participate in de-novo phospholipid biosynthesis, whereas type II PAP enzymes have an established role in lipid signaling. To identify novel human type II PAPs potentially involved in de-novo phospholipid synthesis we used bioinformatics to screen for enzymes with an active site exposed to the cytosolic side of membranes. Two related enzymes, a novel lipid phosphatase related protein (LPRP-A) and a presqualene diphosphate phosphatase (PA-PSP) met this criterion. PA-PSP and LPRP-A have differential tissue and subcellular distribution, and novel yet differential roles in lipid metabolism. Specifically, PA-PSP, but not LPRP-A, was a potent Mg 2+-independent, NEM-insensitive type II PAP. Subcellular fractionation detection indicated that both proteins were associated with membranes, while immunofluorescent deconvolution imaging revealed that these membranes were exclusively from the nuclear envelope and the endoplasmic reticulum. PA-PSP overexpression, but not LPRP-A, accelerated the synthesis of phosphatidylcholine and caused accumulation of triacylglycerol with concomitant decrease in the rate of phosphatidylinositol synthesis. Coexpression of human CTP:phosphocholine cytidylyltransferase-α with PA-PSP enhanced the effect of PA-PSP on phosphatidylcholine levels, yet attenuated its effect on triacylglycerol. Taken together, our studies provide the first evidence that the eukaryotic, ER-resident PA-PSP is a bifunctional enzyme with specific type II PAP activity, and regulates, in addition to type I PAPs, the de-novo biosynthesis of phospholipids and triacylglycerols.