MucA is a small peptide encoded by an overlapping sequence with cdsA that upregulates the biosynthesis of glycolipid MPIase in the cold.

MPIase is a glycolipid involved in protein insertion into and preprotein translocation across the cytoplasmic membranes of E. coli. MPIase is upregulated in the cold conditions to overcome the cold-sensitive protein export. CdsA, a CDP-diacylglycerol synthase, catalyzes the first reaction in MPIase biosynthesis. An open reading frame for a peptide of 50 amino acids is encoded immediately after ispU, a neighboring upstream gene of cdsA, and overlaps cdsA to a large extent. Mutational analysis revealed that the expression of this peptide is essential for upregulation of MPIase in the cold. Consistently, expression of this peptide in trans resulted in cold upregulation of MPIase. We therefore named this peptide MucA after its function (MPIase upregulation in the cold). When the partially purified MucA was added to the reaction of the intermediate in MPIase biosynthesis, a significant increase in the product formation was observed, supporting the function of MucA. The possible role of MucA in MPIase biosynthesis is discussed.

Metabolomic Evaluation of the Central Metabolic Pathways of Mannosylerythritol Lipid Biosynthesis in Moesziomyces antarcticus T-34.

Moesziomyces antarcticus is a basidiomycetous yeast that produces mannosylerythritol lipids (MELs), which have potential applications as bio-based functional materials in various oleochemical industries, the cosmetics, toiletry, agriculture, and pharmaceutical industries. To better understand the MEL producer, we characterized the central metabolic pathways of M. antarcticus strain T-34 grown on glucose or olive oil via metabolomics. The relative fatty acid content was higher in the cells cultured in olive oil compared to glucose, while the acetyl-CoA content was lower in cells cultured in olive oil. The levels of the tricarboxylic acid cycle metabolites citrate/isocitrate, α-ketoglutarate, and succinate were lower in olive oil compared to glucose, while fumarate and malate levels exhibited the opposite pattern. Pyruvate was not detected in olive oil compared to glucose culture. The levels of glycerol, as well as trehalose, myo-inositol, threitol/erythritol, and mannitol/sorbitol, were higher in olive oil compared to glucose cultures. The ATP level was lower in olive oil compared to glucose culture, although the assimilation of fatty acids produced by digestion of olive oil should promote large amounts of ATP production. The possibility that ATP regeneration by respiratory chain complex promote oil utilization and MEL production in M. antarcticus T-34 was found based on the results of this metabolomic analysis.

Heavy Vacuum Gas Oil Upregulates the Rhamnosyltransferases and Quorum Sensing Cascades of Rhamnolipids Biosynthesis in Pseudomonas sp. AK6U.

We followed a comparative approach to investigate how heavy vacuum gas oil (HVGO) affects the expression of genes involved in biosurfactants biosynthesis and the composition of the rhamnolipid congeners in Pseudomonas sp. AK6U. HVGO stimulated biosurfactants production as indicated by the lower surface tension (26 mN/m) and higher yield (7.8 g/L) compared to a glucose culture (49.7 mN/m, 0.305 g/L). Quantitative real-time PCR showed that the biosurfactants production genes rhlA and rhlB were strongly upregulated in the HVGO culture during the early and late exponential growth phases. To the contrary, the rhamnose biosynthesis genes algC, rmlA and rmlC were downregulated in the HVGO culture. Genes of the quorum sensing systems which regulate biosurfactants biosynthesis exhibited a hierarchical expression profile. The lasI gene was strongly upregulated (20-fold) in the HVGO culture during the early log phase, whereas both rhlI and pqsE were upregulated during the late log phase. Rhamnolipid congener analysis using high-performance liquid chromatography-mass spectrometry revealed a much higher proportion (up to 69%) of the high-molecularweight homologue Rha-Rha-C10-C10 in the HVGO culture. The results shed light on the temporal and carbon source-mediated shifts in rhamonlipids' composition and regulation of biosynthesis which can be potentially exploited to produce different rhamnolipid formulations tailored for specific applications.

UFL1 regulates milk protein and fat synthesis-related gene expression of bovine mammary epithelial cells probably via the mTOR signaling pathway.

UFL1 is an ufmylation (a novel post-translational modification) E3 ligase, mainly located in the endoplasmic reticulum (ER), that has emerged as a significant regulator of several physiological and pathological processes. Yet its physiological function in milk synthesis in bovine mammary epithelial cells (BMECs) remains unknown. In this study, we investigated the effects of UFL1 in milk protein and fat synthesis-related gene expression, with a particular emphasis on the role of UFL1 in LPS-treated BMECs. Results showed that UFL1 depletion significantly reduced the expression of milk protein and fat synthesis-related gene and mTOR phosphorylation in both normal and LPS-treated BMECs. Overexpression of UFL1 enhanced the activation of the mTOR and milk protein and fat synthesis-related gene expression. Collectively, these above results strongly demonstrate that UFL1 could regulate milk protein and fat synthesis-related gene expression of BMECs probably via the mTOR signaling pathway.

Anaerobic biosynthesis of rhamnolipids by Pseudomonas aeruginosa: performance, mechanism and its application potential for enhanced oil recovery.

BACKGROUND: Pseudomonas aeruginosa, the rhamnolipids-producer, is one of dominant bacteria in oil reservoirs. Although P. aeruginosa strains are facultative bacteria, the anaerobic biosynthesis mechanism of rhamnolipids is unclear. Considering the oxygen scarcity within oil reservoirs, revealing the anaerobic biosynthesis mechanism of rhamnolipids are significant for improving the in-situ production of rhamnolipids in oil reservoirs to enhance oil recovery. RESULTS: Pseudomonas aeruginosa SG anaerobically produced rhamnolipids using glycerol rather than glucose as carbon sources. Two possible hypotheses on anaerobic biosynthesis of rhamnolipids were proposed, the new anaerobic biosynthetic pathway (hypothesis 1) and the highly anaerobic expression of key genes (hypothesis 2). Knockout strain SGΔrmlB failed to anaerobically produce rhamnolipids using glycerol. Comparative transcriptomics analysis results revealed that glucose inhibited the anaerobic expression of genes rmlBDAC, fabABG, rhlABRI, rhlC and lasI. Using glycerol as carbon source, the anaerobic expression of key genes in P. aeruginosa SG was significantly up-regulated. The anaerobic biosynthetic pathway of rhamnolipids in P. aeruginosa SG were confirmed, involving the gluconeogenesis from glycerol, the biosynthesis of dTDP-L-rhamnose and ß-hydroxy fatty acids, and the rhamnosyl transfer process. The engineered strain P. aeruginosa PrhlAB constructed in previous work enhanced 9.67% of oil recovery higher than the wild-type strain P. aeruginosa SG enhancing 8.33% of oil recovery. CONCLUSION: The highly anaerobic expression of key genes enables P. aeruginosa SG to anaerobically biosynthesize rhamnolipids. The genes, rmlBDAC, fabABG, rhlABRI, rhlC and lasI, are key genes for anaerobic biosynthesis of rhamnolipid by P. aeruginosa. Improving the anaerobic production of rhamnolipids better enhanced oil recovery in core flooding test. This study fills the gaps in the anaerobic biosynthesis mechanism of rhamnolipids. Results are significant for the metabolic engineering of P. aeruginosa to enhance anaerobic production of rhamnolipids.

MiR-485 targets the DTX4 gene to regulate milk fat synthesis in bovine mammary epithelial cells.

MicroRNAs (miRNAs) are mRNA suppressors that regulate a variety of cellular and physiological processes, including cell proliferation, apoptosis, triglyceride synthesis, fat formation, and lipolysis, by post-transcriptional processing. In previous studies, we isolated and sequenced miRNAs from mammary epithelial cells from Chinese Holstein cows with high and low milk fat percentages. MiR-485 was one of the significantly differentially expressed miRNAs that were identified. In the present study, the relationship between the candidate target gene DTX4 and miR-485 was validated by bioinformatics and real-time fluorescent quantitative PCR (qRT-PCR) and Western blot (WB) analyses in bovine mammary epithelial cells (bMECs). The results indicated that miR-485 negatively regulated the mRNA expression of the target gene DTX4. Furthermore, an shRNA interference vector for the target gene DTX4 was constructed successfully, and it increased the triglyceride content and reduced the cholesterol content of transfected cells. These results suggest that miR-485 may affect the contents of triglycerides (TGs) and cholesterol (CHOL) by targeting the DTX4 gene. This study indicates that miR-485 has a role in regulating milk fat synthesis and that miR-485 targets the DTX4 gene to regulate lipid metabolism in bMECs. These findings contribute to the understanding of the functional significance of miR-485 in milk fat synthesis.

Anti-quorum sensing and antibiofilm potential of 1,8-cineole derived from Musa paradisiaca against Pseudomonas aeruginosa strain PAO1.

Pseudomonas aeruginosa is one of the vulnerable opportunistic pathogens associated with nosocomial infections, cystic fibrosis, burn wounds and surgical site infections. Several studies have reported that quorum sensing (QS) systems are controlled the P. aeruginosa pathogenicity. Hence, the targeting of QS considered as an alternative approach to control P. aeruginosa infections. This study aimed to evaluate the anti-quorum sensing and antibiofilm inhibitory potential of Musa paradisiaca against Chromobacterium violaceum (ATCC 12472) and Pseudomonas aeruginosa. The methanol extract of M. paradisiacsa exhibits that better antibiofilm potential against P. aeruginosa. Then, the crude methanol extract was subjected to purify by column chromatography and collected the fractions. The mass-spectrometric analysis of a methanol extract of M. paradisiaca revealed that 1,8-cineole is the major compounds. 1, 8-cineole significantly inhibited the QS regulated violacein production in C. violaceum. Moreover, 1,8-cineole significantly inhibited the QS mediated virulence production and biofilm formation of P. aeruginosa without affecting their growth. The real-time PCR analysis showed the downregulation of autoinducer synthase and transcriptional regulator genes upon 1,8-cineole treatment. The findings of the present study strongly suggested that metabolite of M. paradisiaca impedes P. aeruginosa QS system and associated virulence productions.

Characterization and application of rhamnolipid from Pseudomonas plecoglossicida BP03.

The production of rhamnolipid (glycolipid) biosurfactant was achieved under optimized conditions from newly isolated bacteria (Pseudomonas plecoglossicida BP03) from rice mill effluent. The isolated biosurfactant was structurally characterized using FTIR and NMR spectroscopic studies. The obtained biosurfactant (1·39 g l-1 ) showed a variety of applications including larvicidal and pupicidal activity against malarial vector (Anopheles sunadicus). It also exhibited antimicrobial activity against human pathogens, and possessed potent anti-biofilm activity against Staphylococcus aureus, Bacillus subtilis and Aeromonas hydrophila. The obtained biosurfactant showed a dose-dependent inhibition of exopolymeric substance (EPS) and growth curve in S. aureus. Furthermore, the cytotoxicity assays revealed that the biosurfactant exhibit a cytotoxic potency against the human fibroblastic sarcoma cells Ht-1080. An in silco analysis was also performed using Schrodinger maestro 9.3 against surface protein (SasG) of S. aureus, and the resultant analysis revealed an interactive docking score of -3·4 kcal mol-1 . The obtained result indicates that the synthesized economically viable biosurfactant ensures excellent applications towards various fields.

Biosynthesis and Export of Bacterial Glycolipids.

Complex carbohydrates are essential for many biological processes, from protein quality control to cell recognition, energy storage, and cell wall formation. Many of these processes are performed in topologically extracellular compartments or on the cell surface; hence, diverse secretion systems evolved to transport the hydrophilic molecules to their sites of action. Polyprenyl lipids serve as ubiquitous anchors and facilitators of these transport processes. Here, we summarize and compare bacterial biosynthesis pathways relying on the recognition and transport of lipid-linked complex carbohydrates. In particular, we compare transporters implicated in O antigen and capsular polysaccharide biosyntheses with those facilitating teichoic acid and N-linked glycan transport. Further, we discuss recent insights into the generation, recognition, and recycling of polyprenyl lipids.

The effect of carbon, nitrogen and iron ions on mono-rhamnolipid production and rhamnolipid synthesis gene expression by Pseudomonas aeruginosa ATCC 15442.

Pseudomonas spp. are the main producers of rhamnolipids. These products have applications in pharmaceuticals, cosmetics, food industry and bioremediation. The biosynthesis of rhamnolipids is influenced by nutrient composition, pH and temperature. In this study, the impact of nutrients on the expression levels of rhamnolipid synthesis genes was evaluated in P. aeruginosa ATCC 15442. Glucose and glycerol were used as carbon sources; while, NaNO3, NH4NO3 and yeast extract/peptone were employed as nitrogen sources. The effect of different concentrations of Fe2+ and Fe3+ on rhamnolipid synthesis genes was also evaluated. Highest biosurfactant production was obtained in minimal medium supplemented with glucose, NaNO3 and Fe2+. Two rhamnolipid synthesis genes, rhlA and rhlB, were amplified with PCR. CapLC ESI-Ion trap-MS/MS detected only mono-rhamnolipid Rha-C10-C10 in the extract. Although similar induction levels were recorded in the presence of 0.05 g/L iron ions, the presence of Fe2+ resulted in higher expression levels than Fe3+ at concentrations equivalent to 0.025 and 0.075 g/L.

Improvement in Production of Rhamnolipids Using Fried Oil with Hydrophilic Co-substrate by Indigenous Pseudomonas aeruginosa NJ2 and Characterizations.

Commercialization of biosurfactant remained a challenge due to lack of structural variation and economical process using low-cost materials and low productivity. Improvement in production of biosurfactant using fried oil with hydrophilic co-substrate by an indigenous strain was studied. Microbe isolated from exhaust chimney condensate was screened for utilization of mixed carbon source and then identified as Pseudomonas aeruginosa NJ2 by 16S rDNA gene sequence. FTIR, HPLC, and NMR analyses confirmed that biosurfactant was rhamnolipids. Batch fermentation using mixed substrates improved the cell growth yield to 1.48 g/L (2.34 times) and product yield to 4.28 g/L (3.4 times) with maximum specific growth rate 0.1 h-1 (two times) and specific production rate 0.5 h-1 (13 times) due to higher cell density and direct synthesis of lipid and rhamnose moieties through central metabolic pathways of the two substrates. Increase in carrying capacity and coefficient value (two times) of logistic equation confirmed the significance of mixed substrates. The biosurfactant showed excellent surface active and thermo-chemical stability properties. Economical production of biosurfactant with high yield and productivity could be possible by isolation of mixed carbon source utilizing strain and optimization of waste substrates from oil/soapstock and sugar/corn syrup industries in media.

Human UDP-galactose 4'-epimerase (GALE) is required for cell-surface glycome structure and function.

Glycan biosynthesis relies on nucleotide sugars (NSs), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease. However, how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, here we examined UDP-galactose 4'-epimerase (GALE), which interconverts two pairs of essential NSs. Using immunoblotting, flow cytometry, and LC-MS-based glycolipid and glycan profiling, we found that CRISPR/Cas9-mediated GALE deletion in human cells triggers major imbalances in NSs and dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the cell-surface death receptor FS-7-associated surface antigen. In particular, we observed substantial decreases in total sialic acid, galactose, and GalNAc levels in glycans. These changes also directly impacted cell signaling, as GALE-/- cells exhibited FS-7-associated surface antigen ligand-induced apoptosis. Our results reveal a role of GALE-mediated NS regulation in death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.

Cytotoxic effects of mono- and di-rhamnolipids from Pseudomonas aeruginosa MR01 on MCF-7 human breast cancer cells.

Rhamnolipids produced by P. aeruginosa MR01 were fractionated into mono- and di-rhamnolipids, and their dominant congeners, Rha-C10-C10 and Rha-Rha-C10-C10, were shown by mass spectrometry. Minimum surface tensions and critical micelle concentrations (CMC) were determined as "≃34 mN/m; ≃26.17 mg/l;" and "≃29 mN/m; ≃29.63 mg/l" for mono- and di-rhamnolipids, respectively. Spectrophotometry measurements provided a close approximation of CMC. Contact angle and diameter of wet area were determined for rhamnolipid-containing drops on hydrophobic paper to display their capability for alteration of surface wettability. Wet area measurement is a simple, reliable method not requiring a Drop Shape Analyzer. Cell viabilities determined by MTT assay showed a decline in a dose-dependent manner and estimated IC50 values were 25.87 µg/ml and 31.00 µg/ml for mono- and di-rhamnolipids treating MCF-7 cells for 48 h. Morphological observations using the inverted phase-contrast microscopy and fluorescence microscopy via Hoechst staining revealed the apoptotic characteristics in treated MCF-7 cells. The semi-quantitative RT-PCR method demonstrated that expression of the p53 gene in mRNA levels significantly (P < 0.05) increased when treated with 30 µg/ml of each rhamnolipid compound for 12 h. It can be concluded that rhamnolipids derived from MR01 show significant anticancer potential against MCF-7 cell line and should be further investigated as natural, therapeutic anti-tumor agents.

Recycling of cooking oil fume condensate for the production of rhamnolipids by Pseudomonas aeruginosa WB505.

Rhamnolipids (RLs) are anionic biosurfactants with great application potential. This study explored the possibility of producing RLs from cooking oil fume condensates (COFCs) collected from range hoods. A mutant of Pseudomonas aeruginosa AB93066 was obtained and used to produce RLs from COFCs as a substrate. RL yields in a 7-L fermenter reached 12.3 g/L, and MALDI-TOF MS showed that Rha2-C10-C10 and Rha-C10-C10 are the most abundant (39.6% and 26.4%, respectively) RL components. The critical micellar concentration (CMC) of the RLs was 45.0 mg/L and the surface tension of water decreased from 60.5 to 25.3 mN/m. Using six kinds of common hydrocarbons as indices, the emulsification coefficients of the RLs obtained were found to exceed 60%; in particular, the emulsification coefficient for benzene was 80.3%. COFCs provide an inexpensive alternative as a substrate for RL production, and the synthetic process is relatively harmless and economical.

Identification of a novel loss-of-function mutation of the GLA gene in a Chinese Han family with Fabry disease.

BACKGROUND: Fabry disease is an X-linked recessive lysosomal disorder caused by deficient enzymatic activity of α-galactosidase A (α-Gal A). The insufficient enzymatic activity leads to excessive accumulation of glycosphingolipids, the substrates of the enzyme, in lysosomes in organs and tissues. Mutations in the α-Gal A gene (GLA, Xq22) have been proven to be responsible for Fabry disease. METHODS: In this study, we report a four-generation pedigree with left ventricular hypertrophy and chronic renal failure that was diagnosed by sequencing the GLA gene. An over expression system was constructed to evaluate the function of the detected mutation. RESULTS: We identified a novel mutation in exon 6 of the GLA gene, p.Asn278Lys, which completely co-segregated with the disease phenotype. The protein level of α-Gal A was significantly lower in the variant group than in the wild-type group; additionally, the pharmacological chaperone 1-deoxy-galactonojirimycin (DGJ) effectively normalized the enzyme activity of α-Gal A and its decline at the protein level. CONCLUSIONS: This study is the first to report a novel loss-of-function mutation, p.Asn278Lys, in exon 6 of the GLA gene as a genetic aetiology for Fabry disease. In addition, we analysed the feasibility of DGJ as a therapeutic approach for this particular GLA mutation.

Modification of membrane lipid compositions in single-celled organisms - From basics to applications.

All intact cells, and their organelles, are surrounded by a ∼30 Šhydrophobic film that typically separates the interior from the environment. This film is composed of lipid bilayers that form from a pool of structurally highly diverse, amphipathic lipids. The specific composition and nature of these lipids strongly contributes to many different processes in the cell by influencing membrane structures, membrane protein sorting and functionalities. In this review, we discuss strategies to alter membrane lipid compositions of organelles and plasma membranes in different organisms, focusing on microbial cells. Reflecting the many essential roles of lipids in cellular regulation, we delineate diverse cellular processes affected by membrane lipid modifications and discuss possible applications in a biotechnological and biomedical context. A major motivation for membrane lipid engineering has been the improvement of expression, translocation and activity of heterologous membrane proteins, which can facilitate the biochemical and structural characterization of this challenging class of proteins. Additionally, better expression of membrane proteins or membrane lipid engineering - or a combination of both - led to improved production of high-value compounds and food additives, e.g. polyunsaturated fatty acids and glycolipids, in diverse hosts. More recently it has been shown that diverse cellular pathologies such as cancer and Alzheimer's disease are associated with lipid alterations. Hence, the progress in our understanding of membrane structure, function and protein-lipid interactions, and the resulting possibilities regarding the engineering of membrane lipid composition clearly enable novel nutraceutical and pharmaceutical interventions to be developed. Significant progress in this important area of research is highlighted in this review.

MPDU1 regulates CEACAM1 and cell adhesion in vitro and in vivo.

N-linked glycosylation (NLG) is a co-translational modification that is essential for the folding, stability, and trafficking of transmembrane (TM) and secretory glycoproteins. Efficient NLG requires the stepwise synthesis and en bloc transfer of a 14-sugar carbohydrate known as a lipid-linked oligosaccharide (LLO). The genetics of LLO biosynthesis have been established in yeast and Chinese hamster systems, but human models of LLO biosynthesis are lacking. In this study we report that Kato III human gastric cancer cells represent a model of deficient LLO synthesis, possessing a homozygous deletion of the LLO biosynthesis factor, MPDU1. Kato III cells lacking MPDU1 have all the hallmarks of a glycosylation-deficient cell line, including altered sensitivity to lectins and the formation of truncated LLOs. Analysis of transcription using an expression microarray and protein levels using a proteome antibody array reveal changes in the expression of several membrane proteins, including the metalloprotease ADAM-15 and the cell adhesion molecule CEACAM1. Surprisingly, the restoration of MPDU1 expression in Kato III cells demonstrated a clear phenotype of increased cell-cell adhesion, a finding that was confirmed in vivo through analysis of tumor xenografts. These experiments also confirmed that protein levels of CEACAM-1, which functions in cell adhesion, is dependent on LLO biosynthesis in vivo. Kato III cells and the MPDU1-rescued Kato IIIM cells therefore provide a novel model to examine the consequences of defective LLO biosynthesis both in vitro and in vivo.

Targeting the alternative sigma factor RpoN to combat virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that infects immunocompromised and cystic fibrosis patients. Treatment is difficult due to antibiotic resistance, and new antimicrobials are needed to treat infections. The alternative sigma factor 54 (σ54, RpoN), regulates many virulence-associated genes. Thus, we evaluated inhibition of virulence in P. aeruginosa by a designed peptide (RpoN molecular roadblock, RpoN*) which binds specifically to RpoN consensus promoters. We expected that RpoN* binding to its consensus promoter sites would repress gene expression and thus virulence by blocking RpoN and/or other transcription factors. RpoN* reduced transcription of approximately 700 genes as determined by microarray analysis, including genes related to virulence. RpoN* expression significantly reduced motility, protease secretion, pyocyanin and pyoverdine production, rhamnolipid production, and biofilm formation. Given the effectiveness of RpoN* in vitro, we explored its effects in a Caenorhabditis elegans-P. aeruginosa infection model. Expression of RpoN* protected C. elegans in a paralytic killing assay, whereas worms succumbed to paralysis and death in its absence. In a slow killing assay, which mimics establishment and proliferation of an infection, C. elegans survival was prolonged when RpoN* was expressed. Thus, blocking RpoN consensus promoter sites is an effective strategy for abrogation of P. aeruginosa virulence.

Simultaneous valorization and biocatalytic upgrading of heavy vacuum gas oil by the biosurfactant-producing Pseudomonas aeruginosa AK6U.

Heavy vacuum gas oil (HVGO) is a complex and viscous hydrocarbon stream that is produced as the bottom side product from the vacuum distillation units in petroleum refineries. HVGO is conventionally treated with thermochemical process, which is costly and environmentally polluting. Here, we investigate two petroleum biotechnology applications, namely valorization and bioupgrading, as green approaches for valorization and upgrading of HVGO. The Pseudomonas aeruginosa AK6U strain grew on 20% v/v of HVGO as a sole carbon and sulfur source. It produced rhamnolipid biosurfactants in a growth-associated mode with a maximum crude biosurfactants yield of 10.1 g l-1 , which reduced the surface tension of the cell-free culture supernatant to 30.6 mN m-1 within 1 week of incubation. The rarely occurring dirhamnolipid Rha-Rha-C12 -C12 dominated the congeners' profile of the biosurfactants produced from HVGO. Heavy vacuum gas oil was recovered from the cultures and abiotic controls and the maltene fraction was extracted for further analysis. Fractional distillation (SimDist) of the biotreated maltene fraction showed a relative decrease in the high-boiling heavy fuel fraction (BP 426-565 °C) concomitant with increase in the lighter distillate diesel fraction (BP 315-426 °C). Analysis of the maltene fraction revealed compositional changes. The number-average (Mn) and weight-average (Mw) molecular weights, as well as the absolute number of hydrocarbons and sulfur heterocycles were higher in the biotreated maltene fraction of HVGO. These findings suggest that HVGO can be potentially exploited as a carbon-rich substrate for production of the high-value biosurfactants by P. aeruginosa AK6U and to concomitantly improve/upgrade its chemical composition.

Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats.

To investigate responses of milk protein synthesis and mammary AA metabolism to a graded decrease of postruminal Lys supply, 4 lactating goats fitted with jugular vein, mammary vein, and carotid artery catheters and transonic blood flow detectors on the external pudic artery were used in a 4 × 4 Latin square experiment. Goats were fasted for 24 h and then received a 9-h intravenous infusion of an AA mixture plus glucose. Milk yield was recorded and samples were taken in h 2 to 8 of the infusion period; a mammary biopsy was performed in the last hour. Treatments were graded decrease of lysine content in the infusate to 100 (complete), 60, 30, or 0% as in casein. Lysine-removed infusions linearly decreased milk yield, tended to decrease lactose yield, and tended to increase milk fat to protein ratio. Milk protein content and yield were linearly decreased by graded Lys deficiency. Mammary Lys uptake was concomitantly decreased, but linear regression analysis found no significant relationship between mammary Lys uptake and milk protein yield. Treatments had no effects on phosphorylation levels of the downstream proteins measured in the mammalian target or rapamycin pathway except for a tended quadratic effect on that of eukaryotic initiation factor 2, which was increased and then decreased by graded Lys deficiency. Removal of Lys from the infusate linearly increased circulating glucagon and glucose. Removal of Lys from the infusate linearly decreased arterial and venous concentrations of Lys. Treatments also had a significant quadratic effect on venous Lys, suggesting mechanisms to stabilize circulating Lys at a certain range. The 2 infusions partially removing Lys resulted in a similar 20% decrease, whereas the 0% Lys infusion resulted in an abrupt 70% decrease in mammary Lys uptake compared with that of the full-AA mixture infusion. Consistent with the abrupt decrease, mammary Lys uptake-to-output ratio decreased from 2.2 to 0.92, suggesting catabolism of Lys in the mammary gland could be completely prevented when the animal faced severe Lys deficiency. Mammary blood flow was linearly increased, consistent with the linearly increased circulating nitric oxide by graded Lys deficiency, indicating mechanisms to ensure the priority of the mammary gland in acquiring AA for milk protein synthesis. Infusions with Lys removed increased mammary clearance rate of Lys numerically by 2 to 3 fold. In conclusion, the decreased milk protein yield by graded Lys deficiency was mainly a result of the varied physiological status, as indicated by the elevated circulating glucagon and glucose, rather than a result of the decreased mammary Lys uptake or depressed signals in the mTOR pathway. Mechanisms of Lys deficiency to promote glucagon secretion and mammary blood flow and glucagon to depress milk protein synthesis need to be clarified by future studies.