Robust and flexible continuous unit operations that enable the establishment of intensified bioprocesses is one of the most relevant trends in manufacturing of biopharmaceuticals, including virus-based products. Sulfated cellulose membrane adsorbers (SCMA) are one of the most promising matrices for chromatographic purification of virus particles, like influenza viruses. Here, a three 'column' periodical counter current set-up was used to continuously purify influenza A/PR/8/34 virus particles using SCMA in bind-elute mode. It was possible to recover 67.4% of the HA-activity and to remove 67.4% and 99.8% of the total protein and DNA, respectively. The performance of the continuous process operated over a total of 10 loops, was slightly inferior to was obtained in a comparable batch process. Nevertheless, it was possible to increase the effective usage of binding capacity to 80%, resulting on a productivity of 22.8 kHAU mlmemb-1 min-1. As a proof-of-principle, SCMA were successfully used as matrix for purification of cell-derived influenza virus particles, in continuous mode.

Pseudomonas aeruginosa is an important chassis for production of polyhydroxyalkanoates (PHA) and rhamnolipids (RHL). Advances in the understanding of the biosynthesis metabolism of these biocompounds are crucial for increasing yield. 13C-Metabolic Flux Ratio Analysis (13C-MFA) is a technique to estimate in vivo metabolic fluxes ratios. PHA and RHL are essentially non-growth associated products of biotechnological interest and both contain hydroxyalkanoates (HAs), whose labeling patterns could be accessed by GC-MS. In this study, to reveal the relative contributions of the Entner-Doudoroff (ED) pathway and the non-oxidative Pentose Phosphate (PP) pathway to PHA and RHL production, 13C-MFA was performed in Pseudomonas aeruginosa LFM634 when supplied with labeled glucose. This bacterial strain lacks both functional EMP and the oxidative PP branch. Labeling patterns in HAs were measured. Experiments with [U-13C] glucose indicated a low flux though PP pathway. An optimal design of labeling experiment showed that [6-13C] glucose would be the best substrate to enable an estimation of the ED flux with high accuracy. Results of experiments performed with this isotope indicated that about two-thirds of glyceraldehyde 3-phosphate is recycled through a cyclic ED architecture, suggesting that P. aeruginosa utilizes that cycle to regulate the NADPH/Acetyl-CoA ratio for PHA and RHL biosynthesis.

Clostridium beijerinckii population branches into metabolically diverse cell types in batch cultures. Here, we present a new kinetic model of C. beijerinckii's Acetone-Butanol-Ethanol fermentation that considers three cell types: producers of acids (acidogenic), consumer of acids and producers of solvents (solventogenic), and spores cells. The model accurately recapitulates batch culture data. Also, the model estimates cell type-specific kinetic parameters, which can be helpful to improve the operation of the ABE fermentation and give a framework to study acidogenic and solventogenic metabolic pathways. To exemplify the latter, we used a constraint-based model to study how the ABE pathways are used among acidogenic and solventogenic cell types. We found that among both cell types, glycolytic production of ATP and consumption of NAD+ varies widely during the fermentation, with their maximum production/consumption rates happening when acidogenic and solventogenic growth rates were at their highest. However, acidogenic cells use the ABE pathway to contribute with an extra 12.5% of the total production of ATP, whereas solventogenic cell types use the ABE pathway to supply more than 75% of the demand for NAD+, alternating between the production of lactate and butyrate, being both coupled to the production of NAD+.

Today, there is a great interest in using astaxanthin due to its potential health advantages. Application of different types of nanoparticles (NPs) as stress agents to enhance astaxanthin production in Haematococcus pluvialis, a microalgae strain, has been reported in the literature. In this study, the effect of different concentrations of zinc oxide (ZnO) NPs on the enhancement of astaxanthin production in H. pluvialis was investigated. First, ZnO NPs were synthesized from zinc nitrate as the precursor and sodium hydroxide (chemical method), and peel extract of pomegranate (green method) as reducing agents. To study the cell viability and stimulate the astaxanthin production, H. pluvialis cells were exposed to the different concentrations (i.e. 50, 100, 200, and 400 μg.ml-1) of ZnO NPs. The synthesized powders were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and dynamic light scattering (DLS) methods. The characterization results showed that the pure ZnO NPs were successfully synthesized via both methods with uniform particle size distribution. But, the average particle size of the green synthesized ZnO NPs (about 30 nm) was smaller than that of the chemically synthesized ones (about 80 nm). Maximum astaxanthin production (~ 20 mg.g-1 of dry biomass of H. pluvialis) was achieved at 100 μg.ml-1 of green synthesized ZnO NPs exposure to the H. pluvialis in comparison with the control culture after 15 days. However, ZnO NPs concentration above 200 μg.ml-1 was toxic to the microalgae. From these results, it can be concluded that a specific amount of ZnO NPs could be considered as a worthy candidate for the enhancement of astaxanthin production in H. pluvialis.

Recombinant Pichia pastoris biomass surface-expressing the viral binding protein PmRab7 (YSD-PmRab7) was prepared by fed-batch, aerobic fermentation with methanol induction for 48 h. By cell based ELISA assay, immunofluorescence and flow cytometry, 45% of the YSD-PmRab7 cells were positive for PmRab7. Freeze dried YSD-PmRab7 cells were added to formulated shrimp feed pellets at 0.25 g and 0.5 g per g feed and fed to 2 shrimp groups for 7 days prior to challenge with white spot syndrome virus (WSSV). Controls consisted of 1 shrimp group fed normal pellets and one fed pellets containing P. pastoris carrying an empty gene cassette. At 10 days post challenge, survival in the two control groups was 6.7 ± 6.6%, while it was 26.7 ± 6.6% in the 0.25 g YSD-PmRab7 group and significantly higher (p < 0.05) in the 0.5 g YSD-PmRab7 group at 46.7 ± 10.1%. Nested PCR assays and histopathological analysis revealed significantly lower WSSV replication levels in the 0.5 g YSD-PmRab7 group. The results indicated potential for development of YSD-PmRab7 cells as an oral prophylactic against WSSV in shrimp.

The dopamine transporter (DAT) is targeted in substance use disorders (SUDs), and "non-classical"" DAT inhibitors with low abuse potential are therapeutic candidates. Lobinaline, from Lobelia cardinalis, is an atypical DAT inhibitor lead. Chemical synthesis of lobinaline is challenging; thus, "target-directed evolution" was used for lead optimization. A target protein is expressed in plant cells, and a mutant cell population is selected under conditions where target protein functional inhibition confers a survival advantage. Surviving mutants are "mined" for the targeted activity. Applied to a mutant L. cardinalis cell population expressing the human DAT, we identified 20 mutants overproducing DAT inhibitors. Microanalysis prioritized novel lobinaline derivatives, and we first investigated the more water-soluble lobinaline N-oxide. It inhibited rat synaptosomal [3H]DA uptake with an IC50 similar to lobinaline. Against repeated DA microinjections into the rat striatum, lobinaline produced transient DA clearance reductions. In contrast, lobinaline N-oxide prolongingly increased DA peak amplitudes, particularly in the ventral striatum. Lobinaline N-oxide also produced complex changes in post-peak DA clearance inconsistent with simple DAT inhibition. This unusual DAT interaction may prove therapeutically useful for treating SUDs. This study demonstrates the value of target-directed evolution of plant cells for optimizing lead compounds difficult to synthesize chemically.

Polycyclic aromatic hydrocarbon (PAH) contamination has a negative impact on ecosystems. PAHs are a large group of toxins with two or more benzene rings that are persistent in the environment. Some PAHs can be cytotoxic, teratogenic, and/or carcinogenic. In the bacterium Pseudomonas, PAHs can be modified by dioxygenases, which increase the reactivity of PAHs. We hypothesize that some plant dioxygenases are capable of PAH biodegradation. Herein, we investigate the involvement of Arabidopsis thaliana At1g14130 in the degradation of phenanthrene, our model PAH. The At1g14130 gene encodes Dioxygenase For Auxin Oxidation 1 (AtDAO1), an enzyme involved in the oxidative inactivation of the hormone auxin. Expression analysis using a β-glucuronidase (GUS) reporter revealed that At1g14130 is prominently expressed in new leaves of plants exposed to media with phenanthrene. Analysis of the oxidative state of gain-of-function mutants showed elevated levels of H2O2 after phenanthrene treatments, probably due to an increase in the oxidation of phenanthrene by AtDAO1. Biochemical assays with purified AtDAO1 and phenanthrene suggest an enzymatic activity towards the PAH. Thus, data presented in this study support the hypothesis that an auxin dioxygenase, AtDAO1, from Arabidopsis thaliana contributes to the degradation of phenanthrene and that there is possible toxic metabolite accumulation after PAH exposure.

The present work evaluates the immobilization of LipC12 on different supports in tandem with bioimprinting technique, in order to improve its activity and stability in organic medium. Oleic acid was selected as the bioimprinting molecule. The immobilized LipC12 was applied in the synthesis of pentyl oleate by esterification reaction and in the production of fatty acids, mono, and diglycerides via hydrolysis of triacylglycerols, in n-heptane reaction media. For all immobilized lipase preparations, an increase in the conversion of oleic acid to pentyl oleate was observed when immobilization in tandem with bioimprinting treatment was carried out versus immobilization without bioimprinting. The highest conversions were achieved using LipC12 immobilized on hydrophobic supports. The reuse potential of the immobilized preparations was evaluated. The preparations were used in eight successive cycles of esterification reactions and the best results were obtained for LipC12 immobilized on Immobead 150 and chitosan. The activity for the hydrolysis of soybean oil was improved by bioimprinting treatment only for LipC12 immobilized on commercial polypropylene and Accurel MP-1000. LipC12 immobilized on hydrophilic supports or on Immobead150 could be used to hydrolyze tricaprylin to obtain diglycerides with a high proportion of 1,2-diglycerides in reaction times as short as 30 min.

The screening, identification, and study of the functional properties of cellulolytic xylanolytic bacteria are crucial for the construction of applicable bioprocesses. The thermophilic facultatively anaerobic, xylanolytic bacterial strain DA-C8 (=JCM34211=DSM111723) exhibiting efficient xylan degradation was newly isolated from compost. Strain DA-C8 completely degraded 1% beechwood xylan within 4 days under anaerobic conditions. By 16S rRNA gene sequence homology and phylogenetic tree analysis, strain DA-C8 was closely related to Paenibacillus cisolokensis and Xylanibacillus composti; however, the average nucleotide identity and digital DNA-DNA hybridization values based on genome information and the carbon source utilization properties indicated that strain DA-C8 belongs to Paenibacillus rather than Xylanibacillus. The gene numbers of xylanase and endoglucanase of strain DA-C8 and X. composti were not different; however, strain DA-C8 had higher abundance of α-L-arabinofuranosidase, β-xylosidase, and β-glucosidase than X. composti. Strain DA-C8 showed decreased xylan and corn hull degradation abilities and growth on xylan medium under aerobic conditions. Quantitative PCR showed high expression of xylan and cellulose degradation genes under anaerobic conditions, but the genes were repressed under aerobic conditions, indicating that strain DA-C8 controls polysaccharide degradation depending on the aeration conditions. Strain DA-C8 is a new species of Paenibacillus with a unique polysaccharide degradation system.

Hair graying is processed by the inactivation of tyrosinase caused by the accumulation of oxidative stress and a decrease in the number of melanocytes. Therefore, the purpose of this study was to investigate the effect of SIRT1 gene knockout using the CRISPR/Cas9 system on the protein and gene expressions related to melanogenesis. In this study, the mutation in the SIRT1 knockout(KO) gene was verified by T7EI assay and Sanger DNA sequencing. Furthermore, the expression levels of SIRT1 protein and gene in KO cells were remarkably decreased compared with normal cells. Therefore, the SIRT1 gene KO cell line was successfully established for further study. The KO cells also increased SA-β-galactosidase and decreased melanin production and the scavenging activity of hydrogen peroxide. In particular, the down-regulation of p38 and c-kit as well as the up-regulation of ERK resulted in the inactivation of MITF in the KO cells. Thus, KO cells reduced the expressions of Tyrosinase, Tyrosine hydroxylase, TRP-1 and TRP-2 through the negative modulation of MITF. Furthermore, SIRT1 gene KO cells negatively modulated antioxidant proteins such as Catalase, MnSOD, MsrA and MsrB3 through FOXO1 and Keap1. Therefore, it is suggested that SIRT1 could play a positive role in melanogenesis via MITF and FOXO1.

The present study demonstrates biotechnological applications of the lichen Pleurosticta acetabulum, specifically the production of large amounts of hydrogen even after the lichen exposure to extreme conditions such as a) extreme UVB radiation (1.7 mW/cm2 = 1000 J m-2 min-1) over different time periods (4, 20 & 70 h) and b) combined exposure of the lichen to high intensity UVB radiation and extreme low (-196 °C) or extreme high temperatures (+70 °C). The results highlight that the extremophilic and polyextremophilic behavior of lichens both in dehydrated and in regenerated form, under extreme conditions not necessarily recorded on earth, is compatible with their biotechnological uses. The lichen viability was measured using fluorescence induction techniques (OJIP-test), which record changes in the molecular structure and function of the photosynthetic mechanism, while its ability to produce molecular hydrogen was measured through thermal conductivity gas chromatography (GC-TCD) analysis. Hydrogen is a promising fuel for the future. The exciting result of a lichen micro-ecosystem is its ability to expel its moisture and remain in an inactive state, protecting itself from extreme conditions and maintaining its ability to high yield hydrogen production in a closed system, with the sole addition of water and without the need for additional energy. Our results expand the potential use of lichens for future biotechnological applications in extreme Earth environments, but also in environments on other planets, such as Mars, thus paving the way for astrobiotechnological applications.

GR15 is a short molecule or peptide composed of aliphatic amino acids and possesses to have antioxidant properties. The GR15, 1GGGAFSGKDPTKVDR15 was identified from the protein S-adenosylmethionine synthase (SAMe) expressed during the sulfur departed state of Arthrospira platensis (spirulina or cyanobacteria). The in-silico assessment and the structural features of GR15 showed its antioxidant potency. Real-time PCR analysis found the up-regulation of ApSAMe expression on day 15 against oxidative stress due to 10 mM H2O2 treatment in A. platensis (Ap). The antioxidant activity of GR15 was accessed by the cell-free antioxidant assays such as ABTS, SARS, HRAS and NO; the results showed dose-dependent antioxidant activity. The toxicity assay was performed in both in vitro and in vivo models, in which peptide does not exhibit any toxicity in MDCK cell and zebrafish embryos. The intercellular ROS reduction potential of GR15 peptide was also investigated in both in vitro and in vivo models including LDH assay, antioxidant enzymes (SOD and CAT), and fluorescent staining assay (DCFDA, Hochest and Acridine orange sting) was performed; the results showed that the GR15 peptide was effectively reduced the ROS level. Further, RT-PCR demonstrated that GR15 enhanced the antioxidant property and also up-regulated the antioxidant gene, thus reduced the ROS level in both in vitro and in vivo models. Based on the results obtained from this study, we propose that GR15 has the potential antioxidant ability; hence further research can be directed towards the therapeutic product or drug development against disease caused by oxidative stress.

The production of polyhydroxyalkanoates (PHAs) from waste cooking oil (WCO) by a mixed culture was investigated in the present study at increasing WCO concentrations, temperature and ammonium availability. The PHA production was done in two steps: in the first step, a mixed culture was enriched in PHA-accumulating bacteria from activated sludge in a sequencing batch reactor operated in a feast-famine mode and in the second step the PHA accumulation by the enriched mixed culture was assessed in a batch reactor. In the enrichment step, two substrates, WCO and nonanoic acid were used for enrichment and in the PHA accumulation step only WCO was used. It was not possible to enrich a mixed culture in PHA-accumulating bacteria using WCO as substrate due to the development of filamentous bacteria causing foam formation and bulking in the reactor. However, our results showed that the mixed culture continuously fed with nonanoic acid was enriched in PHA-accumulating bacteria. This enriched culture accumulated both scl- and mcl-PHA using WCO as substrate. The maximum PHA accumulation capacity of this mixed culture from WCO was 38.2% cdw. Increasing the temperature (30-40 ℃) or WCO concentrations (5-20 g/l) increased the PHA accumulation capacity of the mixed culture and the ratios of scl-PHA to mcl-PHA. The presence of ammonium increased PHA accumulation (21.9% cdw) compared to the complete absence of ammonium (5.8% cdw). The thermal characterization of the PHA exhibited the advantageous properties of both scl- and mcl-PHA, i.e., higher melting temperature (152-172 ℃) similar to scl-PHA and a lower degree of crystallinity (12%) similar to mcl-PHA. This is the first study to report the potential of open mixed culture to produce scl- and mcl-PHA from WCO and thus contributing to the understanding of sustainable polymer production.

Indole alkaloid camalexin has potential medicinal properties such as suppressing the viability of leukemic but not normal cells. Camalexin is not produced in plants and an external factor is required to activate its biosynthesis. In this work, we stimulated camalexin biosynthesis in Arabidopsis calli by blocking one of repressors of the jasmonate pathway, the jasmonate ZIM-domain protein 1 (JAZ1) by using amiRNA targeting JAZ1 gene transcripts. Inhibition of the JAZ1 gene led to an increase in camalexin content from trace amounts in control culture to 9 µg/g DW in the jaz1 line without affecting growth. In addition, JAZ1 silencing enhanced tolerance to cold stress with simultaneous increasing camalexin content up to 30 µg/g DW. Real-time quantitative PCR determination of marker gene expression showed that effects caused by the JAZ1 silencing might be realized through crosslinking JA, ROS, and abscisic acid signaling pathways. Thus, targeting the distal components of signaling pathways can be suggested as a tool for bioengineering of secondary metabolism, along with standard techniques for targeting biosynthetic genes or genes encoding transcription factors.