Simultaneous Improvements of Pseudomonas Cell Growth and Polyhydroxyalkanoate Production from a Lignin Derivative for Lignin-Consolidated Bioprocessing.

Cell growth and polyhydroxyalkanoate (PHA) biosynthesis are two key traits in PHA production from lignin or its derivatives. However, the links between them remain poorly understood. Here, the transcription levels of key genes involved in PHA biosynthesis were tracked in Pseudomonas putida strain A514 grown on vanillic acid as the sole carbon source under different levels of nutrient availability. First, enoyl-coenzyme A (CoA) hydratase (encoded by phaJ4) is stress induced and likely to contribute to PHA synthesis under nitrogen starvation conditions. Second, much higher expression levels of 3-hydroxyacyl-acyl carrier protein (ACP) thioesterase (encoded by phaG) and long-chain fatty acid-CoA ligase (encoded by alkK) under both high and low nitrogen (N) led to the hypothesis that they likely not only have a role in PHA biosynthesis but are also essential to cell growth. Third, 40 mg/liter PHA was synthesized by strain AphaJ4C1 (overexpression of phaJ4 and phaC1 in strain A514) under low-N conditions, in contrast to 23 mg/liter PHA synthesized under high-N conditions. Under high-N conditions, strain AalkKphaGC1 (overexpression of phaG, alkK, and phaC1 in A514) produced 90 mg/liter PHA with a cell dry weight of 667 mg/liter, experimentally validating our hypothesis. Finally, further enhancement in cell growth (714 mg/liter) and PHA titer (246 mg/liter) was achieved in strain Axyl_alkKphaGC1 via transcription level optimization, which was regulated by an inducible strong promoter with its regulator, XylR-PxylA, from the xylose catabolic gene cluster of the A514 genome. This study reveals genetic features of genes involved in PHA synthesis from a lignin derivative and provides a novel strategy for rational engineering of these two traits, laying the foundation for lignin-consolidated bioprocessing.IMPORTANCE With the recent advances in processing carbohydrates in lignocellulosics for bioproducts, almost all biological conversion platforms result in the formation of a significant amount of lignin by-products, representing the second most abundant feedstock on earth. However, this resource is greatly underutilized due to its heterogeneity and recalcitrant chemical structure. Thus, exploiting lignin valorization routes would achieve the complete utilization of lignocellulosic biomass and improve cost-effectiveness. The culture conditions that encourage cell growth and polyhydroxyalkanoate (PHA) accumulation are different. Such an inconsistency represents a major hurdle in lignin-to-PHA bioconversion. In this study, we traced and compared transcription levels of key genes involved in PHA biosynthesis pathways in Pseudomonas putida A514 under different nitrogen concentrations to unveil the unusual features of PHA synthesis. Furthermore, an inducible strong promoter was identified. Thus, the molecular features and new genetic tools reveal a strategy to coenhance PHA production and cell growth from a lignin derivative.

Culturable fungal diversity and cellulase production by mixed culture Aspergillus fungi from Sanya mangrove.

Fungi are the most suitable cellulase producers attributing to its ability to produce a complete cellulase system. 33 Genus, 175 Species fungi were isolated from Sanya mangrove, Hainan, China. Using congo red cellulose (CMC) medium, five fungi of cellulose-degrading were selected for further study. Molecular biology and morphological identification showed that all of these five fungi belong to Aspergillus fungi. The cellulase produced by these fungi were monitored during liquid state fermentation. The optimum conditions study for enzyme production illustrated that the highest activities appeared at pH 3.0, 35°C after fermentation for 3 days. Beyond that, the enzyme activity of mixed fungi is 11-26% higher than pure. The study demonstrated that mixed culture improved the hydrolysis of fungi cellulase.

Targeted therapy of brain ischaemia using Fas ligand antibody conjugated PEG-lipid nanoparticles.

The translation of experimental stroke research from the laboratory to successful clinical practice remains a formidable challenge. We previously reported that PEGylated-lipid nanoparticles (PLNs) effectively transport across the blood-brain barrier along with less inflammatory responses. In the present study, PLNs conjugated to Fas ligand antibody that selectively present on brain ischaemic region were used for therapeutic targeting. Fluorescent analysis of the mice brain show that encapsulated 3-n-Butylphthalide (dl-NBP) in PLNs conjugated with Fas ligand antibody effectively delivered to ipsilateral region of ischaemic brain. Furthermore, the confocal immunohistochemical study demonstrated that brain-targeted nanocontainers specifically accumulated on OX42 positive microglia cells in ischaemic region of mice model. Finally, dl-NBP encapsulated nano-drug delivery system is resulted in significant improvements in brain injury and in neurological deficit after ischaemia, with the significantly reduced dosages versus regular dl-NBP. Overall, these data suggests that PLNs conjugated to an antibody specific to the Fas ligand constituted an ideal brain targeting drug delivery system for brain ischaemia.

The effect of lipid nanoparticle PEGylation on neuroinflammatory response in mouse brain.

Nanocarrier-based drug delivery systems have attracted wide interest for the treatment of brain disease. However, neurotoxicity of nanoparticle has limited their therapeutic application. Here we demonstrated that lipid nanoparticles (LNs) accumulated in the brain parenchyma within 3 h of intravenous injection to mice and persisted for more than 24 weeks, coinciding with a dramatic activation of brain microglia. Morphological characteristic of microglial activation also observed in LNs-treated Cx3cr1GFP/+ mice. In vivo study with two-photon confocal microscopy revealed abnormal Ca²âº waves in microglia following LNs injection. The correlated activation of caspase-1, IL-1ß and neurovascular damage following LNs injection was attenuated in P2X7-/- mice. PEGylation of LNs reduced correlated nanoparticles aggregation. Moreover, PEGylation of LNs ameliorated the P2X7/caspase-1/IL-1ß signalling-dependent microglia activation and neurovascular damage. In conclusion, PEGylation of LNs is a promising biomaterial for brain-targeted therapy that inhibits P2X77-dependent neuroinflammatory response.