Sepsis is the principal cause of fatality in the intensive care units worldwide. It involves uncontrolled inflammatory response resulting in multi-organ failure and even death. Micheliolide (MCL), a sesquiterpene lactone, was reported to inhibit dextran sodium sulphate (DSS)-induced inflammatory intestinal disease, colitis-associated cancer and rheumatic arthritis. Nevertheless, the role of MCL in microbial infection and sepsis is unclear. We demonstrated that MCL decreased lipopolysaccharide (LPS, the main cell wall component of Gram-negative bacteria)-mediated production of cytokines (IL-6, TNF-α, MCP-1, etc) in Raw264.7 cells, primary macrophages, dendritic cells and human monocytes. MCL plays an anti-inflammatory role by inhibiting LPS-induced activation of NF-κB and PI3K/Akt/p70S6K pathways. It has negligible impact on the activation of mitogen-activated protein kinase (MAPK) pathways. In the acute peritonitis mouse model, MCL reduced the secretion of IL-6, TNF-α, IL-1β, MCP-1, IFN-β and IL-10 in sera, and ameliorated lung and liver damage. MCL down-regulated the high mortality rate caused by lethal LPS challenge. Collectively, our data illustrated that MCL enabled maintenance of immune equilibrium may represent a potentially new anti-inflammatory and immunosuppressive drug candidate in the treatment of sepsis and septic shock.

BackgroundYarrowia lipolytica, a non-traditional oil yeast, has been widely used as a platform for lipid production. However, the production of other chemicals such as terpenoids in engineered Y. lipolytica is still low. α-Farnesene, a sesquiterpene, can be used in medicine, bioenergy and other fields, and has very high economic value. Here, we used α-farnesene as an example to explore the potential of Y. lipolytica for terpenoid production.ResultsWe constructed libraries of strains overexpressing mevalonate pathway and α-farnesene synthase genes by non-homologous end-joining (NHEJ) mediated integration into the Y. lipolytica chromosome. First, a mevalonate overproduction strain was selected by overexpressing relevant genes and changing the cofactor specificity. Based on this strain, the downstream α-farnesene synthesis pathway was overexpressed by iterative integration. Culture conditions were also optimized. A strain that produced 25.55 g/L α-farnesene was obtained. This is the highest terpenoid titer reported in Y. lipolytica.ConclusionsYarrowia lipolytica is a potentially valuable species for terpenoid production, and NHEJ-mediated modular integration is effective for expression library construction and screening of high-producer strains.

Yarrowia lipolytica is an important oleaginous industrial microorganism used to produce biofuels and other value-added compounds. Although several genetic engineering tools have been developed for Y. lipolytica, there is no efficient method for genomic integration of large DNA fragments. In addition, methods for constructing multigene expression libraries for biosynthetic pathway optimization are still lacking in Y. lipolytica. In this study, we demonstrate that multiple and large DNA fragments can be randomly and efficiently integrated into the genome of Y.lipolytica in a homology-independent manner. This homology-independent integration generates variation in the chromosomal locations of the inserted fragments and in gene copy numbers, resulting in the expression differences in the integrated genes or pathways. Because of these variations, gene expression libraries can be easily created through one-step integration. As a proof of concept, a LIP2 (producing lipase) expression library and a library of multiple genes in the β-carotene biosynthetic pathway were constructed, and high-production strains were obtained through library screening. Our work demonstrates the potential of homology-independent genome integration for library construction, especially for multivariate modular libraries for metabolic pathways in Y. lipolytica, and will facilitate pathway optimization in metabolic engineering applications.

Mycobacterium tuberculosis (Mtb) remains a significant threat to global health as it induces granuloma and systemic inflammatory responses during active tuberculosis. Mtb can induce macrophage pyroptosis, leading to the release of IL-1β and tissue damage, promoting its spread. Here, we established an in vitro Mtb-infected macrophage model to seek an effective antipyroptosis agent. Baicalin, isolated from Radix Scutellariae, was found to reduce pyroptosis in Mtb-infected macrophages. Baicalin could inhibit activation of the PERK/eIF2α pathway and thus downregulates TXNIP expression and subsequently reduces activation of the NLRP3 inflammasome, resulting in reduced pyroptosis in Mtb-infected macrophages. In conclusion, baicalin reduced pyroptosis by inhibiting the PERK/TXNIP/NLRP3 axis and might thus be a new adjuvant host-directed therapy (HDT) drug.

5-Aminolevulinic acid (5-ALA) is a key metabolic intermediate of the heme biosynthesis pathway, which has broad application prospects in agriculture and medicine. However, segregational instability of plasmid-based expression systems and low yield have hampered large-scale manufacture of 5-ALA. In this study, two important genes of the 5-ALA C5 biosynthesis pathway, hemA and hemL, were integrated into Escherichia coli MG1655 for chemically induced chromosomal evolution (CIChE). The highest hemA and hemL copy-number, 98 per genome, was obtained in CIChE strain MG136. The 5-ALA titer of this strain reached 2724 mg/L in optimized condition. Then, after undergoing adaptative evolution and the deletion of recA, strain MG136a ΔrecA::FRT could stably produce 4550 mg/L 5-ALA from glucose, 450 times the amount produced by hemA-hemL single copy strain MG1655-hemAL. This study constructed a plasmid-free E. coli strain for 5-ALA production, which will provide the basis for further manipulation of metabolic regulation and optimization of fermentation.

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