Engineering Escherichia coli to improve tryptophan production via genetic manipulation of precursor and cofactor pathways

Optimizing the supply of biosynthetic precursors and cofactors is usually an effective metabolic strategy to improve the production of target compounds. Here, the combination of optimizing precursor synthesis and balancing cofactor metabolism was adopted to improve tryptophan production in Escherichia coli. First, glutamine synthesis was improved by expressing heterologous glutamine synthetase from Bacillus subtilis and Bacillus megaterium in the engineered Escherichia coli strain KW001, resulting in the best candidate strain TS-1. Then icd and gdhA were overexpressed in TS-1, which led to the accumulation of 1.060 g/L tryptophan. Subsequently, one more copy of prs was introduced on the chromosome to increase the flux of 5-phospho-α-d-ribose 1-diphosphate followed by the expression of mutated serA and thrA to increase the precursor supply of serine, resulting in the accumulation of 1.380 g/L tryptophan. Finally, to maintain cofactor balance, sthA and pntAB, encoding transhydrogenase, were overexpressed. With sufficient amounts of precursors and balanced cofactors, the engineered strain could produce 1.710 g/L tryptophan after 48 h of shake-flask fermentation, which was 2.76-times higher than the titer of the parent strain. Taken together, our results demonstrate that the combination of optimizing precursor supply and regulating cofactor metabolism is an effective approach for high-level production of tryptophan. Similar strategies could be applied to the production of other amino acids or related derivatives

Network Pharmacology Analysis and Experimental Verification on Antiangiogenesis Mechanism of Hedyotis diffusa Willd in Liver Cancer

Purpose. Hedyotis diffusa Willd (HDW) is one of the most well-known herbs used in the therapy of cancer. However, the potential mechanisms of its antiangiogenic effects have not been fully explored. Here, we applied a network pharmacology approach to explore the potential mechanisms of HDW against liver cancer angiogenesis (LCA) and used a mouse orthotopic liver cancer model for experimental verification accordingly. Methods. The effective components, primary active compounds, and possible targets in the therapy of LCA were predicted using network pharmacology and bioinformatics. In vivo testing of the pharmacodynamic foundation of HDW in the treatment of LCA was performed. Hepa1-6 cells were implanted in C57BL/6 mice to establish an orthotopic liver cancer model to evaluate the antitumor and antiangiogenesis effects of the drug. Furthermore, protein levels were evaluated by western blotting, immunofluorescence, and immunohistochemistry. Results. We firstly confirmed the therapeutic effect of HDW on LCA and subsequently screened 7 active compounds from HDW according to their pharmacokinetic properties. Network analysis and enrichment analysis indicated that these compounds exhibit antiangiogenic effect by acting on multiple targets and thereby regulating multiple pathways mainly involved in Akt1, IL-6, IL-1β, IL-17, hypoxia inducible factor-1α (HIF-1α), and tumor necrosis factor-α (TNF-α). Importantly, we preliminarily verified the results of the network pharmacology analysis in vivo. Conclusion. Collectively, our work initially explored the therapeutic mechanism of HDW on tumor angiogenesis, which lays an experimental reference for further exploring its pharmacological action and its clinical application

Glycosaminoglycanomics of Cultured Cells Using a Rapid and Sensitive LC-MS/MS Approach

Glycosaminoglycans (GAGs), a family of polysaccharides widely distributed in eukaryotic cells, are responsible for a wide array of biological functions. Quantitative disaccharide compositional analysis is one of the primary ways to characterize the GAG structure. This structural analysis is typically time-consuming (1–2 weeks) and labor intensive, requiring GAG recovery and multistep purification, prior to the enzymatic/chemical digestion of GAGs, and finally their analysis. Moreover, 10⁵–10⁷ cells are usually required for compositional analysis. We report a sensitive, rapid, and quantitative analysis of GAGs present in a small number of cells. Commonly studied cell lines were selected based on phenotypic properties related to the biological functions of GAGs. These cells were lysed using a commercial surfactant reagent, sonicated, and digested with polysaccharide lyases. The resulting disaccharides were recovered by centrifugal filtration, labeled with 2-aminoacridone, and analyzed by liquid chromatography (LC)-mass spectrometry (MS). Using a highly sensitive MS method, multiple reaction monitoring (MRM), the limit of detection for each disaccharide was reduced to 0.5–1.0 pg, as compared with 1.0–5.0 ng obtained using standard LC-MS analysis. Sample preparation time was reduced to 1–2 days, and the cell number required was reduced to 5000 cells for complete GAG characterization to as few as 500 cells for the characterization of the major GAG disaccharide components. Our survey of the glycosaminoglycanomes of the 20 selected cell lines reveals major differences in their GAG amounts and compositions. Structure–function relationships are explored using these data, suggesting the utility of this method in cellular glycobiology