Carbon competition between cell growth and product synthesis is the bottleneck in efficient N-acetyl glucosamine (GlcNAc) production in microbial cell factories. In this study, a xylose-induced T7 RNA polymerase−P T7 promoter system was introduced in Escherichia coli W3110 to control the GlcNAc synthesis. Meanwhile, an arabinose-induced CRISPR interference (CRISPRi) system was applied to adjust cell growth by attenuating the transcription of key growth-related genes. By designing proper sgRNAs, followed by elaborate adjustment of the addition time and concentration of the two inducers, the carbon flux between cell growth and GlcNAc synthesis was precisely redistributed. Comparative metabolomics analysis results confirmed that the repression of pf kA and zwf significantly attenuated the TCA cycle and the synthesis of related amino acids, saving more carbon for the GlcNAc synthesis. Finally, the simultaneous repression of pf kA and zwf in strain GLA-14 increased the GlcNAc titer by 47.6% compared with that in E. coli without the CRISPRi system in a shake flask. GLA-14 could produce 90.9 g/L GlcNAc within 40 h in a 5 L bioreactor, with a high productivity of 2.27 g/L/h. This dynamic strategy for rebalancing cell growth and product synthesis could be applied in the fermentative production of other chemicals derived from precursors synthesized via central carbon metabolism.
Hepatocellular carcinoma (HCC) is a major subtype of primary liver cancer with a high mortality rate. Pyroptosis and autophagy are crucial processes in the pathophysiology of HCC. Searching for efficient drugs targeting pyroptosis and autophagy with lower toxicity is useful for HCC treatment. Mallotucin D (MLD), a clerodane diterpenoid from Croton crassifolius, has not been previously reported for its anticancer effects in HCC. This study aims to evaluate the inhibitory effects of MLD in HCC and explore the underlying mechanism. We found that the cell proliferation, DNA synthesis, and colony formation of HepG2 cells and the angiogenesis of HUVECs were all greatly inhibited by MLD. MLD caused mitochondrial damage and decreased the TOM20 expression and mitochondrial membrane potential, inducing ROS overproduction. Moreover, MLD promoted the cytochrome C from mitochondria into cytoplasm, leading to cleavage of caspase-9 and caspase-3 inducing GSDMD-related pyroptosis. In addition, we revealed that MLD activated mitophagy by inhibiting the PI3K/AKT/mTOR pathway. Using the ROS-scavenging reagent NAC, the activation effects of MLD on pyroptosis- and autophagy-related pathways were all inhibited. In the HepG2 xenograft model, MLD effectively inhibited tumor growth without detectable toxicities in normal tissue. In conclusion, MLD could be developed as a candidate drug for HCC treatment by inducing mitophagy and pyroptosis via promoting mitochondrial-related ROS production.
Colorectal cancer (CRC) is the leading cause of cancer-related deaths worldwide. Fibromodulin (FMOD) is the main proteoglycan that contributes to extracellular matrix (ECM) remodeling by binding to matrix molecules, thereby playing an essential role in tumor growth and metastasis. There are still no useful drugs that target FMOD for CRC treatment in clinics. Here, we first used public whole-genome expression datasets to analyze the expression level of FMOD in CRC and found that FMOD was upregulated in CRC and associated with poor patient prognosis. We then used the Ph.D.-12 phage display peptide library to obtain a novel FMOD antagonist peptide, named RP4, and tested its anti-cancer effects of RP4 in vitro and in vivo. These results showed that RP4 inhibited CRC cell growth and metastasis, and promoted apoptosis both in vitro and in vivo by binding to FMOD. In addition, RP4 treatment affected the CRC-associated immune microenvironment in a tumor model by promoting cytotoxic CD8+ T and NKT (natural killer T) cells and inhibiting CD25+ Foxp3+ Treg cells. Mechanistically, RP4 exerted anti-tumor effects by blocking the Akt and Wnt/β-catenin signaling pathways. This study implies that FMOD is a potential target for CRC treatment, and the novel FMOD antagonist peptide RP4 can be developed as a clinical drug for CRC treatment.
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