Hypoxia-induced UBE2K promotes the malignant progression of HCC.

BACKGROUND: Hypoxia critically drives malignant tumor development and is characteristic of hepatocellular carcinoma (HCC), where HIF-1α plays a crucial role. The ubiquitin-conjugating enzyme E2K (UBE2K) is known to participate in the advancement of several human cancers. However, the role of UBE2K in HCC or whether it is a hypoxia-responsive gene remains to be further identified. METHOD: We performed a microarray to measure the gene expression differences between normoxia and hypoxia. CoCl2 mimicked the hypoxic condition. The protein and RNA expression of HIF-1α, UBE2K, and Actin in HCC cells were measured by western blotting(WB) and RT-qPCR, respectively. Immunohistochemical (IHC) staining analyzed the expression of UBE2K and HIF-1α in HCC tissues. CCK-8 and colony formation assay evaluated the HCC cell growth. Scratch healing and transwell assays were used to detect the migration capability of the cells. Lipofectamine 3000 was used to transfect the plasmids or siRNAs to HCC cells. RESULTS: We identified UBE2K as a potential hypoxia-responsive gene. Our study showed that hypoxia induced HIF-1α-mediated increase of UBE2K levels in HCC cells, which decreased under HIF-1α deficiency under hypoxia. Further bioinformatics analysis based on UALCAN and GEPIA databases confirmed that UBE2K was highly expressed in HCC tissues and positively associated with HIF-1α expression. Functionally, Hep3B and Huh7 cell proliferation and migration were stimulated upon UBE2K overexpression, while the UBE2K knockdown suppressed such effect. Furthermore, functional rescue experiment proved that depletion of UBE2K inhibited hypoxia-induced cell proliferation and migration in HCC cells. In contrast, enhancing UBE2K levels rescued cell proliferation and migration repression caused by HIF-1α deficiency in hypoxia. CONCLUSION: Our results established UBE2K as a potential hypoxia-inducible gene in HCC cells, positively regulated by HIF-1α in hypoxia. Moreover, UBE2K served as an oncogene and cooperated with HIF-1α to form a functional HIF-1α/UBE2K axis to trigger HCC progression, highlighting a potential application of UBE2K as a therapeutic target for HCC treatment.

High-Level Production of l-Methionine by Dynamic Deregulation of Metabolism with Engineered Nonauxotroph Escherichia coli.

l-Methionine is the only sulfur-containing amino acid among the essential amino acids, and it is mainly produced by the chemical method in industry so far. The fermentation production of l-methionine by genetically engineered strains is an attractive alternative. Due to the complex metabolic mechanism and multilevel regulation of the synthesis pathway in the organism, the fermentation production of l-methionine by genetically engineered strains was still not satisfied. In this study, the biosynthesis pathway of l-methionine was regulated based on the previous studies. As the competitive pathway and an essential amino acid for cell growth, the biosynthesis pathway of l-lysine was first repaired by complementation of the lysA gene in situ on the genome and then replaced the in situ promoter with the dynamically regulated promoter PfliA to construct a nonauxotroph strain. In addition, the central metabolic pathway and l-cysteine catabolism pathway were further modified to promote the cell growth and enhance the l-methionine production. Finally, the l-methionine fermentation yield in a 5 L bioreactor reached 17.74 g/L without adding exogenous amino acids. These strategies can effectively balance the contradiction between cell growth and l-methionine production and alleviate the complexity of fermentation operation and the cost with auxotroph strains, which provide a reference for the industrial production of l-methionine by microbial fermentation.

[Advances in the biosynthesis of L-homoserine and its derivatives by metabolic engineering of Escherichia coli].

L-homoserine and its derivatives (O-succinyl-L-homoserine and O-acetyl-L-homoserine) are precursors for the biosynthesis of L-methionine, and various C4 compounds (isobutanol, γ-butyrolactone, 1, 4-butanediol, 2, 4-dihydroxybutyric acid) and L-phosphinothricin. Therefore, the fermentative production of L-homoserine and its derivatives became the research hotspot in recent years. However, the low fermentation yield and conversion rate, and the unclear regulation mechanism for the biosynthesis of L-homoserine and its derivatives, hamper the development of an efficient production process for L-homoserine and its derivatives. This review summarized the advances in the biosynthesis of L-homoserine and its derivatives by metabolic engineering of Escherichia coli from the aspects of substrate uptake, redirection of carbon flow at the key nodes, recycle of NADPH and export of target products. This review may facilitate subsequent metabolic engineering and biotechnological production of L-homoserine and its derivatives.