Genome-Wide Identification, Characterization, and Transcriptional Profile of the HECT E3 Ubiquitin Ligase Gene Family in the Hard-Shelled Mussel Mytilus coruscus Gould.

The homologous E6-AP carboxy-terminal structural domain (HECT) contained in E3 ubiquitin ligases (E3s) is a key factor in protein degradation and maintenance of cellular homeostasis in animals. However, the functional roles and evolutionary aspects of the HECT gene family in bivalve mussels remain unclear and warrant further investigation. In this study, we identified 22 HECT genes within the genome of Mytilus coruscus Gould, all containing a conserved HECT structural domain derived from dispersed repeats, distributed unevenly across 11 chromosomes. Phylogenetic analysis classified M. coruscus HECT genes into six major classes, with amino acid sequences within the same evolutionary clade displaying similar conserved motifs. Homology analysis with HECT genes of four bivalve species revealed that M. coruscus and Mytilus galloprovincialis possessed the largest number of homologous gene pairs, showing a significant correlation between the two in the evolution of the HECT gene family. Homology analysis with HECT genes of four bivalve species revealed that M. coruscus and M. galloprovincialis possessed the largest number of homologous gene pairs, showing a significant correlation between the two in the evolution of the HECT gene family. M. coruscus exhibited pronounced and specific expression in gills and blood tissues. Notably, Mco_UPL3 gene expression was significantly upregulated after 12 h of acute heat stress (33 °C) and 24 h of Vibrio injection (0.4 OD). Gene ontology analysis of the HECT genes in M. coruscus revealed that it is primarily enriched in protein modification and degradation functions. This suggests that HECT genes may play a key role in protein degradation and immunomodulation in M. coruscus. These findings offer valuable insights for the breeding of stress-tolerant traits in M. coruscus. In summary, our data shed light on the potential functions of HECT E3 ligases in response to heat stress and Vibrio infection, providing practical guidance for enhancing resilience through breeding in M. coruscus.

[Molecular engineering of transketolase from Escherichia coli and tartaric semialdehyde biosynthesis].

Transketolase (EC 2.2.1.1, TK) is a thiamine diphosphate-dependent enzyme that catalyzes the transfer of a two-carbon hydroxyacetyl unit with reversible C-C bond cleavage and formation. It is widely used in the production of chemicals, drug precursors, and asymmetric synthesis by cascade enzyme catalysis. In this paper, the activity of transketolase TKTA from Escherichia coli K12 on non-phosphorylated substrates was enhanced through site-directed saturation mutation and combined mutation. On this basis, the synthesis of tartaric semialdehyde was explored. The results showed that the optimal reaction temperature and pH of TKTA_M (R358I/H461S/R520Q) were 32 ℃ and 7.0, respectively. The specific activity on d-glyceraldehyde was (6.57±0.14) U/mg, which was 9.25 times higher than that of the wild type ((0.71±0.02) U/mg). Based on the characterization of TKTA_M, tartaric acid semialdehyde was synthesized with 50 mmol/L 5-keto-d-gluconate and 50 mmol/L non-phosphorylated ethanolaldehyde. The final yield of tartaric acid semialdehyde was 3.71 g with a molar conversion rate of 55.34%. Hence, the results may facilitate the preparation of l-(+)-tartaric acid from biomass, and provide an example for transketolase-catalyzed non-phosphorylated substrates.