Biotechnology Journal最新文献

Enhancing nitrogen assimilation and coordinating carbon-energy metabolism in the autotrophic chassis Cupriavidus necator remain key challenges for maximizing single-cell protein (SCP) production from CO₂. In this study, multilevel engineering was performed on C. necator. First, glnA was overexpressed to enhance nitrogen assimilation, which increased crude protein content to 72.75% in the best-performing strain, GS13. Preliminary physiological characterization suggested that this increase correlated with enhanced heme level. Second, glcR was disrupted to alter carbon allocation toward amino acid precursor metabolism, and epr-1 was deleted to inhibit extracellular protein degradation during the secretion process. The double-knockout strain Δepr-1ΔglcR achieved a high crude protein content of 70.13%. However, when the GS13 module was introduced into this background, OD₆₀₀ increased to 13.12 while crude protein content decreased to 68.05%, indicating a non-additive interaction between the engineered modules. Overall, our study provided improved strains and a modular framework for future SCP optimization and mechanistic studies of carbon-nitrogen coordination in C. necator.

Aromatic noncanonical amino acids (aromatic ncAAs) are valuable building blocks for pharmaceuticals, agrochemicals, food additives, and specialty chemicals, yet their sustainable and stereoselective synthesis remains challenging. Ammonia-lyases (ALs) have emerged as powerful biocatalysts for the one-step asymmetric hydroamination of arylacrylic acids, and recent advances in protein engineering have substantially enhanced their catalytic performance including efficiency, selectivity, promiscuity, and productivity. This review integrates catalytic mechanisms with the deployment of engineering strategies to illustrate how aromatic amino acid ammonia-lyases (AALs) have evolved into versatile biocatalysts for aromatic ncAAs synthesis. Particular attention is devoted to the engineering of AALs (including phenylalanine, tyrosine, histidine ammonia lyase) and (methyl)aspartate ammonia lyases, highlighting progress in substrate scope expansion, catalytic promiscuity, selectivity modulation, and hybrid catalytic systems. Current limitations and emerging opportunities are further assessed to delineate future directions for next-generation ALs engineering. The engineering strategies and current states of ALs summarized herein provide a conceptual basis for the efficient development of ALs for sustainable aromatic ncAAs biomanufacturing.

Recombinant adeno-associated virus (rAAV) vectors are essential for gene therapy, but their production generates empty and partial capsids as product-related impurities that can compromise efficacy and safety. While empty capsids have been partially characterized, the isolation and study of partial capsids remain hindered by a lack of robust separation methods. Here, we present an optimized iodixanol density gradient ultracentrifugation strategy enabling high-resolution separation and enrichment of empty, partial, and full AAV capsids. Key innovations include expanding the 40% iodixanol layer to create a broader intermediate-density zone and implementing controlled bottom-puncture fractionation. Applied to a rAAV2 preparation, this method enriched partial capsids from 18.9% in the starting material to approximately 94.9% in the target fraction (determined by AUC integration under the equal response assumption). For a rAAV2 variant with a low baseline of partial capsids (1.3%), over 20-fold enrichment was achieved, reaching 30.1% purity as determined by AUC integration under the equal response assumption. This approach provides partial-capsid-enriched fractions, enabling detailed characterization of partial capsids, supporting critical quality attribute assessment, and facilitating the development of safer, more effective rAAV-based therapeutics.

Calpain is a family of cysteine proteases whose role in sea cucumber autolysis was previously unreported. In this study, specific inhibition of calpain notably alleviated autolysis in the respiratory tree and intestine, significantly reduced body wall hardness loss. During an 8 h autolysis period, calpain activity in the body wall continuously declined, whereas activity in the respiratory tree and intestine peaked at 2 h. The contents of calpain 5 & 9 were highest in the respiratory tree and remained relatively stable, whereas these two isoforms showed distinct temporal patterns in the intestine and body wall. Immunohistochemistry revealed high calpain expression in the muscle layers and at the mucosal fold tips of the intestine and respiratory tree, but sparse distribution in the body wall connective tissue. After 4 h of autolysis, disintegration of the intestinal serosal layer, disorganization of the respiratory tree cellular structure, and destruction of the epidermis and endodermis of the body wall were observed. Co-immunoprecipitation coupled with mass spectrometry identified 40 calpain-interacting proteins, and fluorescence colocalization confirmed direct interaction with tubulin. This first report establishes calpain involvement in sea cucumber autolysis, clarifying its dynamic activity, tissue distribution, and interacting proteins, and suggests calpain may act by degrading tubulin.

Recombinant adeno-associated virus (rAAV) vectors have emerged as leading platforms for gene therapy owing to their safety, long-term transgene expression, and broad tissue tropism. However, rAAV production in HEK293 cells via triple-plasmid transfection remains limited by low yield, partly due to inefficient nuclear import of plasmids. In this study, we enhanced rAAV production by promoting plasmid nuclear delivery. DNA-targeting sequence (DTS) insertion alone did not improve plasmid nuclear import or rAAV yield, whereas combining DTS insertion with tubacin, an HDAC6 inhibitor, significantly increased nuclear plasmid accumulation and rAAV productivity. Among the three plasmids, inserting the 5NF sequence into pHelper produced the greatest effect. Under tubacin treatment, pHelper-5NF increased rAAV5 yield from 1.08 × 10¹¹ to 2.44 × 10¹¹ vg/mL (2.26-fold), without altering the full capsid percentage. This improvement was associated with increased Rep/Cap expression and viral DNA replication. Moreover, the pHelper-5NF enhanced rAAV production across multiple capsid serotypes, including rAAV1, rAAV2, rAAV6, rAAV8, rAAV9, and rAAVrh10. Unexpectedly, insertion of 5NF into two or three plasmids did not further increase rAAV yield via triple-plasmid transfection. These results identify pHelper-5NF combined with HDAC6 inhibition as an effective strategy to enhance triple-plasmid transfection-based rAAV manufacturing.