ABSTRACT Microbial cell factory development requires iterative optimization of enzymes, pathway fluxes, and cell traits to achieve efficient and robust production of target chemicals. However, the complexity of intracellular metabolic networks often limits predictable identification and control of engineering targets. Genetically encoded metabolite‐responsive RNA biosensors based on aptamer recognition and riboswitch‐ or ribozyme‐mediated regulation have emerged as versatile tools for linking intracellular metabolite levels to measurable or selectable outputs. Their rapid signal transduction, compact genetic architecture, and low metabolic burden make them attractive for high‐throughput screening and dynamic regulation. This Review adopts a hierarchical engineering framework that examines RNA biosensor applications across progressively broader levels, from enzyme engineering to pathway‐level regulation and host‐level cellular trait engineering. We further discuss their molecular organization and technological evolution, including ligand recognition, regulatory coupling, sensor tuning, and computational approaches. Finally, challenges associated with sensor robustness, host dependency, scalability, and translation into production‐relevant phenotypes are discussed together with opportunities in cell‐free characterization and data‐driven design. This hierarchical perspective highlights RNA biosensors as engineering interfaces connecting intracellular sensing with interventions across multiple scales of microbial cell factory engineering.
ABSTRACT Lactiplantibacillus plantarum has emerged as a promising synthetic biology chassis owing to its food‐grade safety, probiotic properties, and adaptability to the gastrointestinal tract. Its engineering potential has expanded rapidly in live biotherapeutics, functional foods, biosensors, and animal health. However, its broader application is still limited by the lack of standardized genetic parts, versatile engineering tools, and industrially compatible chassis. This review summarizes recent advances in the synthetic biology toolbox of L. plantarum , including expression elements, genome engineering strategies, and representative applications. Particular emphasis is placed on the development of genetic components, conventional and CRISPR‐based engineering technologies, and their applications in medicine, food biotechnology, biosensing, and animal nutrition. Current challenges and future opportunities, including standardized genetic parts, precision genome engineering, AI‐assisted strain design, and high‐performance chassis development, are also discussed. This review provides a comprehensive framework for developing next‐generation programmable probiotic cell factories based on L. plantarum .
ABSTRACT 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 CO 2 . 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 Δ epr‐1 Δ glcR achieved a high crude protein content of 70.13%. However, when the GS13 module was introduced into this background, OD 600 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 .
ABSTRACT 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.
ABSTRACT 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.
