Plant bioreactor is a new production platform for expression of recombinant protein, which is one of the cores of molecular farming. In this study, the anti DYKDDDDK (FLAG) antibody was recombinantly expressed in tobacco (Nicotiana benthamiana) and purified. FLAG antibody with high affinity was obtained after immunizing mice for several times and its sequence was determined. Based on this, virus vectors expressing heavy chain (HC) and light chain (LC) inoculated into Nicotiana benthamiana leaves by using Agrobacterium-mediated delivery. Accumulation of the HC and LC was analyzed by SDS/PAGE followed by Western blotting probed with specific antibodies from 2 to 9 days postinfiltration (dpi). Accumulation of the FLAG antibody displayed at 3 dpi, and reached a maximum at 5 dpi. It was estimated that 66 mg of antibody per kilogram of fresh leaves could be obtained. After separation and purification, the antibody was concentrated to 1 mg/mL. The 1:10 000 diluted antibody can probe with 1 ng/mL FLAG fused antigen well, indicating the high affinity of the FLAG antibody produced in plants. In conclusion, the plant bioreactor is able to produce high affinity FLAG antibodies, with the characteristics of simplicity, low cost and highly added value, which contains enormous potential for the rapid and abundant biosynthesis of antibodies.
Animals ; Mice ; Antibodies ; Nicotiana/genetics* ; Agrobacterium/genetics* ; Bioreactors ; Blotting, Western

The human oral microbiome harbors one of the most diverse microbial communities in the human body, playing critical roles in oral and systemic health. Recent technological innovations are propelling the characterization and manipulation of oral microbiota. High-throughput sequencing enables comprehensive taxonomic and functional profiling of oral microbiomes. New long-read platforms improve genome assembly from complex samples. Single-cell genomics provides insights into uncultured taxa. Advanced imaging modalities including fluorescence, mass spectrometry, and Raman spectroscopy have enabled the visualization of the spatial organization and interactions of oral microbes with increasing resolution. Fluorescence techniques link phylogenetic identity with localization. Mass spectrometry imaging reveals metabolic niches and activities while Raman spectroscopy generates rapid biomolecular fingerprints for classification. Culturomics facilitates the isolation and cultivation of novel fastidious oral taxa using high-throughput approaches. Ongoing integration of these technologies holds the promise of transforming our understanding of oral microbiome assembly, gene expression, metabolites, microenvironments, virulence mechanisms, and microbe-host interfaces in the context of health and disease. However, significant knowledge gaps persist regarding community origins, developmental trajectories, homeostasis versus dysbiosis triggers, functional biomarkers, and strategies to deliberately reshape the oral microbiome for therapeutic benefit. The convergence of sequencing, imaging, cultureomics, synthetic systems, and biomimetic models will provide unprecedented insights into the oral microbiome and offer opportunities to predict, prevent, diagnose, and treat associated oral diseases.
Humans ; Phylogeny ; Biomimetics ; Dysbiosis ; Homeostasis ; Mass Spectrometry

As human microbiome research advances, a large body of evidence shows that microorganisms are closely related to human health. Probiotics were discovered and used as foods or dietary supplements with health benefits in the last century. Microorganisms have shown broader application prospects in human health since the turn of the century, owing to the rapid development of technologies such as microbiome analysis, DNA synthesis and sequencing, and gene editing. In recent years, the concept of "next-generation probiotics" has been proposed as new drugs, and microorganisms are considered as "live biotherapeutic products (LBP)". In a nutshell, LBP is a living bacterial drug that can be used to prevent or treat certain human diseases and indications. Because of its distinct advantages, LBP has risen to the forefront of drug development research and has very broad development prospects. This review introduces the varieties and research advances on LBP from a biotechnology standpoint, followed by summarizing the challenges and opportunities for LBP clinical implementations, with the aim to facilitate LBP development.
Humans ; Probiotics ; Dietary Supplements ; Bacteria ; Drug Development ; Biotechnology

Synthetic plastics have been widely used in various fields of the national economy and are the pillar industry. However, irregular production, plastic product use, and plastic waste piling have caused long-term accumulation in the environment, contributing considerably to the global solid waste stream and environmental plastic pollution, which has become a global problem to be solved. Biodegradation has recently emerged as a viable disposal method for a circular plastic economy and has become a thriving research area. In recent years, important breakthroughs have been made in the screening, isolation, and identification of plastic-degrading microorganisms/enzyme resources and their further engineering, which provide new ideas and solutions for treating microplastics in the environment and the closed-loop bio-recycling of waste plastics. On the other hand, the use of microorganisms (pure cultures or consortia) to further transform different plastic degradants into biodegradable plastics and other compounds with high added value is of great significance, promoting the development of a plastic recycling economy and reducing the carbon emission of plastics in their life cycle. We edited a Special Issue on the topic of "Biotechnology of Plastic Waste Degradation and Valorization", focusing on the researches progress in three aspects: Mining microbial and enzyme resources for plastic biodegradation, Design and engineering of plastic depolymerase, and biological high-value transformation of plastic degradants. In total, 16 papers have been collected in this issue including reviews, comments, and research articles, which provide reference and guidance for further development of plastic waste degradation and valorization biotechnology.
Biodegradable Plastics ; Biodegradation, Environmental ; Biotechnology

In recent years, the petroleum-based plastic pollution problem has been causing global attention. The idea of "degradation and up-cycling of plastics" was proposed for solving the environmental pollution caused by non-degradable plastics. Following this idea, plastics would be firstly degraded and then reconstructed. Polyhydroxyalkanoates (PHA) can be produced from the degraded plastic monomers as a choice to recycle among various plastics. PHA, a family of biopolyesters synthesized by many microbes, have attracted great interest in industrial, agricultural and medical sectors due to its biodegradability, biocompatibility, thermoplasticity and carbon neutrality. Moreover, the regulations on PHA monomer compositions, processing technology, and modification methods may further improve the material properties, making PHA a promising alternative to traditional plastics. Furthermore, the application of the "next-generation industrial biotechnology (NGIB)" utilizing extremophiles for PHA production is expected to enhance the PHA market competitiveness, promoting this environmentally friendly bio-based material to partially replace petroleum-based products, and achieve sustainable development with carbon-neutrality. This review summarizes the basic material properties, plastic upcycling via PHA biosynthesis, processing and modification methods of PHA, and biosynthesis of novel PHA.
Polyhydroxyalkanoates ; Plastics ; Biotechnology ; Petroleum ; Carbon

Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.
Bacillus subtilis/metabolism* ; Vitamin K 2/metabolism* ; Bioreactors/microbiology* ; Membrane Microdomains/metabolism*

Natural plant-derived diterpenoids are a class of compounds with diverse structures and functions. These compounds are widely used in pharmaceuticals, cosmetics and food additives industries because of their pharmacological properties such as anticancer, anti-inflammatory and antibacterial activities. In recent years, with the gradual discovery of functional genes in the biosynthetic pathway of plant-derived diterpenoids and the development of synthetic biotechnology, great efforts have been made to construct a variety of diterpenoid microbial cell factories through metabolic engineering and synthetic biology, resulting in gram-level production of many compounds. This article summarizes the construction of plant-derived diterpenoid microbial cell factories through synthetic biotechnology, followed by introducing the metabolic engineering strategies applied to improve plant-derived diterpenoids production, with the aim to provide a reference for the construction of high-yield plant-derived diterpenoid microbial cell factories and the industrial production of diterpenoids.
Diterpenes/metabolism* ; Biotechnology ; Metabolic Engineering ; Biosynthetic Pathways/genetics* ; Plants/genetics* ; Synthetic Biology

As an essential amino acid, l-tryptophan is widely used in food, feed and medicine sectors. Nowadays, microbial l-tryptophan production suffers from low productivity and yield. Here we construct a chassis E. coli TRP3 producing 11.80 g/L l-tryptophan, which was generated by knocking out the l-tryptophan operon repressor protein (trpR) and the l-tryptophan attenuator (trpL), and introducing the feedback-resistant mutant aroG^fbr. On this basis, the l-tryptophan biosynthesis pathway was divided into three modules, including the central metabolic pathway module, the shikimic acid pathway to chorismate module and the chorismate to tryptophan module. Then we used promoter engineering approach to balance the three modules and obtained an engineered E. coli TRP9. After fed-batch cultures in a 5 L fermentor, tryptophan titer reached to 36.08 g/L, with a yield of 18.55%, which reached 81.7% of the maximum theoretical yield. The tryptophan producing strain with high yield laid a good foundation for large-scale production of tryptophan.
Escherichia coli/metabolism* ; Tryptophan ; Metabolic Engineering ; Bioreactors ; Metabolic Networks and Pathways

Adipic acid is a high-value-added dicarboxylic acid which is primarily used in the production of nylon-66 for manufacturing polyurethane foam and polyester resins. At present, the biosynthesis of adipic acid is hampered by its low production efficiency. By introducing the key enzymes of adipic acid reverse degradation pathway into a succinic acid overproducing strain Escherichia coli FMME N-2, an engineered E. coli JL00 capable of producing 0.34 g/L adipic acid was constructed. Subsequently, the expression level of the rate-limiting enzyme was optimized and the adipic acid titer in shake-flask fermentation increased to 0.87 g/L. Moreover, the supply of precursors was balanced by a combinatorial strategy consisting of deletion of sucD, over-expression of acs, and mutation of lpd, and the adipic acid titer of the resulting E. coli JL12 increased to 1.51 g/L. Finally, the fermentation process was optimized in a 5 L fermenter. After 72 h fed-batch fermentation, adipic acid titer reached 22.3 g/L with a yield of 0.25 g/g and a productivity of 0.31 g/(L·h). This work may serve as a technical reference for the biosynthesis of various dicarboxylic acids.
Escherichia coli/metabolism* ; Metabolic Engineering ; Bioreactors ; Fermentation ; Adipates/metabolism*

To investigate the bioelectrochemical enhanced anaerobic ammonia oxidation (anammox) nitrogen removal process, a bioelectrochemical system with coupled anammox cathode was constructed using a dual-chamber microbial electrolysis cell (MEC). Specifically, a dark incubation batch experiment was conducted at 30 ℃ with different influent total nitrogen concentrations under an applied voltage of 0.2 V, and the enhanced denitrification mechanism was investigated by combining various characterization methods such as cyclic voltammetry, electrochemical impedance spectroscopy and high-throughput sequencing methods. The results showed that the total nitrogen removal rates of 96.9%±0.3%, 97.3%±0.4% and 99.0%±0.3% were obtained when the initial total nitrogen concentration was 200, 300 and 400 mg/L, respectively. In addition, the cathode electrode biofilm showed good electrochemical activity. High-throughput sequencing results showed that the applied voltage enriched other denitrifying functional groups, including Denitratisoma, Limnobacter, and ammonia oxidizing bacteria SM1A02 and Anaerolineaceae, Nitrosomonas europaea and Nitrospira, besides the anammox bacteria. These electrochemically active microorganisms comprised of ammonium oxidizing exoelectrogens (AOE) and denitrifying electrotrophs (DNE). Together with anammox bacteria Candidatus Brocadia, they constituted the microbial community structure of denitrification system. Enhanced direct interspecies electron transfer between AOE and DNE was the fundamental reason for the further improvement of the total nitrogen removal rate of the system.
Denitrification ; Wastewater ; Anaerobic Ammonia Oxidation ; Nitrogen ; Oxidation-Reduction ; Bioreactors/microbiology* ; Ammonium Compounds ; Bacteria/genetics* ; Microbiota ; Sewage