Rheumatoid arthritis (RA) can be viewed as a disease of barrier failure, in which CX3CR1⁺/TREM2⁺ synovial tissue resident macrophages that form the lining barrier over cartilage and bone become fragmented and disorganized. However, how to therapeutically rebuild this barrier, and how macrophage states transition during repair, remain unclear. We engineered TR-Ab19, a mouse-selective agonistic antibody against TREM2, as a precision tool to initiate and interrogate barrier repair in vivo. TR-Ab19 engages TREM2 linked downstream signaling and redirects synovial macrophages from Clec4d⁺ inflammatory/proliferative programs toward TREM2⁺CX3CR1⁺Aqp1⁺ barrier-like states, thereby rebuilding the lining barrier. Across collagen-induced arthritis (CIA) and serum-transfer arthritis (STA) models, TR-Ab19 reduces synovitis, preserves cartilage and bone microarchitecture, limits osteoclastogenesis, and attenuates systemic cytokines and B cell abnormalities. Single-cell RNA-seq with trajectory and cell cell communication analyses reveal a TREM2 dependent shift toward a barrier-dominant macrophage ecosystem. Together, these findings establish antibody-mediated reprogramming of resident synovial macrophages as a barrier-centered strategy for RA and provide a framework for instructing macrophage niches in chronic inflammation. ### Competing Interest Statement X-D. Sun is an inventor on a patent application covering TR-Ab19 and holds a potential translation interest in its future clinical development; all other authors declare they have no competing interests. National Natural Science Foundation of China, No. 52272278, 92159305, 52402347 National Postdoctoral Program for Innovative Talents, No. BX20230190

Microbial cell factories for sustainable bioproduction often suffer from trade-offs between cell growth and product synthesis. Spatiotemporal regulation of metabolic pathways offers a potential solution to this challenge. In this study, we engineered synthetic membraneless organelles (MLOs) based on liquid-liquid phase separation (LLPS) in the halophilic bacterium Halomonas bluephagenesis, a robust chassis for next-generation industrial biotechnology (NGIB) operated under open unsterile conditions. Using a tandem Arginine-Glycine-Glycine (RGG) domain as an intrinsically disordered protein scaffold, dynamic protein condensates were constructed capable of recruiting cargo proteins via coiled-coil peptide interactions. Compartmentalization of the 3-hydroxypropionic acid (3HP) biosynthetic enzymes AdhP and AldD into MLOs enhanced 3HP production by up to 91.3%. Furthermore, by fusing the LLPS-promoting WGR-1 peptide to the RGG scaffold, high-rigidity MLOs (hMLOs) were constructed enabling functional insulation of essential endogenous enzymes. Recruitment of bacterial fission ring protein FtsZ to hMLOs resulted in the cell elongation, while sequestration of citrate synthase (GltA) or respiratory chain enzyme IspB led to significant improvement in PHB production. The GltA- and IspB- recruited H. bluephagenesis TP03-2 and TP04-2 grown in shake flasks presented a 22.5% and 16.7% increase on PHB synthesis, a 6.5% and 6.8% increase on glucose-to-PHB conversion rate, respectively. A 5% increase on glucose conversion efficiency was observed for H. bluephagenesis TP03-2 incubated in a 7L bioreactor. A versatile MLO-based toolbox is established for engineering non-model industrial Halomonas.

Isodon rubescens is a widely used traditional folk medicine in China. In this study, 13 natural products were isolated using a two-step ethanol extraction. Notably, flavonoid glycosides 2 and 4, and polymeric caffeic acids 7 and 8, were identified for the first time from the secondary extraction residues, highlighting the continued presence of bioactive compounds with potential for further development. Oridonin (9), the major active compound, was quantified in nine commercial preparations using quantitative NMR (qNMR). Its concentration in the drop pill (0.92 mg/g) was comparable to that in the raw extract (0.90 mg/g). The three tablet samples showed varying levels: Tablet 3 (2.66 mg/g) > Tablet 1 (1.22 mg/g) > Tablet 2 (1.09 mg/g). Capsule, tea bag, and syrup samples could not be quantified due to low concentrations.

Background Hepatitis B virus (HBV ) infection poses a critical threat to public health burden worldwide. The covalently closed circular DNAs (cccDNAs) of HBV was known to form microchromosomes and interact with host epigenetic factors. The aim of the present study is to elucidate the mechanisms of ZNF638, as a transcription factor participating viral epigenetic regulations, for its role in the suppression of HBV cccDNA transcription. Methods Using chromatin immunoprecipitation and nuclear HBV cccDNA pulldown assays to determine ZNF638 binding sites. ZNF638's regulatory effects on HBV in HBV cell models are examined by FISH-IF, qRT-PCR, and ELISA assays. The effect of ZNF638 deficiency on HBV inhibition through HBV-targeting siRNA delivery was evaluated in an in vivo model of HBV transgenic mice. Results It was found that ZNF638 was able to bind the preS and S gene regions of HBV, repressing the transcription of cccDNA upon increased modification of H3K9me3 mediated by SETDB1. HUSH complex was demonstrated to involve in the epigenetic silencing of cccDNA transcription. Subcutaneous injection of ZNF638 siRNA significantly compromised the efficacy of HBV RNAi therapy in vivo. Conclusions We conclude that ZNF638 is a repressive host factor to inhibit the transcription of HBV DNAs and potently attenuate HBV infection. The binding of ZNF638 to specific regions for recruiting HUSH complex to write H3K9me3 histone marks is required for the epigenetic silencing of cccDNA transcription

Poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P (3HB-co-4HB) or P34HB) is a promising biopolyester for applications in food packaging, medical sutures, drug delivery, and tissue engineering due to its tunable thermomechanical properties. However, industrial-scale production of P34HB from glucose remains challenging. In this study, scalable P34HB production by engineered Halomonas bluephagenesis was developed. A de novo 4HB synthesis pathway was introduced into an endogenous toxin-antitoxin plasmid, enabling stable expression in the absence of antibiotics. Promoter engineering and pathway optimization fine-tuned 4HB molar ratio in P34HB from 18 to 39 mol%. The engineered H. bluephagenesis WR3 demonstrated successful scale-up from 7-L to 100-L and 5000-L bioreactors, achieving a maximum cell dry weight (CDW) of 72 g/L and 84% P (3HB-co-30 mol% 4HB) from the cultures in 7-L bioreactor. In further scale-up studies, H. bluephagenesis WR3 maintained comparable 4HB ratios, producing 69 g/L and 71 g/L CDW containing 74% and 61% P34HB copolymer in 100-L and 5000-L scale bioreactors, respectively. The amorphous P (3HB-co-30 mol% 4HB) exhibited high ductility, with an elongation at break of over 800% and a Young's modulus of 164 MPa. Additionally, morphology engineering and a controllable cell lysis were applied to enhance downstream processing efficiency. The optimized H. bluephagenesis WR25 produced 97 g/L CDW containing 83% P (3HB-co-20 mol% 4HB) in 7-L bioreactor, and 83 g/L CDW with 80% P34HB in 100-L bioreactor, while maintaining a consistently high glucose to P34HB conversion efficiency of 37%. This study provides a robust and cost-effective platform for industrial P34HB production from glucose harboring the toxin-antitoxin stable plasmid encoded with the 4HB pathway.

Lignocellulose is the most abundant terrestrial biomass type, and lignocellulose hydrolysate has the potential to replace glucose for microbial fermentation. Halomonas bluephagenesis has significant advantages in producing bioplastics polyhydroxyalkanoates (PHA), but there is relatively little research on the use of lignocellulose hydrolysate for this strain. In present study, H. bluephagenesis was engineered to use xylose and lignocellulose hydrolysate to produce PHB. Firstly, four xylose metabolism pathways were established. Secondly, several xfp genes were compared and genes in pathway I (xylA and xfp gene) were integrated into the genome. Thirdly, H. bluephagenesis was found to be able to utilize glucose and xylose simultaneously. H. bluephagenesis T39 containing xylA and xfp generated 15 g/L CDW containing 76 wt% PHB when cultured in lignocellulose hydrolysate, and it was grown to 62 g/L CDW containing 67 wt% PHB in a 7 L bioreactor. H. bluephagenesis T43 harboring xylA was found able to synthesize P(3HB-4HB-3HV) containing 3-hydroxybutyrate (3HB), 4-hydroxybutyrte (4HB) and 3-hydroxyvalerate (3HV) when grown on lignocellulose hydrolysate.

Rheumatoid arthritis (RA) is a chronic autoimmune disease and the integrity of CX3CR1+ synovial macrophage barrier significantly impacts its progression. However, the mechanisms driving the dynamic changes of this macrophage barrier remain unclear. Traditional drug therapies for RA have substantial limitations. Mesenchymal stem cells (MSCs)-based cell therapy, especially adipose-derived stem cells (ADSCs), hold therapeutic promise. Nevertheless, the underlying therapeutic mechanism of ADSCs, especially their interactions with CX3CR1+ macrophages, require further investigation. To explore the interaction between ADSCs and CX3CR1+ synovial macrophages during barrier reconstruction, underlying the therapeutic mechanism of ADSCs and the mechanisms on the dynamic changes of the macrophage barrier, scRNA-seq analysis was conducted 4 days after ADSCs injection in serum transfer-induced arthritis model mice. The roles of mitochondria transfer and ADSCs transplantation were also explored. Bulk RNA-seq analysis was performed after the co-culture of ADSCs and CX3CR1+ synovial macrophages. To study the in vivo fate of ADSCs, bulk RNA-seq was performed on ADSCs retrieved at 0, 2, 4, and 7 days post-injection. Intra-articular injection of ADSCs effectively attenuated the pathological progression of mice with serum transfer-induced arthritis. ADSCs gradually adhered to CX3CR1+ macrophages, facilitating the restore of the macrophage barrier, while the absence of this barrier greatly weakened the therapeutic effect of ADSCs. scRNA-seq analysis revealed an Atf3high Ccl3high subset of CX3CR1+ macrophages with impaired oxidative phosphorylation that increased during RA progression. ADSCs-mediated reduction of this subset appeared to be linked to mitochondrial transfer, and transplantation of isolated ADSCs-derived mitochondria also proved effective in treating RA. Both bulk RNA-seq and scRNA-seq analyses revealed multiple interaction mechanisms between ADSCs and CX3CR1+ macrophages, including Cd74/Mif axis and GAS6/MERTK axis, which contribute to barrier restoration and therapeutic effects. Furthermore, bulk RNA-seq analysis showed that ADSCs primarily contribute to tissue repair and immune regulation subsequently. Our results suggest that ADSCs ameliorated the energy metabolism signature of CX3CR1+ lining macrophages and may promote barrier restoration through mitochondria transfer. In addition, we elucidated the fate of ADSCs and the therapeutic potential of mitochondria in RA treatment.

The dynamic regulation of gene circuits using efficient, low-toxicity, and cost-effective inducers has profound implications for the production of a wide range of biological products. Halomonas bluephagenesis, a next generation industrial biotechnology chassis able to grown under open unsterile condition while remains contamination free, was equipped with a synthetic and lactose-induced T7-like induction system against the carbon catabolite repression. This system exhibited comparable efficiency and robustness to the T7-like systems induced by IPTG for the Halomonas with an induction fold change exceeding 350-fold, while offering cost-saving advantages. The lactose-controlled genetic circuit was used to induce heterologous genes for production of acetoin, lysine and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB), forming 2.20 g/L of acetoin, 4.23 g/L of lysine and 5.90 g/L poly(3-hydroxybutyrate-co-9.14 mol% 4-hydroxybutyrate) in shake flasks, respectively, exhibiting an increase of 16 % and 23 % and 26 % for the bioproducts, respectively, compared to the titers achieved under IPTG induction controls. Additionally, lactose induced cell elongation has proven effective for enhanced intracellular polyhydroxyalkanoates (PHA) accumulation, achieving 17 % and 5 % increase in cell dry weight (CDW) and its poly-3-hydroxybutyrate (PHB) content compared to IPTG induction did. Lactose induction in recombinant H. bluephagenesis cultured in a bioreactor resulted in 35 % and 10 % increases in CDW and its PHB content compared to IPTG induction did, respectively. This study demonstrated that the lactose induction system is highly effective combined with cost reduction compared to IPTG for H. bluephagenesis.

Synthetic RNA-based switches provide distinctive merits in modulating gene expression. Simple and flexible RNA-based switches are crucial for advancing the field of gene regulation, paving the way for innovative tools that can sense and manipulate cellular processes. In this research, we have developed programmable ribozymes that are capable of suppressing gene expression in response to specific, endogenously expressed trigger RNAs. We engineer ribozymes by introducing upstream antisense sequences (anti-ribozymes) to inhibit the self-cleaving activity of the hammerhead ribozyme and open the expression of the target gene. The trigger RNA is designed to recognize and bind to complementary sequences within the anti-ribozymes, thereby inhibiting their ability to direct protein synthesis. The anti-ribozyme performance is optimized by regulating the essential sequence modules that play a crucial role in determining the specificity and efficiency of the anti-ribozyme's interaction with its trigger RNA. By applying this switch mechanism to various ribozyme designs, we have shown that it is possible to achieve control over gene expression across a wide range of trigger RNAs. By exploiting these programmable anti-ribozymes, we aim to create a powerful tool for controlling gene expression in mammalian cells, which could have important implications for basic research, disease diagnosis, and therapeutic interventions.

Nexilin F-actin binding protein (NEXN) is crucial for myocardial structural integrity, but its role in HCC progression is unclear. This study aimed to elucidate the role of NEXN in HCC. NEXN was weakly expressed in HCC, and its reduction correlated with poorer overall, disease-free, and progression-free survival. Overexpressing NEXN inhibited cell proliferation, colony formation, migration, and invasion in vitro, as well as tumor formation in vivo. NEXN overexpression downregulated the expression of mesenchymal markers and partially upregulated E-cadherin expression. Mechanistically, NEXN overexpression reduced β-catenin nuclear accumulation. Furthermore, by binding to MYOCD, NEXN co-regulated the EMT in HCC through the WNT/β-catenin signaling pathway. In conclusion, the diminished expression of NEXN interacts with MYOCD, influencing poor HCC prognosis by promoting EMT via the NEXN-MYOCD-β-catenin signaling axis. These findings provide a preclinical foundation for the development of metastasis-targeting inhibitors within the WNT pathway.

Breast cancer is the most common type of cancer among women. It is well-established that microRNAs (miRNAs) play a critical role in cancer development by either degrading messenger RNA (mRNA) or inhibiting its translation, thereby suppressing the expression of specific genes. In this study, we found that the expression level of miR-142-3p was significantly lower in breast cancer cells and tissues than in normal breast epithelial cells and adjacent tissues. We demonstrated that miR-142-3p could inhibit the proliferation, migration, epithelial-mesenchymal transition (EMT), and stemness of MCF 7 breast cancer cells, while also promoting apoptosis. Further investigation revealed that miR-142-3p directly targets CXCL12 and regulates its expression. Silencing CXCL12 (using CXCL12 siRNA) suppressed the migration, EMT, and stemness of MCF 7 cells, and these effects were reversed by inhibition of miR-142-3p. Additionally, we observed alterations in β-catenin protein levels, suggesting that miR-142-3p may modulate the WNT/β-catenin signaling pathway through targeting CXCL12 in MCF 7 cells. Subsequent experiments indicated that miR-142-3p also plays a crucial role in overcoming paclitaxel resistance in MCF 7/PTX cells. SOX2 protein levels, which are associated with paclitaxel resistance, proliferation, migration, and EMT, were higher in MCF 7/PTX cells compared to MCF 7 cells. Overall, our findings suggest that miR-142-3p influences breast cancer progression by targeting the CXCL12/WNT/β-catenin pathway, thereby affecting cell migration, EMT, stemness, and paclitaxel resistance.

Background: Hepatocellular carcinoma (HCC) cell development was investigated in relation to the regulation of Sec61 translocon alpha 1 subunit (SEC61A1) by microRNA (miR)-18a-5p. Materials and Methods: After collection of clinical samples, the transfection of interfering vectors of miR-18a-5p or SEC61A1 was into HCC cells to figure out their roles in development of HCC. The Pearson test and starBase analyzed the association and target prediction of miR-18a-5p. Subsequently, through the dual luciferase reporter experiment, SEC61A1 can be regulated via miR-18a-5p. The salvage experiment revealed that miR-18a-5p influence the degradation process of Hepatocellular carcinoma through combining SEC61A1. Results: In HCC cells, we found that SEC61A1 was elevated and miR-18a-5p was downregulated. HCC cells deteriorating was considerably slowed down by the enhanced miR-18a-5p level. The outcomes demonstrated that miR-18a-5p can interact and regulate SEC61A1. Additionally, the effects of earlier therapy on HCC cell proliferation can be restored by overexpressing SEC61A1. Conclusion: Overall, in HCC cells and tissues miR-18a-5p was significantly downregulated, and inhibited the proliferative ability of HCC cells by targeting SEC61A1.

Microbial poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyvalerate), abbreviated as P(3HB-4HB-3HV) or P34HBHV, is a flexible polyhydroxyalkanoate (PHA) material ranging from softness to elasticity depending on the ratios of various monomers. Halomonas bluephagenesis, as the chassis of the next generation industrial biotechnology (NGIB) able to grow contamination free under open unsterile conditions. The resulting recombinants of H. bluephagenesis became capable of efficiently synthesizing P34HBHV utilizing glucose as the sole carbon source. Engineered H. bluephagenesis H1 (encoding ogdA, sucD, 4hbD, orfZ, scpA and scpB in chromosomes) transformed with a plasmid containing PHA synthesis genes phaC and phaA and its derivative H29 produced up to 92 % P(3HB-co-8.85 %4HB-co-8.47 %3HV) and 72 % P(3HB-co-13.21 %4HB-co-11.97 %3HV) in cell dry weight (CDW), respectively, in shake flasks. In bioreactor cultivation, H. bluephagenesis H39 constructed by integrating the 4hbD, phaC and phaA genes into the genome of H. bluephagenesis H1 achieved 95 g/L CDW with 69 % P(3HB-co-10.49 %4HB-co-3.54 %3HV), while H. bluephagenesis H43, further optimized with lpxM deletion, reached 73 g/L CDW with 78 % P(3HB-co-10.35 %4HB-co-4.54 %3HV) in a 100 L bioreactor. For the first time, H. bluephagenesis was successfully engineered to generate stable and hyperproductive derivative strains for pilot production of P(3HB-4HB-3HV) with customizable monomer ratios from glucose as the sole carbon source.

Microbial polyhydroxyalkanoates (PHA) are promising for wide applications including food and medical packaging, drug delivery systems, coatings and bone scaffolds. The diverse properties of PHA are dependent on the variety of hydroxy fatty acid monomers. Microbial synthesis of homopolymers has been challenging except for poly(3-hydroxybutyrate) (PHB). In this study, an engineered metabolic pathway in Halomonas bluephagenesis was constructed to produce poly(5-hydroxyvalerate) (P5HV) or poly(delta-valerolactone) (PVL). PHA synthase PhaCBP-MCPF4 (PhaCun) and 4-hydroxybutyrate CoA-transferase (AbfT) were identified as suitable for the artificial metabolic pathway. Deletion on the endogenous phaCAB for poly-3-hydroxybutyrate (PHB) synthesis was crucial to reduce 3-hydroxybutyric acid (3HB) monomer ratio in P(3HB-5HV) copolymer from 36% to 0.2% when expressing abfT and phaCun on plasmid pWJL55, or from 38% to 18% when expressing abfT and phaCun on genome. Additionally, deletion of the endogenous acyl-CoA thioesterase gene tesB enhanced 5HV molar ratio from 75% to 81% in P(3HB-5HV), as it removed the CoA moiety from 5HV-CoA. Subsequently, deletion of endogenous fadB gene encoding enoyl-CoA hydratase formed a near PVL homopolymer. The resulted H. bluephagenesis JL03 was grown to 23 g/L dry cell weight containing 58% PVL after 44 h cultivation in a 7-L bioreactor. The PVL exhibited better mechanical properties compared to chemically synthesized PVL, with an elongation at break of 521%, a Young's modulus of 293 MPa, and a higher molecular weight of 149 kDa.

Mesenchymal stem cells (MSCs) are widely used in regenerative medicine, including the treatment of pulmonary fibrosis. However, implanted MSCs disappear within days, constraining therapeutic efficacy, which is largely attributed to nutrient deprivation. In this study, we established glycogen metabolism engineering strategies in mammalian cells. By expressing a functionally optimized glycogen synthase (GYSmut), MSCs could accumulate large amounts of glycogen rapidly as a reserve substance. Glycogen engineering significantly improved the survival of MSCs during starvation both in vitro and in vivo, enhancing cell viability post-implantation and their therapeutic efficacy in pulmonary fibrosis. Glycogen-engineered MSCs may serve as chassis cells for further applications. Our research highlights the importance of glucose metabolism regulation in cell-based therapy and demonstrates the great potential for the metabolic engineering of MSCs and other therapeutic cells.

Background: Diabetic wounds are major clinical challenges, often complicated by oxidative stress and free radical generation. Hydrogen (H2), a selective antioxidant, offers potential as a therapeutic agent for chronic diabetic wounds. However, its precise mechanisms remain underexplored. Objective: This study aimed to investigate the protective effects of H2 on high glucose-induced oxidative damage and apoptosis in human skin cells. Methods: HaCaT keratinocytes and HSF fibroblasts were treated with high glucose or AGEs. Cell viability, oxidative stress markers, inflammatory cytokines, and apoptosis were analyzed. AGEs/RAGE/NF-κB signaling was evaluated via Western blot. Results: H2 treatment significantly reduced ROS, MDA, IL-1β, and TNF-α levels, while enhancing SOD and GSH activity. It also inhibited AGEs/RAGE/NF-κB signaling and apoptosis. Conclusion: Hydrogen therapy protects against oxidative stress and inflammation induced by high glucose or AGEs, offering potential as an adjunctive treatment for diabetic wound healing.

OBJECTIVES:To study the molecular mechanisms of LDH-loaded si-NEAT1 for regulating paclitaxel resistance and tumor-associated macrophage (TAM) polarization in breast cancer. METHODS:qRT-PCR and Western blotting were used to detect the expression of lncRNA NEAT1, miR-133b, and PD-L1 in breast cancer SKBR3 cells and paclitaxel-resistant SKBR3 cells (SKBR3-PR). The effects of transfection with si-NEAT1 and miR-133b mimics on MRP, MCRP and PD-L1 expressions and cell proliferation, migration and apoptosis were investigated using qRT-PCR, Western blotting, scratch and Transwell assays, and flow cytometry. Rescue experiments were conducted using si-NEAT1 and miR-133b inhibitor. Human THP-1 macrophages were cultured in the presence of conditioned media (CM) derived from SKBR3 and SKBR3-PR cells with or with si-NEAT1 transfection for comparison of IL-4-induced macrophage polarization by detecting the surface markers. LDH@si-NEAT1 nanocarriers were constructed, and their effects on MRP, MCRP and PD-L1 expressions and cell behaviors of the tumor cells were examined. THP-1 cells were treated with the CM from LDH@si-NEAT1-treated tumor cells, and the changes in their polarization were assessed. RESULTS:SKBR3-PR cells showered significantly upregulated NEAT1 and PD-L1 expressions and lowered miR-133b expression as compared with their parental cells. Transfection with si-NEAT1 and miR-133b mimics inhibited viability, promoted apoptosis and enhanced MRP and BCRP expressions in SKBR3-PR cells. NEAT1 knockdown obvious upregulated miR-133b and downregulated PD-L1, MRP and BCRP expressions. The CM from SKBR3-PR cells obviously promoted M2 polarization of THP-1 macrophages, which was significantly inhibited by CM from si-NEAT1-transfected cells. Treatment with LDH@si-NEAT1 effectively inhibited migration and invasion, promoted apoptosis, and reduced MRP, BCRP and PD-L1 expressions in the tumor cells. The CM from LDH@si-NEAT1-treated SKBR3-PR cells significantly downregulated Arg-1, CD163, IL-10, and PD-L1 and upregulated miR-133b expression in THP-1 macrophages. CONCLUSIONS:LDH@si-NEAT1 reduces paclitaxel resistance of breast cancer cells and inhibits TAM polarization by targeting the miR-133b/PD-L1 axis.

Polyhydroxyalkanoates (PHAs) are a family of biodegradable polyesters accumulated by various microorganisms. The halophilic microorganism Halomonas bluephagenesis has been developed as a production platform strain for various PHAs, chemicals and proteins under open nonsterile and continuous conditions. For the first time, H. bluephagenesis has been engineered to synthesize a copolymer of a short chain length PHA (SCL PHA) and a long chain length PHA (LCL PHA) called, SCL-co-LCL PHA. This study engineered H. bluephagenesis for synthesizing SCL-co-LCL PHA via beta-oxidation inhibition and expression of heterologous PHA synthase (PhaC). Sodium oleate was utilized as a structurally related carbon source due to its low cost, long carbon chain length, unsaturated bonds and sustainability. The engineered H. bluephagenesis successfully synthesized copolymers of 3-hydroxybutanoic acid (3HB) and 3-hydroxy-9-octadecenoic acid (3H9Od) on sodium oleate. It could be grown to 8 g L-1 cell dry weight (CDW) containing 51 wt% P(3HB-co-12 mol% 3H9Od) in shake flasks, and 28 g L-1 cell dry weight (CDW) containing 53 wt% P(3HB-co-6 mol% 3H9Od) in 7 L fermenters. Subsequently, the thermal properties of this new PHA were characterized. Finally, chemical click reactions were applied to modify P3HB3H9Od into an organogel, and rare-earth modified fluorescent PHA was successfully prepared.

Bacillus subtilis, as a probiotic feed additive, has been increasingly applied in livestock and poultry farming. In the present study, the environmental tolerance of a strain of Bacillus subtilis, isolated from a goat farm, was investigated. This article conducts a series of experiments on the obtained strains. The results demonstrated that the isolated strain exhibits strong tolerance to high temperatures, acidic and alkaline conditions, and high concentrations of bile salts. Furthermore, its self-aggregation rate exceeded 60% after 24 h. Whole-genome sequencing revealed that the genomes of the isolated strains were functionally annotated, identifying genes associated with amino acid metabolism, vitamin biosynthesis, and other metabolic pathways. Based on this genomic analysis, the present study further evaluated the anti-inflammatory effects of a Bacillus subtilis strain isolated in a murine model of colitis induced by dextran sulfate sodium (DSS). The analyses revealed that the DSS-treated group exhibited significantly reduced expression of intestinal tight junction proteins ZO-1 and Occludin, along with elevated expression of pro-inflammatory cytokines, compared to the PBS control group. Following oral administration of 1 × 108 CFU/mL Bacillus subtilis isolated strain suspension, the DSS-treated mice showed increased expression of ZO-1 and Occludin and decreased levels of pro-inflammatory cytokines. These results indicate that the isolated strain of Bacillus subtilis has a protective effect against colitis and demonstrates probiotic potential.

Abstract Most bacterial cells are 1–2 microns in size, limiting intracellular products like polyhydroxyalkanoates (PHA) accumulation. Cell size is regulated by key genes such as mreB and minCD, which encode cellular skeleton protein and control cell fission ring location, respectively. Their expression changes significantly affect microbial growth. This study successfully redesigns the ClpXP protein degradation system by deleting the sspB gene and using mutated SsrA tags with different degradation rates to control MreB degradation. Dynamic degradation of MreB allows non‐model bacterium Halomonas bluephagenesis to grow normally and increase cell size simultaneously. Combined with overexpression of minCD, H. bluephagenesis with progressive MreB degradation increases the cell size further, albeit with a reduced growth rate. H. bluephagenesis CYL0307, with the PHB granule‐associated protein PhaP1 deleted and phaABRe overexpressed in the MreB‐degraded strain, increases cell volume more than nine times compared to the original strain. CYL0307 produces 149 g L−1 cell dry weight containing 82% PHB after 44 h in a 5000 L bioreactor, with cells containing single large PHB granules, simplifying recovery and purification. These results provide a post‐translational gene regulation method in H. bluephagenesis and a strategy for enhancing PHB production via morphological engineering.