ß-Carotene is an orange fat-soluble compound, which has been widely used in fields such as food, medicine and cosmetics owing to its anticancer, antioxidant and cardiovascular disease prevention properties. Currently, natural ß-carotene is mainly extracted from plants and algae, which cannot meet the growing market demand, while chemical synthesis of ß-carotene cannot satisfy the pursuit for natural products of consumers. The ß-carotene production through microbial fermentation has become a promising alternative owing to its high efficiency and environmental friendliness. With the rapid development of synthetic biology and in-depth study on the synthesis pathway of ß-carotene, microbial fermentation has shown promising applications in the ß-carotene synthesis. Accordingly, this review aims to summarize the research progress and strategies of natural carotenoid producing strain and metabolic engineering strategies in the heterologous synthesis of ß-carotene by engineered microorganisms. Moreover, it also summarizes the adoption of inexpensive carbon sources to synthesize ß-carotene as well as proposes new strategies that can further improve the ß-carotene production.
Biological Products , beta Carotene , Fermentation , Carotenoids , Antioxidants
Familial non-medullary thyroid carcinoma (FNMTC) is a type of thyroid cancer characterized by genetic susceptibility, representing approximately 5% of all non-medullary thyroid carcinomas. While some cases of FNMTC are associated with familial multi-organ tumor predisposition syndromes, the majority occur independently. The genetic mechanisms underlying non-syndromic FNMTC remain unclear. Initial studies utilized SNP linkage analysis to identify susceptibility loci, including the 1q21 locus, 2q21 locus, and 4q32 locus, among others. Subsequent research employed more advanced techniques such as Genome-wide Association Study and Whole Exome Sequencing, leading to the discovery of genes such as IMMP2L, GALNTL4, WDR11-AS1, DUOX2, NOP53, MAP2K5, and others. But FNMTC exhibits strong genetic heterogeneity, with each family having its own pathogenic genes. This is the first article to provide a chromosomal landscape map of susceptibility genes associated with non-syndromic FNMTC and analyze their potential associations. It also presents a detailed summary of variant loci, characteristics, research methodologies, and validation results from different countries.
The mechanism by which mammalian liver cell responses are coordinated during tissue homeostasis and perturbation is poorly understood, representing a major obstacle in our understanding of many diseases. This knowledge gap is caused by the difficulty involved with studying multiple cell types in different states and locations, particularly when these are transient. We have combined Stereo-seq (spatiotemporal enhanced resolution omics-sequencing) with single-cell transcriptomic profiling of 473,290 cells to generate a high-definition spatiotemporal atlas of mouse liver homeostasis and regeneration at the whole-lobe scale. Our integrative study dissects in detail the molecular gradients controlling liver cell function, systematically defining how gene networks are dynamically modulated through intercellular communication to promote regeneration. Among other important regulators, we identified the transcriptional cofactor TBL1XR1 as a rheostat linking inflammation to Wnt/ß-catenin signaling for facilitating hepatocyte proliferation. Our data and analytical pipelines lay the foundation for future high-definition tissue-scale atlases of organ physiology and malfunction.
The remarkable regenerative ability of the skin, governed by complex molecular mechanisms, offers profound insights into the skin repair processes and the pathogenesis of various dermatological conditions. This understanding, derived from studies in human skin and various model systems, has not only deepened our knowledge of skin regeneration but also facilitated the development of skin substitutes in clinical practice. Recent research highlights the crucial role of lymphatic vessels in skin regeneration. Traditionally associated with fluid dynamics and immune modulation, these vessels are now recognized for interacting with skin stem cells and coordinating regeneration. This Mini Review provides an overview of recent advancements in basic and translational research related to skin regeneration, focusing on the dynamic interplay between lymphatic vessels and skin biology. Key highlights include the critical role of stem cell-lymphatic vessel crosstalk in orchestrating skin regeneration, emerging translational approaches, and their implications for skin diseases. Additionally, the review identifies research gaps and proposes potential future directions, underscoring the significance of this rapidly evolving research arena.
Background: Familial non-medullary thyroid carcinoma (FNMTC) is a genetically predisposed disease with unclear genetic mechanisms. This makes research on susceptibility genes important for the diagnosis and treatment options. Methods: This study included a five-member family affected by papillary thyroid carcinoma. The candidate genes were identified through whole-exome sequencing and Sanger sequencing in family members, other FNMTC patients, and sporadic non-medullary thyroid carcinoma patients. The pathogenicity of the mutation was predicted using in silico tools. Cell phenotype experiments in vitro and models of lung distant metastasis in vivo were conducted to confirm the characteristics of the mutation. Transcriptome sequencing and mechanistic validation were employed to compare the disparities between PAK4 wild-type (WT) and PAK4 mutant (MUT) cell lines. Results: This mutation alters the protein structure, potentially increasing instability by affecting hydrophobicity, intra-molecular hydrogen bonding, and phosphorylation sites. It specifically promotes phosphorylated PAK4 nuclear translocation and expression in thyroid tissue and cell lines. Compared with the WT cells line, PAK4 I417T demonstrates enhanced proliferation, invasiveness, accelerated cell division, and inhibition of cell apoptosis in vitro. In addition, it exhibits a significant propensity for metastasis in vivo. It activates tumor necrosis factor signaling through increased phosphorylation of PAK4, JNK, NFκB, and c-Jun, unlike the WT that activates it via the PAK4-NFκ-MMP9 axis. In addition, PAK4 MUT protein interacts with matrix metalloproteinase (MMP)3 and regulates MMP3 promoter activity, which is not observed in the WT. Conclusions: Our study identified PAK4: c.T1250C: p.I417T as a potential susceptibility gene for FNMTC. The study concludes that the mutant form of PAK4 exhibits oncogenic function, suggesting its potential as a novel diagnostic molecular marker for FNMTC.
D-glucuronic acid is a kind of glucose derivative, which has excellent properties such as anti-oxidation, treatment of liver disease and hyperlipidemia, and has been widely used in medicine, cosmetics, food and other fields. The traditional production methods of D-glucuronic acid mainly include natural extraction and chemical synthesis, which can no longer meet the growing market demand. The production of D-glucuronic acid by biocatalysis has become a promising alternative method because of its high efficiency and environmental friendliness. This review describes different production methods of D-glucuronic acid, including single enzyme catalysis, multi-enzyme cascade, whole cell catalysis and co-culture, as well as the intervention of some special catalysts. In addition, some feasible enzyme engineering strategies are provided, including the application of enzyme immobilized scaffold, enzyme mutation and high-throughput screening, which provide good ideas for the research of D-glucuronic acid biocatalysis.
Engineering , Biocatalysis , Catalysis , Coculture Techniques , Glucuronic Acid
Carotenoids, as a type of tetraterpene compound, have been widely used in food, medical, and health areas owing to their antioxidant, immune enhancement, and disease risk reduction effects. Rhodosporidium toruloides is a promising oleaginous red yeast that can industrially synthesize carotenoids. In this study, the effects of different light exposure times and intervals on carotenoid production by R. toruloides Z11 were first investigated. Results showed that a higher carotenoid content (1.29 mg/g) can be achieved when R. toruloides Z11 was exposed to light for 12 h per day, which was increased by 1.98 times compared with that of dark cultivation. Transcriptome profiling revealed that light stress could effectively promote the gene expression levels of GGPS1 and AL1 in the carotenoid biosynthesis pathway and phr in the DNA photolysis pathway of R. toruloides. This work will provide a molecular foundation to further improve the production efficiency of carotenoids by genetic engineering.
Basidiomycota , Rhodotorula , Genetic Engineering , Rhodotorula/genetics , Carotenoids/metabolism , Basidiomycota/genetics , Basidiomycota/metabolism
Spatial transcriptomics analysis allows the examination of the biological characteristics and spatial distribution of individual lung cells at a single-cell resolution. However, due to the presence of cavities in the alveoli of the lungs, it is challenging to section them for spatial transcriptomics experiments. Here, we present a protocol for acquiring high-quality fresh mouse lung spatial transcriptomics data. We describe steps for lung perfusion, acquiring frozen slices, collecting cDNA from lung sections, and data analysis. For complete details on the use and execution of this protocol, please refer to Jiang et al.1.
Data Analysis , Gene Expression Profiling , Animals , Mice , DNA, Complementary , Perfusion , Lung
Familial non-medullary thyroid carcinoma (FNMTC) accounts for 3% to 9% of all thyroid cancer cases, yet its genetic mechanisms remain unknown. Our study aimed to screen and identify novel susceptibility genes for FNMTC. Whole-exome sequencing (WES) was conducted on a confirmed FNMTC pedigree, comprising four affected individuals across two generations. Variants were filtered and analyzed using ExAC and 1000 Genomes Project, with candidate gene pathogenicity predicted using SIFT, PolyPhen, and MutationTaster. Validation was performed through Sanger sequencing in affected pedigree members and sporadic patients (TCGA database) as well as general population data (gnomAD database). Ultimately, we identified the mutant PPP4R3A (NC_000014.8:g.91942196C>T, or NM_001366432.2(NP_001353361.1):p.(Asp409Asn), based on GRCH37) as an FNMTC susceptibility gene. Subsequently, a series of functional experiments were conducted to investigate the impact of PPP4R3A and its Asp409Asn missense variant in thyroid cancer. Our findings demonstrated that wild-type PPP4R3A exerted tumor-suppressive effects via the Akt-mTOR-P70 S6K/4E-BP1 axis. However, overexpression of the PPP4R3A Asp409Asn mutant resulted in loss of tumor-suppressive function, ineffective inhibition of cell invasion, and even promotion of cell proliferation and migration by activating the Akt/mTOR signaling pathway. These results indicated that the missense variant PPP4R3A Asp409Asn is a candidate susceptibility gene for FNMTC, providing new insights into the diagnosis and intervention of FNMTC.
Gel materials are appealing due to their diverse applications in biomedicine, soft electronics, sensors, and actuators. Nevertheless, the existing synthetic gels are often plagued by feeble network structures and inherent defects associated with solvents, which compromise their mechanical load-bearing capacity and cast persistent doubts about their reliability. Herein, combined with attractive deep eutectic solvent (DES), a stepwise-enhanced strategy is presented to fabricate ultrarobust eutectogels. It focuses on the continuous modulation and optimization of polymer networks through complementary annealing and solvent exchange processes, which drives a progressive increase in both quantity and mass of the interconnected polymer chains at microscopic scale, hence contributing to the evolutionary enhancement of network structure. The resultant eutectogel exhibits superb mechanical properties, including record-breaking strength (31.8 MPa), toughness (76.0 MJ m-3 ), and Young's modulus (25.6 MPa), together with exceptional resistance ability to tear and crack propagation. Moreover, this eutectogel is able to be further programmed through photolithography to in situ create patterned eutectogel for imparting specific functionalities. Enhanced by its broad applicability to various DES combinations, this stepwise-enhanced strategy is poised to serve as a crucial template and methodology for the future development of robust gels.
As an amino acid derivative and a typical compatible solute, ectoine can assist microorganisms in resisting high osmotic pressure. Own to its long-term moisturizing effects, ectoine shows extensive applications in cosmetics, medicine and other fields. With the rapid development of synthetic biology and fermentation engineering, many biological strategies have been developed to improve the ectoine production and simplify the production process. Currently, the microbial fermentation has been widely used for large scaling ectoine production. Accordingly, this review will introduce the metabolic pathway for ectoine synthesis and also comprehensively evaluate both wild-type and genetically modified strains for ectoine production. Furthermore, process parameters affecting the ectoine production efficiency and adoption of low cost substrates will be evaluated. Lastly, future prospects on the improvement of ectoine production will be proposed.
Amino Acids, Diamino , Amino Acids, Diamino/chemistry , Amino Acids, Diamino/metabolism , Fermentation , Metabolic Networks and Pathways
Background: Current guidelines lack a standardized management for patients with family history of thyroid carcinoma (fTC),particularly benign thyroid neoplasm (fBTN). Our objective was to investigate the influence of various family histories on the selection of surgical approaches and disease-free survival (DFS). Methods: A cohort study was conducted involving 2261 patients diagnosed with differentiated thyroid carcinoma including those with fTC (n=224), fBTN (n=122), and individuals without a family history of thyroid carcinoma (nfTC; n=1915). Clinicopathological characteristics were collected. DFS was estimated using Kaplan-Meier analysis and factors affecting DFS were identified using Cox proportional hazard model. Results: Compared to nfTC, small tumor size, clinically lymph node-positive, extrathyroidal extension, vascular invasion, Hashimoto's disease and nodular goiter were more common in fTC and fBTN groups. They had lower T stage and a lower rate of good response to TSH suppression therapy but received more radioiodine therapy. It is worth noting that fTC is associated with male, bilateral and multifocal tumors, as well as central lymph node metastasis and distant metastasis. Both fTC (aHR = 2.45, 95% CI=1.11-5.38; P = 0.03) and fBTN (aHR = 3.43, 95% CI=1.27-9.29; P = 0.02) were independent predictors of DFS in patients who underwent lobectomy, but not total thyroidectomy. For 1-4 cm thyroid carcinomas with clinically node-negative, fTC was identified as an independent predictor, whereas fBTN was not. Conclusion: Our findings indicate that a family history, particularly of malignancy, is associated with a more aggressive disease. Family history does not affect the prognosis of patients who undergo total thyroidectomy, but it may increase the risk of postoperative malignant events in those who have a lobectomy. Additionally, it may be necessary to monitor individuals with a family history of benign thyroid neoplasms.
Adenoma, Oxyphilic , Thyroid Neoplasms , Humans , Male , Disease-Free Survival , Cohort Studies , Iodine Radioisotopes , Retrospective Studies , Thyroid Neoplasms/genetics , Thyroid Neoplasms/surgery , Thyroid Neoplasms/diagnosis
Exposure to ultraviolet (UV) rays is a known risk factor for skin cancer, which can be notably mitigated through the application of sun care products. However, escalating concerns regarding the adverse health and environmental impacts of synthetic anti-UV chemicals underscore a pressing need for the development of biodegradable and eco-friendly sunscreen ingredients. Mycosporine-like amino acids (MAAs) represent a family of water-soluble anti-UV natural products synthesized by various organisms. These compounds can provide a two-pronged strategy for sun protection as they not only exhibit a superior UV absorption profile but also possess the potential to alleviate UV-induced oxidative stresses. Nevertheless, the widespread incorporation of MAAs in sun protection products is hindered by supply constraints. Delving into the biosynthetic pathways of MAAs can offer innovative strategies to overcome this limitation. Here, we review recent progress in MAA biosynthesis, with an emphasis on key biosynthetic enzymes, including the dehydroquinate synthase homolog MysA, the adenosine triphosphate (ATP)-grasp ligases MysC and MysD, and the nonribosomal peptide synthetase (NRPS)-like enzyme MysE. Additionally, we discuss recently discovered MAA tailoring enzymes. The enhanced understanding of the MAA biosynthesis paves the way for not only facilitating the supply of MAA analogs but also for exploring the evolution of this unique family of natural sunscreens. ONE-SENTENCE SUMMARY: This review discusses the role of mycosporine-like amino acids (MAAs) as potent natural sunscreens and delves into recent progress in their biosynthesis.
Amino Acids , Sunscreening Agents , Amino Acids/chemistry , Sunscreening Agents/chemistry , Sunscreening Agents/pharmacology , Oxidative Stress , Ultraviolet Rays
Botulinum neurotoxin (BoNT) shows high lethality and toxicity, marking it as an important biological threat. The only effective post-exposure therapy is botulinum antitoxin; however, such products have great potential for improvement. To prevent or treat BoNT, monoclonal antibodies (mAbs) are promising agents. Herein, we aimed to construct a bispecific antibody (termed LUZ-A1-A3) based on the anti-BoNT/A human monoclonal antibodies (HMAb) A1 and A3. LUZ-A1-A3 binds to the Hc and L-HN domains of BoNT/A, displaying potent neutralization activity against BoNT/A (124 × higher than that of HMAb A1 or HMAb A3 alone and 15 × higher than that of the A1 + A3 combination). LUZ-A1-A3 provided effective protection against BoNT/A in an in vivo mouse model. Mice were protected from infection with 500 × LD50 of BoNT/A by LUZ-A1-A3 from up to 7 days before intraperitoneal administration of BoNT/A. We also demonstrated the effective therapeutic capacity of LUZ-A1-A3 against BoNT/A in a mouse model. LUZ-A1-A3 (5 µg/mouse) neutralized 20 × LD50 of BoNT/A at 3 h after intraperitoneal BoNT/A administration and complete neutralized 20 × LD50 of BoNT/A at 0.5 h after intraperitoneal BoNT/A administration. Thus, LUZ-A1-A3 is a promising agent for the pre-exposure prophylaxis and post-exposure treatment of BoNT/A.
Botulinum Toxins, Type A , Botulism , Humans , Mice , Animals , Serogroup , Antibodies, Monoclonal/pharmacology , Disease Models, Animal , Lethal Dose 50 , Botulism/prevention & control
Nitro aromatics have broad applications in industry, agriculture, and pharmaceutics. However, their industrial production is faced with many challenges including poor selectivity, heavy pollution and safety concerns. Nature provides multiple strategies for aromatic nitration, which opens the door for the development of green and efficient biocatalysts. Our group's efforts focused on a unique bacterial cytochrome P450 TxtE that originates from the biosynthetic pathway of phytotoxin thaxtomins, which can install a nitro group at C4 of l-Trp indole ring. TxtE is a Class I P450 and its reaction relies on a pair of redox partners ferredoxin and ferredoxin reductase for essential electron transfer. To develop TxtE as an efficient nitration biocatalyst, we created artificial self-sufficient P450 chimeras by fusing TxtE with the reductase domain of the bacterial P450BM3 (BM3R). We evaluated the catalytic performance of the chimeras with different lengths of the linker connecting TxtE and BM3R domains and identified one with a 14-amino-acid linker (TB14) to give the best activity. In addition, we demonstrated the broad substrate scope of the engineered biocatalyst by screening diverse l-Trp analogs. In this chapter, we provide a detailed procedure for the development of aromatic nitration biocatalysts, including the construction of P450 fusion chimeras, biochemical characterization, determination of catalytic parameters, and testing of enzyme-substrate scope. These protocols can be followed to engineer other P450 enzymes and illustrate the processes of biocatalytic development for the synthesis of nitro chemicals.
Cytochrome P-450 Enzyme System , Ferredoxins , Ferredoxins/metabolism , Cytochrome P-450 Enzyme System/metabolism , Biocatalysis , Amino Acids/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism
Bis (2-hydroxyethyl) terephthalate (BHET) is one of the main compounds produced by enzymatic hydrolysis or chemical depolymerization of polyethylene terephthalate (PET). However, the lack of understanding on BHET microbial metabolism is a main factor limiting the bio-upcycling of PET. In this study, BHET-degrading strains of Rhodococcus biphenylivorans GA1 and Burkholderia sp. EG1 were isolated and identified, which can grow with BHET as the sole carbon source. Furthermore, a novel esterase gene betH was cloned from strain GA1, which encodes a BHET hydrolyzing esterase with the highest activity at 30 °C and pH 7.0. In addition, the co-culture containing strain GA1 and strain EG1 could completely degrade high concentration of BHET, eliminating the inhibition on strain GA1 caused by the accumulation of intermediate metabolite ethylene glycol (EG). This work will provide potential strains and a feasible strategy for PET bio-upcycling.
Phthalic Acids , Rhodococcus , Esterases , Phthalic Acids/metabolism , Hydrolysis , Polyethylene Terephthalates/chemistry , Polyethylene Terephthalates/metabolism , Rhodococcus/metabolism
Artificial multi-enzyme cascades bear great potential for bioconversion of C1 compounds to value-added chemicals. Over the past decade, massive efforts have been devoted to constructing multi-enzyme cascades to produce glycolic acid, rare functional sugars and even starch from C1 compounds. However, in contrast to traditional fermentation utilizing C1 compounds with the expectation of competitive economic performance in future industrialization, multi-enzyme cascades systems in the proof-of-concept phase are facing the challenges of upscaling. Here, we offered an overview of the recent advances in the construction of in vitro multi-enzyme cascades and whole-cell transformation using C1 compounds as substrate. In addition, the existing challenges and possible solutions were also discussed aiming to combine the strengths of in vitro and in vivo multi-enzyme cascades systems for upscaling.
As the most abundant and renewable natural resource in the world, lignocellulose is a promising alternative to fossil energy to relieve environmental concerns and resource depletion. However, due to its recalcitrant structure, strains with efficient degradation capability still need exploring. In this study, a fungus was successfully isolated from decayed wood and named as Trichoderma asperellum LYS1 by phylogenetic and draft genomic analysis. The further investigations showed that strain LYS1 had an outstanding performance on lignocellulose degradation, especially for hemicellulose-rich biomass. After the analysis of encoded CAZymes, mainly on GH family, a large amount of genes coding ß-glucosidase and xylanase may contribute to the high degradation of cellulose and hemicellulose. Collectively, the results generated in this study demonstrated that T. asperellum LYS1 is a potential cell factory for lignocellulose biorefinery.
Cellulase , Trichoderma , Cellulase/genetics , Cellulase/metabolism , Biomass , Phylogeny
Microbial synthesis of target chemicals usually involves multienzymatic reactions in vivo, especially for compounds with a long metabolic pathway. However, when various genes are introduced into one single strain, it leads to a heavy metabolic burden. In contrast, the microbial coculture system can allocate metabolic pathways into different hosts, which will relieve the metabolic burdens. Escherichia coli is the most used chassis to synthesize biofuels and chemicals owing to its well-known genetics, high transformation efficiency, and easy cultivation. Accordingly, cocultures containing the cooperative E. coli with other microbial species have received great attention. In this review, the individual applications and boundedness of different combinations will be summarized. Additionally, the strategies for the self-regulation of population composition, which can help enhance the stability of a coculture system, will also be discussed. Finally, perspectives for the cocultures will be proposed.
Escherichia coli , Metabolic Engineering , Escherichia coli/genetics , Escherichia coli/metabolism , Coculture Techniques , Metabolic Networks and Pathways , Biofuels
The 3' untranslated region (UTR) of Senecavirus A (SVA) was predicted to harbor two hairpin structures, hairpin-I and -II. The former is composed of two internal loops, one terminal loop and three stem regions; the latter comprises one internal loop, one terminal loop and two stem regions. In this study, we constructed a total of nine SVA cDNA clones, which contained different point mutations within a stem-formed motif in the hairpin-I or -II, for rescuing replication-competent viruses. Only three mutants were successfully rescued and moreover genetically stable during at least five serial passages. Computer-aided prediction showed these three mutants bearing either a wild-type or a wild-type-like hairpin-I in their individual 3' UTRs. Neither wild-type nor wild-type-like hairpin-I could be computationally predicted to exist in 3' UTRs of the other six unviable "viruses". The results suggested that the wild-type or wild-type-like hairpin-I was necessary in the 3' UTR for SVA replication.
RNA, Viral , Virus Replication , 3' Untranslated Regions , Base Sequence , RNA, Viral/genetics , RNA, Viral/chemistry , Cell Line , Nucleic Acid Conformation
