Journal of biotechnology最新文献

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, and classifying the developmental stages of HCC can help with early prognosis and treatment. This study aimed to investigate diagnostic and prognostic molecular signatures underlying the progression of HCC, including tumor initiation and growth, and to classify its developmental stages based on gene expression levels. We integrated data from two cancer systems, including 78 patients with Edmondson-Steiner (ES) grade and 417 patients with TNM stage cancer. Functional profiling was performed using identified signatures. Using a multinomial logistic regression model (MLR), we classified controls, early-stage HCC, and advanced-stage HCC. The model was validated in three independent cohorts comprising 45 patients (neoplastic stage), 394 patients (ES grade), and 466 patients (TNM stage). Multivariate Cox regression was employed for HCC prognosis prediction. We identified 35 genes with gradual upregulation or downregulation in both ES grade and TNM stage patients during HCC progression. These genes are involved in cell division, chromosome segregation, and mitotic cytokinesis, promoting tumor cell proliferation through the mitotic cell cycle. The MLR model accurately differentiated controls, early-stage HCC, and advanced-stage HCC across multiple cancer systems, which was further validated in various independent cohorts. Survival analysis revealed a subset of five genes from TNM stage (HR: 3.27, p < 0.0001) and three genes from ES grade (HR: 7.56, p < 0.0001) that showed significant association with HCC prognosis. The identified molecular signature not only initiates tumorigenesis but also promotes HCC development. It has the potential to improve clinical diagnosis, prognosis, and therapeutic interventions for HCC. This study enhances our understanding of HCC progression and provides valuable insights for precision medicine approaches.

Lactic acid bacteria (LAB) are known to exhibit various beneficial roles in fermentation, serving as probiotics, and producing a plethora of valuable compounds including antimicrobial activity such as bacteriocin-like inhibitory substance (BLIS) that can be used as biopreservative to improve food safety and quality. However, the yield of BLIS is often limited, which poses a challenge to be commercially competitive with the current preservation practice. Therefore, the present work aimed to establish an optimised two-plasmid CRISPR/Cas9 system to redirect the carbon flux away from lactate towards compounds with antimicrobial activity by disrupting lactate dehydrogenase gene (ldh) on various strains of LAB. The lactic acid-deficient (ldhΔ) strains caused a metabolic shift resulting in increased inhibitory activity against selected foodborne pathogens up to 78 % than the wild-type (WT) strain. The most significant effect was depicted by Enterococcus faecalis-ldh∆ which displayed prominent bactericidal effects against all foodborne pathogens as compared to the WT that showed no antimicrobial activity. The present work provided a framework model for economically important LAB and other beneficial bacteria to synthesise and increase the yield of valuable food and industrial compounds. The present work reported for the first time that the metabolism of selected LAB can be manipulated by modifying ldh to attain metabolites with higher antimicrobial activity.

Enzymatic production of nucleotide sugars on a multigram scale presents a challenge, as only a few processes have been reported for large-scale nucleotide sugar production. They rely primarily on batch synthesis and employ exceptional amounts of enzymes. This study introduces a novel approach for the multigram-scale production of nucleotide sugars with a continuous fed-batch membrane reactor. We successfully synthesized five main nucleotide sugars: UDP-Gal, UDP-GalNAc, UDP-GlcA, GDP-Man, and CMP-Neu5Ac on a multigram scale. Efficient biocatalyst utilization results in high performance, including space-time yield (STY, g*L⁻¹h⁻¹), total turnover number (TTN, g product per g enzyme), and an efficient product formation rate (g/h) suitable for industrially relevant bioprocesses. The established continuous-fed batch reactor system produced up to 8.2 g CMP-Neu5Ac in three consecutive productions in less than 15 h with satisfying TTNs of 91 g_Product/g_Enzyme. Continuous production of UDP-GlcA over 28 h resulted in a final product amount of 14.8 g and TTN of 493 g_P/g_E. This process enables the production of nucleotide sugars with stable product formation, requiring minimal technical equipment for multigram quantities of nucleotide sugars at the laboratory scale. Notably, the system exhibited robustness and flexibility, allowing its application to various enzymatic nucleotide sugar synthesis cascades.

This research delved into the effects of nutrient limitation on the level of sporulation and the cadmium adsorption capacity of the bacterium Bacillus sp. isolated from the rhizosphere of endemic soils in the Region of Valparaiso, Chile. The bacteria were subjected to nitrogen limitation in fed-batch mode and were compared to bacteria grown in batch culture without nutrient limitation. The cultures were carried out in a 3 L bioreactor with an external nitrogen supply of ammonium at a flow of 0.123 L h⁻¹. The specific maximum growth rate was 0.42 h⁻¹ in batch and 0.45 h⁻¹ in the exponential phase of the fed-batch.

The analysis of sporulation did not show any significant difference between the biomass coming from the fed-batch and batch cultures.

It was found that maximum cadmium adsorption capacity varied with culture strategy. The dry biomass grown without nutrient limitation exhibited a maximum adsorption capacity for cadmium of 65.0 mg_Cd g⁻¹_biomass. Conversely, the limited biomass achieved a lower cadmium adsorption capacity of approximately 36.0 mg_Cd g⁻¹_biomass. FTIR analysis showed that nitrogen limitation induced changes in the composition of the outer cell wall, specifically an increase of deacetlylated polysaccharides, reducing the relative amount of secondary amines and proteins from the peptidoglycan matrix. Amino groups from acetylated polysaccharides and proteins have been associated elsewhere with greater cadmium affinity, which could explain the poor results obtained with the nitrogen-restricted biomass. This study shows that new physiological states displaying different adsorption capabilities were effectively obtained by engineering the cell coverage of the bacteria using varying culture strategies. The fed-batch culture proved to be a valuable tool for studying PGPR strains for biosorption and other applications. Exploring diverse nutrient limitations and other pollutants in this bacterium and other members of the PGPR family offer great opportunities to tailor biosorption strategies based on specific conditions, ultimately contributing to sustainable environmental solutions.

Vanillin is an inhibitor of lignocellulose hydrolysate, which can reduce the ability of Saccharomyces cerevisiae to utilize lignocellulose, which is an important factor limiting the development of the ethanol fermentation industry. In this study, mutants of vanillin-tolerant yeast named H6, H7, X3, and X8 were bred by heavy ion irradiation (HIR) combined with adaptive laboratory evolution (ALE). Phenotypic tests revealed that the mutants outperformed the original strain WT in tolerance, growth rate, genetic stability and fermentation ability. At 1.6 g/L vanillin concentration, the average OD₆₀₀ value obtained for mutant strains was 0.95 and thus about 3.4-fold higher than for the wild-type. When the concentration of vanillin was 2.0 g/L, the glucose utilization rate of the mutant was 86.3 % within 96 h, while that of the original strain was only 70.0 %. At this concentration of vanillin, the mitochondrial membrane potential of the mutant strain recovered faster than that of the original strain, and the ROS scavenging ability was stronger. We analyzed the whole transcriptome sequencing map and the whole genome resequencing of the mutant, and found that DEGs such as FLO9, GRC3, PSP2 and SWF1, which have large differential expression multiples and obvious mutation characteristics, play an important role in cell flocculation, rDNA transcription, inhibition of DNA polymerase mutation and protein palmitoylation. These functions can help cells resist vanillin stress. The results show that combining HIR with ALE is an effective mutagenesis strategy. This approach can efficiently obtain Saccharomyces cerevisiae mutants with improved vanillin tolerance, and provide reference for obtaining robust yeast strains with lignocellulose inhibitor tolerance.

D-allulose, a naturally occurring monosaccharide, is present in small quantities in nature. It is considered a valuable low-calorie sweetener due to its low absorption in the digestive tract and zero energy for growth. Most of the recent efforts to produce D-allulose have focused on in vitro enzyme catalysis. However, microbial fermentation is emerging as a promising alternative that offers the advantage of combining enzyme manufacturing and product synthesis within a single bioreactor. Here, a novel approach was proposed for the efficient biosynthesis of D-allulose from glycerol using metabolically engineered Escherichia coli. FbaA, Fbp, AlsE, and A6PP were used to construct the D-allulose synthesis pathway. Subsequently, PfkA, PfkB, and Pgi were disrupted to block the entry of the intermediate fructose-6-phosphate (F6P) into the Embden−Meyerhof−Parnas (EMP) and pentose phosphate (PP) pathways. Additionally, GalE and FryA were inactivated to reduce D-allulose consumption by the cells. Finally, a fed-batch fermentation process was implemented to optimize the performance of the cell factory. As a result, the titer of D-allulose reached 7.02 g/L with a maximum yield of 0.287 g/g.

This study addresses the challenges posed by rainfall variability, leading to water deficits during critical stages of crop growth, resulting in a drastic reduction of cotton yield. In a comprehensive evaluation, thirty cotton genotypes, including five Gossypium arboreum (wild) and twenty-five Gossypium hirsutum (cultivated), were grown under rainfed and irrigated conditions. Drought tolerance indices (DTI) were evaluated, categorizing genotypes based on their resilience. Further, in-vitro screening at the seedling stage (20 days) under PEG-induced drought identified tolerant genotypes exhibiting elevated levels of free proline (19.07±5.31 mg.g⁻¹⁰⁰fr.wt.), amino acids (34.59±6.51 mg.g⁻¹⁰⁰fr.wt.), soluble proteins (13.73±2.65 mg.g⁻¹fr.wt.), and glycine betaine (10.95±3.62 mg.g⁻¹⁰⁰fr.wt.), in their leaves, positively correlating (p<0.001) with relative water content (87.70±3.45 %). Molecular analysis using drought-specific simple sequence repeats (SSR) markers revealed significant genetic variability in a cotton genotypes, with lower observed and higher expected heterozygosity. F statistics exposed a higher level of gene flow corresponding to low differentiation among populations. Among the genotypes group, wild GAM-67 and cultivated Deviraj emerged as the most potent, exhibiting the higher DTI and diverse gene pools. Study exhibited higher total gene diversity in drought-tolerant wild GAM-67 (0.8501) and greater expected heterozygosity (0.626) and gene flow (0.6731) in cultivated Deviraj, underlining their robust genetic adaptability to drought conditions. The integrated approach of field evaluations, in-vitro screening, and molecular analyses explained substantial genetic diversity, with the identification of promising genotypes displaying higher drought tolerance indices, elevated levels of stress-related biochemical osmolytes, and pronounced genetic adaptability, thereby contributing to the advancement of breeding initiatives for enhanced drought resilience in cotton.

The degummed wastewater from silk processing contains a huge amount of amino acids and polypeptides from sericin. The silk degumming water is far from being exploited fully. Sericin in the degumming water is generally wasted and causes environmental pollution. In this study, simulated silk degumming water was hydrolyzed by alkaline protease to produce abundant amino acids and polypeptides. After enzymatic hydrolysis, the maximum free amino groups concentration in the silk degumming water was approximately 54 mM. It facilitated the recycling of silk degumming water for the production of melanin-like amino acid surfactants as raw materials. 4-Tert-butylcatechol was used as the starting material to generate o-quinone via oxidation by ceric ammonium nitrate. o-Quinone was coupled with free amino groups in enzymatic hydrolysates of silk degumming water to synthesize a sericin-based amino acid surfactant as hydrophobic and hydrophilic group, respectively. Through the green and simple synthesis route, the product was characterized to have a novel melanin-like structure. The product exhibited superior surface-active properties by lowering the surface tension to 32.39 mN m⁻¹. Furthermore, it demonstrated good foaming ability and foam stability, with the initial foam volume of 37 mL and the foam half-life time of more than 25 min. The product owned a good emulsification ability in the oil-water emulsion with delamination time of 297 s and 291 s for emulsion formed by soybean oil and liquid paraffin, respectively. The wetting time of the canvas sheet was only 134 s. Consequently, the product showed low surface tension, good foaming, emulsifying, and wetting properties.

ApxII is a vaccine antigen used to protect against porcine contagious pleuropneumonia, which is a significant threat to the pig industry. Here, we aimed to improve the proteolytic degradation stability of ApxII during its secretion by establishing a complete screening process of stable variants through bioinformatics and site-directed mutagenesis. We employed a combination of semi-rational and rational design strategies to create 34 single-point variants of ApxII. Among them, R114E and T115D variants exhibited better stability without compromising antigen activity. Furthermore, we constructed a multi-site variant, R114E/T115D, which demonstrated the best stability, activity, and yield. Protein stability and molecular dynamic analysis indicated that the greater solubility and lower structural expansion coefficient might explain the increased stability of R114E/T115D. Additionally, site T115 was identified as a key point of truncated ApxII stability. The R114E/T115D variant, with its proven stability and intact antigenic activity, holds promising prospects for industrial-scale applications in the prevention of porcine contagious pleuropneumonia.

The development of an environmentally benign method for the synthesis of nanoparticles has been facilitated by green chemistry. "Green synthesis" uses a range of biological elements like microbes, plants, and other biodegradable materials to produce NPs. Active biomolecules that are secreted by natural strains and present in the plant extracts serve as both reducing and capping/stabilizing agents. Microorganisms' intracellular enzymes can reduce metal ions, which explains how NPs might potentially nucleate. Plant-based synthesis of nanomaterials is particularly promising owing to abundant resources, simplicity of synthesis, and low cost. Silver nanoparticles (AgNPs) are attracting great attention in the research community due to their wide variety of applications in chemistry, food technology, microbiology, and biomedicine. Recent years have seen a large amount of research on the bio-genic synthesis of AgNPs employing biomaterials like plant extract and bacteria as reducing agents. Herein we discuss a thorough overview of the plant-based synthesis of silver nanoparticles (AgNPs), characterization approaches, applications, and toxicity. The review covers the green chemistry and nanotechnology elements of producing AgNPs, including a thorough discussion of the plant extract mediated synthesis, detailed formation mechanism, and a well-balanced emphasis on hazards and advantages. Based on current developments, the optimisation strategies, applications, and interdisciplinary characteristics are also covered in detail.