Applied Microbiology and Biotechnology最新文献

Strong sugar tolerance and high bioethanol yield of yeast under high-gravity fermentation have caused great attention in the bioethanol industry. In this study, Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) technology was used to knock out S. cerevisiae GPD2, FPS1, ADH2, DLD3, ERG5, NTH1, and AMS1 to construct engineering strain S. cerevisiae GFADENA. Under high-gravity fermentation with 400 g/L of sucrose, S. cerevisiae GFADENA produced 135 g/L ethanol, which increased 17% compared with the wild-type strain. In addition, S. cerevisiae GFADENA produced 145 g/L of ethanol by simultaneous saccharification and fermentation (SSF) using 400 g/L of corn syrup with a sugar-ethanol conversion rate of 41.1%. Further, the targeted metabolomics involving energy, amino acid, and free fatty acid metabolisms were performed to unravel its molecular mechanisms. The deletion of seven genes in S. cerevisiae GFADENA caused a more significant effect on energy metabolism compared with amino acid and free fatty acid metabolisms based on the significantly different metabolites. Two metabolites α-ketoglutaric acid and fructose-1,6-bisphosphate were the most significantly different upregulation and downregulation metabolites, respectively (p < 0.05). Functions of metabolism, environmental information processing, and genetic information processing were related to sucrose tolerance enhancement and ethanol production increase in S. cerevisiae GFADENA by the regulation of significantly different metabolites. This study provided an effective pathway to increase ethanol yield and enhance sucrose tolerance in S. cerevisiae through bioengineering modification.

• S. cerevisiae GFADENA with gene deletion was constructed by the CRISPR-Cas9 approach

• S. cerevisiae GFADENA could produce ethanol using high-gravity fermentation condition

• The ethanol yield of 145 g/L was produced using 400 g/L corn syrup by the SSF method

Degradation and erosion of soil is a significant threat to global food security and overall agricultural productivity. This issue is exacerbated by climate change and intensive human activity, meaning that the development of sustainable solutions for those problems is critical. Microbially induced calcite precipitation (MICP) offers a promising approach to stabilise soil particles; however, its applicability at low temperatures remains limited. In our study, we introduce a novel two-strain system combining the type strain for biocementation experiments, Sporosarcina pasteurii DSM 33, and Sporosarcina sp. ANT_H38, a novel, psychrotolerant strain obtained from the Antarctic. The novel strain enabled enhanced biocementation performance when combined with the type strain. Biocementation experiments showed a 3.5-fold increase in soil cohesion, while maintaining a similar internal friction angle compared to the type strain alone (10.7 kPa vs 34.12 kPa; 0.55 kPa for untreated soil). The increased cohesion significantly reduces susceptibility to erosion, offering a practical and sustainable solution. Furthermore, to better understand the mechanisms driving this process, we conducted a comprehensive bioinformatic analysis of the ANT_H38 genome, revealing unique cold-adaptive genes, as well as urease genes, which are evolutionarily distant from other Sporosarcina ureases. Those results provide valuable insights into the strain’s functional adaptations, particularly under low-temperature conditions. Overall, our study addresses a critical issue, offering a robust, nature-based solution that enhances soil resilience through MICP. Performed laboratory work confirms the potential of the system for real-world applications, while the comprehensive bioinformatic analysis provides the much needed context and information regarding the possible mechanisms behind the process.

• Antarctic Sporosarcina sp. ANT_H38 contains unique urease genes

• Two-strain ANT_H38/DSM33 system effectively stabilises soil at low temperatures

• Two-strain system has potential for stopping soil erosion and desertification

Fungal spores are usually dispersed by wind, water, and animal vectors. Climate change is accelerating the spread of pathogens to new regions. While well-studied vectors like bark beetles and moths contribute to pathogen transmission, other, less-recognized animal species play a crucial role at different scales. Small-scale dispersers, such as mites, rodents, squirrels, and woodpeckers, facilitate fungal spread within trees or entire forest regions. On a larger scale, birds contribute significantly to long-distance fungal dispersal, potentially aiding the establishment of invasive species across continents. These vectors remain underexplored and are often overlooked in fungal disease studies and are therefore called cryptic vectors. Understanding the full range of dispersal mechanisms is critical as climate change drive shifts in species distributions and increases vector activity. Expanding monitoring and detection tools to include these hidden carriers will improve our ability to track the distribution of fungal pathogens. Integrating targeted research, innovative technologies, and collaborative efforts across disciplines and borders is essential for enhancing disease management and mitigating fungal disease’s ecological and economic impacts.

• Cryptic animal vectors play a critical role in fungal spore dispersal across forests and continents.

• Climate change accelerates fungal pathogen spread by altering species distributions, increasing vector activity, and facilitating long-distance dispersal.

• Innovative monitoring tools, like eDNA sampling and predictive modelling, are essential to uncover cryptic vector contributions and mitigate fungal disease impacts.

Advances in the ethanol fermentation process are essential to improving the performance of bioethanol production. Fed-batch fermentation is a promising approach to increase the final ethanol titer, which benefits the recovery in the bioethanol industry’s downstream process. However, the development of feeding strategies, a crucial control variable in the fed-batch approach, is limited. Thus, in the present work, different modes of substrate delivery—fixed feeding, adapted feeding—were investigated in fed-batch cultures of Saccharomyces cerevisiae in a 5-L bioreactor. Evolved gas production, which was positively correlated with glucose consumption, was used to adjust the sugar feed rate in fed-batch fermentations under an adapted feeding strategy. The adapted feeding strategy enhanced ethanol productivity by 21% compared to the fixed feeding strategy, in which the sugar feed rate was stable, and the ethanol titer reached 91 g/L (~ 11.5%, v/v) at the end of fermentation. Moreover, cell biomass accumulation and cell growth rate were significantly improved when using the adapted feeding strategy. The effect of nitrogen availability on the performance of the adapted feeding strategy was further explored using a low-nitrogen content medium. The results showed that, even under low nitrogen feeding conditions (N/C = 0.046:10), the adapted feeding strategy maintained the same ethanol productivity as nitrogen-rich medium feeding. Overall, these results suggest that sugar delivery with low nitrogen content using the adapted feeding strategy could help reduce medium costs and improve the productivity of current facilities in the ethanol industry.Future work will integrate adapted feeding strategies with other fermentation approaches to improve ethanol production.

• Novel continuous sugar delivery was developed for fed-batch ethanol fermentation.

• The adapted feeding strategy improved ethanol productivity by 21%.

• The final ethanol concentration reached 91 g/L (11.5%, v/v) with no residual sugar.

In this study, the biological applications of cultivation methods related to cultivar selection, vegetative growth, and reproductive development in Lentinula edodes cultivation are briefly reviewed to clarify the current situation and inform future developments. The current cultivars widely used in the main production areas are derived from wild strains distributed in northern Asia. The most effective techniques for cultivar identification are molecular markers identified in two nuclear genome datasets and one mitochondrial genome dataset. The current stage of cultivar breeding is at the junction of Breeding 3.0 (biological breeding) and Breeding 4.0 (intelligent breeding). Plant breeder’s rights and patents have different emphases on new breeding variety protection, with the former being the most utilized globally. L. edodes is mostly produced on synthetic logs filled with sawdust substrates. Hardwood sawdust comprises approximately 80% of the substrates. The vegetative growth of L. edodes on synthetic logs involves two distinct stages of mycelial colonization and browning. Mycelia mainly perform glycolysis, tricarboxylic acid cycle, and respiratory metabolism reactions to produce energy and intermediates for synthesizing the structural components of hyphae in the vegetative colonization stage. Upon stimulation by physiological and environmental pressures after colonization, mycelia trigger gluconeogenesis, autophagy, and secondary metabolism, increase metabolic flux of pentose phosphate pathway, activate the glyoxylate cycle, and accumulate melanin on the surface of logs to ensure growth and survival. Sexually competent mycelia can form hyphal knots as a result of reprogrammed hyphal branching patterns after a period of vegetative growth (which varies by cultivar) and stimulation by specific environmental factors. Under a genetically encoded developmental program, hyphal knots undergo aggregation, tissue differentiation, primordium formation, meiosis in the hymenium, stipe elongation, basidiospore production and maturation, and cap expansion to form mature fruiting bodies. Growers can achieve good fruiting body shape and high yield by regulating the number of young fruiting bodies and adjusting specific environmental factors.

• Cultivar selection becomes less with the increasing technological requirement of L. edodes cultivation.

• L. edodes mycelia showed different biological events in the mycelial colonization and browning stages.

• Specific cultivar breading may be the next milestone in L. edodes cultivation.

Pseudomonas aeruginosa was used to synthesize anisotropic gold nanoparticles from the unusually reducible aryldiazonium gold (III) salt of the chemical formula [HOOC-4-C₆H₄N≡N]AuCl₄ (abbreviated as DS-AuCl₄). We investigated the effect of bacterial cell density, temperature, and pH on the AuNP synthesis. The bacterial cell density of 6.0 × 10⁸ CFU/mL successfully reduced 0.5 mM DS-AuCl₄ salt to AuNPs after incubation at 37 °C (24 h), 42 °C (24 h), and 25 °C (48 h). Transmission electron microscopy (TEM) images revealed the formation of spherical, triangle, star, hexagon, and truncated triangular morphologies for the AuNPs synthesized using P. aeruginosa bacteria. The average size of AuNPs synthesized at 25 °C (48 h), 37 °C (24 h), and 42 °C (24 h) was 39.0 ± 9.1 nm, 26.0 ± 8.1 nm, and 36.7 ± 7.7 nm, respectively. The average size of AuNPs synthesized at pH 3.7, 7.0, and 12.7 was 36.7 ± 7.7 nm, 14.7 ± 3.8 nm, and 7.3 ± 2.5 nm, respectively, with the average size decreasing at a pH of 12.7. The reduction of the DS-AuCl₄ salt was confirmed using X-ray photoelectron spectroscopy (XPS). The significant peaks for C1s, Au4f doublet, N1s, and O1s are centered at 285, 84–88, 400, and 532 eV. The ability of inactivated bacteria (autoclave-dead and mechanically lysed bacteria), peptidoglycan, and lipopolysaccharides to reduce the DS-AuCl₄ salt to AuNPs was also investigated. Anisotropic AuNPs were synthesized using inactivated bacteria and peptidoglycan but not using lipopolysaccharides. The AuNPs demonstrated biocompatibility with human RBCs and were safe, with no antibacterial activities against Escherichia coli and Staphylococcus aureus. This is the first report demonstrating the synthesis of AuNPs using aryldiazonium gold(III) salts with P. aeruginosa. These AuNPs are promising candidates for exploring potential applications in nanomedicine and drug delivery.

• Anisotropic AuNPs were synthesized using P. aeruginosa bacteria.

• Dead and lysed bacterial residues synthesized anisotropic AuNPs.

• AuNPs are hemocompatible.

Cysteine, a common amino acid used in food, cosmetic, and pharmaceutical industries, has a growth inhibitory effect. This growth inhibition by cysteine poses a problem, as the production of cysteine using microbial cells results in decreased cell growth and cysteine productivity. The underlying mechanism of growth inhibition by cysteine is unclear. This study aims to understand the mechanism of growth inhibition by cysteine in Corynebacterium glutamicum. To do this, cysteine-resistant mutants of C. glutamicum were isolated based on adaptive laboratory evolution (ALE) and their characteristics were analyzed. Genome resequencing revealed that mutations in the open reading frame of the ilvN gene encoding the regulatory small subunit of acetohydroxyacid synthase (AHAS), which is involved in branched-chain amino acid biosynthesis, were found in ALE cell populations and the isolated cysteine-resistant mutants. The ilvN mutations which are responsible for increased valine production resulted in improved cell growth in the presence of cysteine. Moreover, the addition of valine to the culture medium mitigated growth inhibition by cysteine, whereas the addition of leucine and isoleucine showed a slight mitigation. Additionally, the activity of AHAS from C. glutamicum was inhibited by cysteine, whereas AHAS from the strains carrying ilvN mutations exhibited resistance to cysteine. These results indicate that growth inhibition by cysteine is caused by perturbations in the biosynthesis of branched-chain amino acids, particularly valine in C. glutamicum. Furthermore, the cysteine-resistant mutants obtained by ALE demonstrated enhanced cysteine production as production hosts, suggesting that cysteine resistance is a useful phenotype for cysteine production in C. glutamicum.

• Cysteine-resistant mutants of C. glutamicum obtained by ALE were analyzed.

• Perturbation of valine biosynthesis by cysteine results in growth inhibition in C. glutamicum.

• Cysteine resistance is a useful phenotype for cysteine production by C. glutamicum.

Bacterial DNA methylases are a diverse group of enzymes which have been pivotal in the development of technologies with applications including genetic engineering, bacteriology, biotechnology and agriculture. This review describes bacterial DNA methylase types, the main technologies for targeted methylation or demethylation and the recent roles of these enzymes in molecular and synthetic biology. Bacterial methylases can be exocyclic or endocyclic and can exist as orphan enzymes or as a part of the restriction-modifications (R-M) systems. As a group, they display a rich diversity of sequence-specificity. Additional technologies for targeting methylation involve using fusion proteins combining a methylase and a DNA-binding protein (DNBP) such as a zinc-finger (ZF), transcription activator-like effector (TALE) or CRISPR/dCas9. Bacterial methylases have contributed significantly to the creation of novel DNA assembly techniques, to the improvement of bacterial transformation and to crop plant engineering. Future studies to define the characteristics of more bacterial methylases have potential to identify new tools of value in synthetic and molecular biology and with widespread applications.

• Bacterial methylases can be used to direct methylation to specific sequences in target DNA

• DNA methylation using bacterial methylases has been applied to improve DNA assembly and to increase the efficiency of bacterial transformation

• Site-selective methylation using bacterial methylases can alter plant gene expression and phenotype

One of the central dogmas of the Catholic Church is the belief in the Eucharistic presence of Jesus Christ, which requires no scientific confirmation because it concerns a supernatural reality. Since the early Middle Ages, however, instances have been recorded improperly referred to as Eucharistic miracles, which believers consider eyewitness testimony to a real transubstantiation. Changes in the structure, number, or an unexpected bloody red colour of the Host were often regarded as supernatural phenomena, but the Church officials themselves, aware of the possibility of a biological basis for these changes, showed far-reaching restraint. The author’s team on the basis of analyses of 25 actual cases undertook to prepare research procedures that make it possible to separate phenomena that are difficult to interpret scientifically, from those associated with contamination. None of these cases revealed the actual proof of existence of human blood, human material other than single epidermal cells, and erythrocytes (probably very low-level contamination). In one case, insignificant amount of human male genetic material was observed, probably as a result of DNA transferred from a person to a host via contact with the host itself. In several specimens, a variety of microbial and fungal material was identified (Brevundimonas intermedia, Serratia marcescens, Epicoccum spp,. Fusarium spp.), including species producing reddish-pink or orange-reddish pigments (Epicoccum spp., Fusarium spp.). Based on the experience gained in this study, a complete procedure suitable for reliable examination of similar cases in the future is suggested.

• The unusual appearance of the tested hosts can be explained by biological contamination.

• Blood-like marks result from the presence of pigment-producing species.

• A complete multidisciplinary procedure for investigating ‘miracle cases’ is proposed.

Wood decay fungi and bacteria play a crucial role in natural ecosystems, contributing to the decomposition of lignocellulosic materials and nutrient cycling. However, their activity poses significant challenges in timber durability, impacting industries reliant on wood as a construction material. This review examines the diversity of microorganisms damaging timber used indoors and outdoors. Additionally, traditional and advanced methods for microbial identification are discussed, with a focus on DNA-based, culture-independent sequencing methods whose importance has increased massively in recent years. It also provides an overview of the various options for wood protection, starting from wood protection by design, to chemical wood preservation and wood modification methods. This should illustrate how important it is to combine an ecological understanding of the decay organisms, precise identification and innovative wood protection methods in order to achieve a long-term and thus resource-saving use of wood.

• Fungi and bacteria play a crucial role in the decomposition of timber wood.

• Traditional and advanced DNA-based methods for microbial identification are discussed.

• An overview of the various options for wood protection is provided.