Engineering Microbiology最新文献

Bacillus subtilis plays an important role in fundamental and applied research, and it has been widely used as a cell factory for the production of enzymes, antimicrobial materials, and chemicals for agriculture, medicine, and industry. However, genetic manipulation tools for B. subtilis have low efficiency. In this work, our goal was to develop a simple recombineering system for B. subtilis. We showed that genome editing can be achieved in B. subtiliis 1A751 through co-expression of YqaJ/YqaK, a native phage recombinase pair found in B. subtilis 168, and the competence master regulator ComK using a double-stranded DNA substrate with short homology arms (100 bp) and a phosphorothioate modification at the 5′-end. Efficient gene knockouts and large DNA insertions were achieved using this new recombineering system in B. subtilis 1A751. As far as we know, this is the first recombineering system using the native phage recombinase pair YqaJ/YqaK in B. subtilis. In conclusion, this new recombineering system provides a simple and fast tool for genetic manipulation of B. subtilis, and it will promote studies of genome function, construction of production strains, and genome mining in B. subtilis.

The overuse and misuse of traditional antimicrobial drugs have led to their weakened effectiveness and the emergence of pathogenic bacterial resistance. Consequently, there has been growing interest in alternative options such as antimicrobial peptides (AMPs) in the pharmaceutical industry. Microcin J25 (MccJ25) has gained significant attention for its potent inhibitory effect on a diverse range of pathogens. Its unique rotaxane structure provides exceptional stability against extreme thermal, pH, and protease degradation, including chymotrypsin, trypsin, and pepsin. Given its remarkable stability and diverse bioactivity, we aim to provide an overview of the physicochemical properties, the mechanism underlying its antimicrobial activity, and the critical functional residues of MccJ25. Additionally, we have summarized the latest strategies for the heterologous expression of MccJ25, and its potential medical use and other applications.

The widespread presence of iron and sulfur compounds such as pyrite in marine waterlogged archeological wood (WAW) can cause irreversible damage to the safety of its preservation. This issue has been a longstanding concern for cultural heritage conservation communities. In this study, we examined the distribution and phase composition of Fe and sulfur compounds in wood samples obtained from the Nanhai I shipwreck using ESEM-EDS, micro-Raman spectroscopy, and an X-ray diffractometer. The removal of iron from WAW samples of the Nanhai I shipwreck using Acidithiobacillus ferrooxidans (A. ferrooxidans) was evaluated using conductivity and ICP-AES analysis. The results showed that A. ferrooxidans effectively improved the removal of iron from WAW. The degradation of fresh healthy wood during treatment was also analyzed using infrared spectroscopy, and the results showed that the treatment had little effect on the samples over a short period. This study demonstrates, for the first time, the feasibility of iron extraction from marine WAW by A.ferrooxidans. This was also the first attempt in China to apply biological oxidation to the removal of iron from marine archeological materials.

β-glucosidases play an important role in the synthesis of cellulase in fungi, but their molecular functions and mechanisms remain unknown. We found that the 10 putative β-glucosidases investigated in Trichoderma reesei facilitate cellulase production, with cel3j being the most crucial. Transcriptional analysis revealed that the most affected biological processes in △cel3j strain were cellulase synthesis, ribosome biogenesis, and RNA polymerases. Moreover, CEL3J was unconventionally transported through the endoplasmic reticulum, bypassing the Golgi apparatus, whereas cel3j overexpression altered cellulase secretion from conventional to unconventional, likely owing to the activated unconventional protein secretion pathway (UPS), as indicated by the upregulation of genes related to UPS. The mTORC1-GRASP55 signaling axis may modulate the unconventional secretion of CEL3J and cellulase. The transcriptional levels of genes associated with DNA replication, the cell cycle, and meiosis were noticeably affected by overexpressing cel3j. These data give new clues for exploring the roles of β-glucosidases and the molecular mechanisms of their unconventional secretion in fungi.

AB₅-type toxins are a group of secreted protein toxins that are central virulence factors for bacterial pathogens such as Shigella dysenteriae, Vibrio cholerae, Bordetella pertussis, and certain lineages of pathogenic Escherichia coli and Salmonella enterica. AB₅ toxins are composed of an active (A) subunit that manipulates host cell biology in complex with a pentameric binding/delivery (B) subunit that mediates the toxin's entry into host cells and its subsequent intracellular trafficking. Broadly speaking, all known AB₅-type toxins adopt similar structural architectures and employ similar mechanisms of binding, entering and trafficking within host cells. Despite this, there is a remarkable amount of diversity amongst AB₅-type toxins; this includes different toxin families with unrelated activities, as well as variation within families that can have profound functional consequences. In this review, we discuss the diversity that exists amongst characterized AB₅-type toxins, with an emphasis on the genetic and functional variability within AB₅ toxin families, how this may have evolved, and its impact on human disease.

Type III CRISPR-Cas10 systems employ multiple immune activities to defend their hosts against invasion from mobile genetic elements (MGEs), including DNase and cyclic oligoadenylates (cOA) synthesis both of which are hosted by the type-specific protein Cas10. Extensive investigations conducted for the activation of Cas accessory proteins by cOAs have revealed their functions in the type III immunity, but the function of the Cas10 DNase in the same process remains elusive. Here, Lactobacillus delbrueckii subsp. Bulgaricus type III-A (Ld) Csm system, a type III CRISPR system that solely relies on its Cas10 DNase for providing immunity, was employed as a model to investigate the DNase function. Interference assay was conducted in Escherichia coli using two plasmids: pCas carrying the LdCsm system and pTarget producing target RNAs. The former functioned as a de facto “CRISPR host element” while the latter, mimicking an invading MGE. We found that, upon induction of immune responses, the fate of each genetic element was determined by their copy numbers: plasmid of a low copy number was selectively eliminated from the E. coli cells regardless whether it represents a de facto CRISPR host or an invader. Together, we reveal, for the first time, that the immune mechanisms of Cas10 DNases are of two folds: the DNase activity is capable of removing low-copy invaders from infected cells, but it also leads to abortive infection when the invader copy number is high.

The metabolic engineering of Saccharomyces cerevisiae has great potential for enhancing the production of high-value chemicals and recombinant proteins. Recent studies have demonstrated the effectiveness of dynamic regulation as a strategy for optimizing metabolic flux and improving production efficiency. In this review, we provide an overview of recent advancements in the dynamic regulation of S. cerevisiae metabolism. Here, we focused on the successful utilization of transcription factor (TF)-based biosensors within the dynamic regulatory network of S. cerevisiae. These biosensors are responsive to a wide range of endogenous and exogenous signals, including chemical inducers, light, temperature, cell density, intracellular metabolites, and stress. Additionally, we explored the potential of omics tools for the discovery of novel responsive promoters and their roles in fine-tuning metabolic networks. We also provide an outlook on the development trends in this field.

Gene editing technology involves the modification of a specific target gene to obtain a new function or phenotype. Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-mediated technologies have provided an efficient tool for genetic engineering of cells and organisms. Here, we review the three emerging gene editing tools (ZFNs, TALENs, and CRISPR-Cas) and briefly introduce the principle, classification, and mechanisms of the CRISPR-Cas systems. Strategies for gene editing based on endogenous and exogenous CRISPR-Cas systems, as well as the novel base editor (BE), prime editor (PE), and CRISPR-associated transposase (CAST) technologies, are described in detail. In addition, we summarize recent developments in the application of CRISPR-based gene editing tools for industrial microorganism and probiotics modifications. Finally, the potential challenges and future perspectives of CRISPR-based gene editing tools are discussed.

7-Dehydrocholesterol (7-DHC), a key pharmaceutical intermediate in the production of vitamin D₃, has a wide range of applications. To explore fermentative synthesis of 7-DHC, a 7-DHC-producing Saccharomyces cerevisiae strain was constructed by blocking the competitive pathway, eliminating rate-limiting steps, altering global regulation, and pathway compartmentalization. After blocking the competitive pathway by disrupting ERG5 and ERG6 and introducing DHCR24 from Gallus gallus, S. cerevisiae produced 139.72 mg/L (17.04 mg/g dry cell weight, hereafter abbreviated as DCW) 7-DHC. Subsequent alteration of global regulation by deleting ROX1 and overexpressing UPC2-1 increased 7-DHC production to 217.68 mg/L (37.56 mg/g DCW). To remove the accumulated squalene, the post-squalene pathway was strengthened by co-overexpression of P_GAL1-driven ERG11 and P_GAL10-driven ERG1, which improved 7-DHC titer and yield to 281.73 mg/L and 46.78 mg/g DCW, respectively, and reduced squalene content by 90.12%. We surmised that the sterol precursors in the plasma membrane and peroxisomes may not be accessible to the pathway enzymes, thus we re-localized DHCR24p and Erg2p-GGGGS-Erg3p to the plasma membrane and peroxisomes, boosting 7-DHC production to 357.53 mg/L (63.12 mg/g DCW). Iron supplementation further increased 7-DHC production to 370.68 mg/L in shake flasks and 1.56 g/L in fed-batch fermentation. This study demonstrates the power of global regulation and subcellular relocalization of key enzymes to improve 7-DHC synthesis in yeast.

The methylotrophic yeast Pichia pastoris (also known as Komagataella phaffii) is widely used as a yeast cell factory for producing heterologous proteins. Recently, it has gained attention for its potential in producing chemicals from inexpensive feedstocks, which requires efficient genetic engineering platforms. This review provides an overview of the current advances in developing genetic tools for metabolic engineering of P. pastoris. The topics cover promoters, terminators, plasmids, genome integration sites, and genetic editing systems, with a special focus on the development of CRISPR/Cas systems and their comparison to other genome editing tools. Additionally, this review highlights the prospects of multiplex genome integration, fine-tuning gene expression, and single-base editing systems. Overall, the aim of this review is to provide valuable insights into current genetic engineering and discuss potential directions for future efforts in developing efficient genetic tools in P. pastoris.