Engineering Microbiology最新文献

Hexokinase II (Hxk2) is a master protein in glucose-mediated transcriptional repression signaling pathway. Degrading Hxk2 through an auxin-inducible protein degradation previously doubled sesquiterpene (nerolidol) production at gram-per-liter levels in Saccharomyces cerevisiae. Global transcriptomics/proteomics profiles in Hxk2-deficient background are important to understanding genetic and molecular mechanisms for improved nerolidol production and guiding further strain optimization. Here, proteomic responses to Hxk2 depletion are investigated in the yeast strains harboring a GAL promoters-controlled nerolidol synthetic pathway, at the exponential and ethanol growth phases and in GAL80-wildtype and gal80Δ backgrounds. Carbon metabolic pathways and amino acid metabolic pathways show diversified responses to Hxk2 depletion and growth on ethanol, including upregulation of alternative carbon catabolism and respiration as well as downregulation of amino acid synthesis. De-repression of GAL genes may contribute to improved nerolidol production in Hxk2-depleted strains. Seventeen transcription factors associated with upregulated genes are enriched. Validating Ash1-mediated repression on the RIM4 promoter shows the variation on the regulatory effects of different Ash1-binding sites and the synergistic effect of Ash1 and Hxk2-mediated repression. Further validation of individual promoters shows that HXT1 promoter activities are glucose-dependent in hxk2Δ background, but much weaker than those in HXK2-wildtype background. In summary, inactivating HXK2 may relieve glucose repression on respiration and GAL promoters for improved bioproduction under aerobic conditions in S. cerevisiae. The proteomics profiles provide a better genetics overview for a better metabolic engineering design in Hxk2-deficient backgrounds.

The increasing shortage of fossil resources and environmental pollution has renewed interest in the synthesis of value-added biochemicals from methanol. However, most of native or synthetic methylotrophs are unable to assimilate methanol at a sufficient rate to produce biochemicals. Thus, the performance of methylotrophs still needs to be optimized to meet the demands of industrial applications. In this review, we provide an in-depth discussion on the properties of natural and synthetic methylotrophs, and summarize the natural and synthetic methanol assimilation pathways. Further, we discuss metabolic engineering strategies for enabling microbial utilization of methanol for the bioproduction of value-added chemicals. Finally, we highlight the potential of microbial engineering for methanol assimilation and offer guidance for achieving a low-carbon footprint for the biosynthesis of chemicals.

Recombineering is a valuable technique for generating recombinant DNA in vivo, primarily in bacterial cells, and is based on homologous recombination using phage-encoded homologous recombinases, such as Redαβγ from the lambda phage and RecET from the Rac prophage. The recombineering technique can efficiently mediate homologous recombination using short homologous arms (∼50 bp) and is unlimited by the size of the DNA molecules or positions of restriction sites. In this review, we summarize characteristics of recombinases, mechanism of recombineering, and advances in recombineering for DNA manipulation in Escherichia coli and other bacteria. Furthermore, the broad applications of recombineering for mining new bioactive microbial natural products, and for viral mutagenesis, phage genome engineering, and understanding bacterial metabolism are also reviewed.

China has a rich history of cultivating medicinal plants, whose root microbial communities closely interact with the medicinal plants, thereby influencing their growth, health, and medicinal properties. Currently, researchers widely use 16S rRNA gene amplicon sequencing to study these root microbial communities. However, publicly available sequence datasets often lack essential sample information or contain errors, impeding the reuse of the datasets in the future. In this study, we aimed to create a united, reliable, and readily usable source of 16S rRNA gene sequences for medicinal plant root microbiomes. We compiled a catalog of 1392 microbiome samples for 58 medicinal plants from 58 studies, and manually provided essential sample information based on the experimental setup described in the associated papers. We then processed the sequences using a custom pipeline, generating a united catalog of operational taxonomic units (OTUs) and conducting taxonomic classification. We also predicted the ecological functions of the communities for each sample. Finally, we used this dataset, to compare the rhizosphere bacterial communities of Pseudostellaria heterophylla from Fujian and Guizhou Provinces, revealing significant differences in the community composition of the same plant from different geographic locations. By providing a comprehensive and united catalog of amplicon sequences and OTUs for medicinal plant root bacterial communities, this study offers an invaluable resource for future comparative studies and data mining.

The use of non-food lignocellulosic biomass to produce ethanol fits into the strategy of a global circular economy with low dependence on fossil energy resources. Xylose is the second most abundant sugar in lignocellulosic hydrolysate, and its utilization in fermentation is a key issue in making the full use of raw plant materials for ethanol production and reduce production costs. Saccharomyces cerevisiae is the best ethanol producer but the organism is not a native xylose user. In recent years, great efforts have been made in the construction of xylose utilizing S. cerevisiae strains by metabolic and evolutionary engineering approaches. In addition, managing global transcriptional regulation works provides an effective means to increase the xylose utilization capacity of recombinant strains. Here we review the common strategies and research advances in the research field in order to facilitate the researches in xylose metabolism and xylose-based fermentation.

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.