Engineering Microbiology最新文献

African swine fever virus (ASFV) infection poses enormous threats and challenges to the global pig industry; however, no effective vaccine is available against ASFV, attributing to the huge viral genome (approximately189 kb) and numerous encoding products (>150 genes) due to the limited understanding on the molecular mechanisms of viral pathogenesis. Elucidating the host-factor/viral-protein interaction network will reveal new targets for developing novel antiviral therapies. Using proteomic analysis, we identified 255 cellular proteins that interact with the ASFV-encoded pE301R protein when transiently expressed in HEK293T cells. Gene ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) database enrichment, and protein-protein interaction (PPI) network analyses revealed that pE301R-interacting host proteins are potentially involved in various biological processes, including protein translation and folding, response to stimulation, and mitochondrial transmembrane transport. The interactions of two putative cellular proteins (apoptosis inducing factor mitochondria associated 1 (AIFM1) and vimentin (VIM)) with pE301R-apoptosis inducing factor have been verified by co-immunoprecipitation. Our study revealed the inhibitory role of pE301R in interferon (IFN) induction that involves VIM sequestration by pE301R, identified interactions between ASFV pE301R and cellular proteins, and predicted the potential function of pE301R and its associated biological processes, providing valuable information to enhance our understanding of viral protein function, pathogenesis, and potential candidates for the prevention and control of ASFV infection.

Peptaibiotics are linear or cyclic peptide antibiotics characterized by the non-proteinogenic amino acid, alpha-aminoisobutyric acid. They exhibit a wide range of bioactivity against various pathogens. This report presents a comprehensive review of analytical methods for Trichoderma cultivation, production, isolation, screening, purification, and characterization of peptaibiotics, along with their bioactivity. Numerous techniques are currently available for each step, and we focus on describing the most commonly used and recently developed chromatographic and spectroscopic techniques. Investigating peptaibiotics requires efficient culture media, growth conditions, and isolation and purification techniques. The combination of chromatographic and spectroscopic tools offers a better opportunity for characterizing and identifying peptaibiotics. The evaluation of the chemical and biological properties of this compound has also been explored concerning its potential application in pharmaceutical and other industries. This review aims to summarize available data on the techniques and tools used to screen and purify peptaibiotics from Trichoderma fungi and bioactivity against various pathogens.

The recent discovery of the CRISPR-Cas-mediated acquired immunity system highlights the fact that our knowledge of phage/virus defense mechanisms encoded in bacterial and archaeal genomes is far from complete. Indeed, new prokaryotic immune systems are now continually being discovered. A recent report described a novel glycosylase that recognizes α-glycosyl-hydroxymethyl cytosin (α-Glu-hmC), a modified base observed in the T4 phage genome, where it produces an abasic site, thereby inhibiting the phage propagation.

Microbial natural products and their derivatives have been developed as a considerable part of clinical drugs and agricultural chemicals. Marine microbial natural products exhibit diverse chemical structures and bioactivities with substantial potential for the development of novel pharmaceuticals. However, discovering compounds with new skeletons from marine microbes remains challenging. In recent decades, multiple approaches have been developed to discover novel marine microbial natural products, among which heterologous expression has proven to be an effective method. Facilitated by large DNA cloning and comparative metabolomic technologies, a few novel bioactive natural products from marine microorganisms have been identified by the expression of their biosynthetic gene clusters (BGCs) in heterologous hosts. Heterologous expression is advantageous for characterizing gene functions and elucidating the biosynthetic mechanisms of natural products. This review provides an overview of recent progress in heterologous expression-guided discovery, biosynthetic mechanism elucidation, and yield optimization of natural products from marine microorganisms and discusses the future directions of the heterologous expression strategy in facilitating novel natural product exploitation.

The bacterium Escherichia coli (E. coli) is one of the most widely used chassis microbes employed for the biosynthesis of numerous valuable chemical compounds. In the past decade, the metabolic engineering of E. coli has undergone significant advances, although further productivity improvements will require extensive genome modification, multi-dimensional regulation, and multiple metabolic-pathway coordination. In this context, clustered regularly interspaced short palindromic repeats (CRISPR), along with CRISPR-associated protein (Cas) and its inactive variant (dCas), have emerged as notable recombination and transcriptional regulation tools that are particularly useful for multiplex metabolic engineering in E. coli. In this review, we briefly describe the CRISPR/Cas9 technology in E. coli, and then summarize the recent advances in CRISPR/dCas9 interference (CRISPRi) systems in E. coli, particularly the strategies designed to effectively regulate gene repression and overcome retroactivity during multiplexing. Moreover, we discuss recent applications of the CRISPRi system for enhancing metabolite production in E. coli, and finally highlight the major challenges and future perspectives of this technology.

Recombineering is an essential tool for molecular biologists, allowing for the facile and efficient manipulation of bacterial genomes directly in cells without the need for costly and laborious in vitro manipulations involving restriction enzymes. The main workhorses behind recombineering are bacteriophage proteins that promote the single-strand annealing (SSA) homologous recombination pathway to repair double-stranded DNA breaks. While there have been several reviews examining recombineering methods and applications, comparatively few have focused on the mechanisms of the proteins that are the key players in the SSA pathway: a 5′→3′ exonuclease and a single-strand annealing protein (SSAP or “annealase”). This review dives into the structures and functions of the two SSA recombination systems that were the first to be developed for recombineering in E. coli: the RecET system from E. coli Rac prophage and the λRed system from bacteriophage λ. By comparing the structures of the RecT and Redβ annealases, and the RecE and λExo exonucleases, we provide new insights into how the structures of these proteins dictate their function. Examining the sequence conservation of the λExo and RecE exonucleases gives more profound insights into their critical functional features. Ultimately, as recombineering accelerates and evolves in the laboratory, a better understanding of the mechanisms of the proteins behind this powerful technique will drive the development of improved and expanded capabilities in the future.

The modularity of carbohydrate-active enzymes facilitates that enzymes with different functions have similar fragments. However, because of the complex structure of the enzyme active sites and the epistatic effects of various mutations on enzyme activity, it is difficult to design enzymes with multiple mutation sites using conventional methods. In this study, we designed multi-point mutants by fragment replacement in the donor-acceptor binding pocket of Actinobacillus pleuropneumoniae N-glycosyltransferase (ApNGT) to obtain novel properties. Candidate fragments were selected from a customized glycosyltransferase database. The stability and substrate-binding energy of the three fragment replacement mutants were calculated in comparison with wild-type ApNGT, and mutants with top-ranking stability and middle-ranking substrate-binding energy were chosen for priority experimental verification. We found that a mutant called F13, which increased the glycosylation efficiency of the natural substrate by 1.44 times, the relative conversion of UDP-galactose by 14.2 times, and the relative conversion of UDP-xylose from almost 0 to 78.6%. Most importantly, F13 mutant acquired an entirely new property, the ability to utilize UDP-glucuronic acid. On one hand, this work shows that replacing similar fragments in the donor-acceptor binding pocket of the enzyme might provide new ideas for designing mutants with new properties; on the other hand, F13 mutant is expected to play an important role in targeted drug delivery.

Saccharomyces cerevisiae is an excellent microbial cell factory for producing valuable recombinant proteins because of its fast growth rate, robustness, biosafety, ease of operability via mature genomic modification technologies, and the presence of a conserved post-translational modification pathway among eukaryotic organisms. However, meeting industrial and market requirements with the current low microbial production of recombinant proteins can be challenging. To address this issue, numerous efforts have been made to enhance the ability of yeast cell factories to efficiently produce proteins. In this review, we provide an overview of recent advances in S. cerevisiae engineering to improve recombinant protein production. This review focuses on the strategies that enhance protein production by regulating transcription through promoter engineering, codon optimization, and expression system optimization. Additionally, we describe modifications to the secretory pathway, including engineered protein translocation, protein folding, glycosylation modification, and vesicle trafficking. Furthermore, we discuss global metabolic pathway optimization and other relevant strategies, such as the disruption of protein degradation, cell wall engineering, and random mutagenesis. Finally, we provide an outlook on the developmental trends in this field, offering insights into future directions for improving recombinant protein production in S. cerevisiae.

In this study, a combined system consisting of an anaerobic membrane bioreactor (AnMBR) and flow-through biofilm reactor/CANON (FTBR/CANON) was developed to simultaneously remove carbon and nitrogen from synthetic livestock wastewater. The average removal efficiencies of total nitrogen (TN) were 64.2 and 76.4% with influent ammonium (NH₄⁺-N) concentrations of approximately 200 and 500 mg/L, respectively. The COD removal efficiencies were higher than 98.0% during the entire operation. Mass balance analysis showed that COD and TN were mainly removed by the AnMBR and FTBR/CANON, respectively. The anammox process was the main nitrogen removal pathway in the combined system, with a contribution of over 80%. High functional bacterial activity was observed in the combined system. Particularly, an increase in the NH₄⁺-N concentration considerably improved the anammox activity of the biofilm in the FTBR/CANON. 16S rRNA high-throughput sequencing revealed that Methanosaeta, Candidatus Methanofastidiosum, and Methanobacterium were the dominant methanogens in the AnMBR granular sludge. In the CANON biofilm, Nitrosomonas and Candidatus Kuenenia were identified as aerobic and anaerobic ammonium-oxidizing bacteria, respectively. In summary, this study proposes a combined AnMBR and FTBR/CANON process targeting COD and nitrogen removal, and provides a potential alternative for treating high-strength wastewater.

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.