Engineering Microbiology最新文献

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.

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.

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 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.

The fungus-growing termite is considered a distinct ecological niche because it involves a tripartite symbiosis between the termite host, gut microflora, and the in vitro fungus Termitomyces, which has led to the expansion of highly organized and complex societies among termite colonies. Tripartite symbiosis in fungus-growing termites may promote unique microbes with distinctive metabolic pathways that may serve as valuable resources for developing novel antimicrobial therapeutic options. Recent research on complex tripartite symbioses has revealed a plethora of previously unknown natural products that may have ecological roles in signaling, communication, or defense responses. Natural products produced by symbionts may act as crucial intermediaries between termites and their pathogens by providing direct protection through their biological activities. Herein, we review the state-of-the-art research on both microbes and natural products originated from fungus-growing termite tripartite symbiosis, highlighting the diversity of microbes and the uniqueness of natural product classes and their bioactivities. Additionally, we emphasize future research prospects on fungus-growing termite related microorganisms, with a particular focus on their potential roles in bioactive product discovery.

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.

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.

Natural product biosynthesis is controlled at multiple levels. Characterization of naturally occurring promoters has facilitated the study of the synthetic biology of natural products. Herein, we report the discovery of two high-yield actinomycin D (ActD)-producing streptomycetes and the identification of a strong bidirectional acmN2p promoter from the ActD gene clusters and its application in heterologous expression of three core genes involved in the bacterial alkaloid bohemamine biosynthesis, providing a good example for identification of new promoters for synthetic biological applications.