Engineering Microbiology最新文献

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.

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.

Owing to the rapid advancement of genome engineering technologies, the scale of genome engineering has expanded dramatically. Genome editing has progressed from one genomic alteration at a time that could only be employed in few species, to the simultaneous generation of multiple modifications across many genomic loci in numerous species. The development and recent advances in multiplex automated genome engineering (MAGE)-associated technologies and clustered regularly interspaced short palindromic repeats and their associated protein (CRISPR-Cas)-based approaches, together with genome-scale synthesis technologies offer unprecedented opportunities for advancing genome-scale engineering in a broader range. These approaches provide new tools to generate strains with desired phenotypes, understand the complexity of biological systems, and directly evolve a genome with novel features. Here, we review the recent major advances in genome-scale engineering tools developed for Escherichia coli, focusing on their applications in identifying essential genes, genome reduction, recoding, and beyond.

Industrial manufacturing of bioproducts, especially bioethanol, can benefit from high-temperature fermentation, which requires the use of thermotolerant yeast strains. Mitochondrial activity in yeast is closely related to its overall metabolism. However, the mitochondrial respiratory changes in response to adaptive thermotolerance are still poorly understood and have been rarely utilized for developing thermotolerant yeast cell factories. Here, adaptive evolution and transcriptional sequencing, as well as whole-genome-level gene knockout, were used to obtain a thermotolerant strain of Saccharomyces cerevisiae. Furthermore, thermotolerance and bioethanol production efficiency of the engineered strain were examined. Physiological evaluation showed the boosted fermentation capacity and suppressed mitochondrial respiratory activity in the thermotolerant strain. The improved fermentation produced an increased supply of adenosine triphosphate required for more active energy-consuming pathways. Transcriptome analysis revealed significant changes in the expression of the genes involved in the mitochondrial respiratory chain. Evaluation of mitochondria-associated gene knockout confirmed that ADK1, DOC1, or MET7 were the key factors for the adaptive evolution of thermotolerance in the engineered yeast strain. Intriguingly, overexpression of DOC1 with TEF1 promoter regulation led to a 10.1% increase in ethanol production at 42 °C. The relationships between thermotolerance, mitochondrial activity, and respiration were explored, and a thermotolerant yeast strain was developed by altering the expression of mitochondrial respiration-related genes. This study provides a better understanding on the physiological mechanism of adaptive evolution of thermotolerance in yeast.

Iron is essential for bacterial survival, and most bacteria capture iron by producing siderophores. Burkholderiales bacteria produce various types of bioactive secondary metabolites, such as ornibactin and malleobactin siderophores. In this study, the genome analysis of Burkholderiales genomes showed a putative novel siderophore gene cluster crb, which is highly similar to the ornibactin and malleobactin gene clusters but does not have pvdF, a gene encoding a formyltransferase for N-δ‑hydroxy-ornithine formylation. Establishing the bacteriophage recombinase Redγ-Redαβ7029 mediated genome editing system in a non-model Burkholderiales strain Paraburkholderia caribensis CICC 10960 allowed the rapid identification of the products of crb gene cluster, caribactins A-F (1–6). Caribactins contain a special amino acid residue N-δ‑hydroxy-N-δ-acetylornithine (haOrn), which differs from the counterpart N-δ‑hydroxy-N-δ-formylornithine (hfOrn) in ornibactin and malleobactin, owing to the absence of pvdF. Gene inactivation showed that the acetylation of hOrn is catalyzed by CrbK, whose homologs probably not be involved in the biosynthesis of ornibactin and malleobactin, showing possible evolutionary clues of these siderophore biosynthetic pathways from different genera. Caribactins promote biofilm production and enhance swarming and swimming abilities, suggesting that they may play crucial roles in biofilm formation. This study also revealed that recombineering has the capability to mine novel secondary metabolites from non-model Burkholderiales species.

Large-scale genome-mining analyses have revealed that microbes potentially harbor a huge reservoir of uncharacterized natural product (NP) biosynthetic gene clusters (BGCs), and this has spurred a renaissance of novel drug discovery. However, the majority of these BGCs are often poorly or not at all expressed in their native hosts under laboratory conditions, and thus are regarded as silent/orphan BGCs. Currently, connecting silent BGCs to their corresponding NPs quickly and on a large scale is particularly challenging because of the lack of universal strategies and enabling technologies. Generally, the heterologous host-based genome mining strategy is believed to be a suitable alternative to the native host-based approach for prioritization of the vast and ever-increasing number of uncharacterized BGCs. In the last ten years, a variety of methods have been reported for the direct cloning of BGCs of interest, which is the first and rate-limiting step in the heterologous expression strategy. Essentially, each method requires that the following three issues be resolved: 1) how to prepare genomic DNA; 2) how to digest the bilateral boundaries for release of the target BGC; and 3) how to assemble the BGC and the capture vector. Here, we summarize recent reports regarding how to directly capture a BGC of interest and briefly discuss the advantages and disadvantages of each method, with an emphasis on the notion that direct cloning is very beneficial for accelerating genome mining research and large-scale drug discovery.

Deciphering gene function is fundamental to engineering of microbiology. The clustered regularly interspaced short palindromic repeats (CRISPR) system has been adapted for gene repression across a range of hosts, creating a versatile tool called CRISPR interference (CRISPRi) that enables genome-scale analysis of gene function. This approach has yielded significant advances in the design of genome-scale CRISPRi libraries, as well as in applications of CRISPRi screening in medical and industrial microbiology. This review provides an overview of the recent progress made in pooled and arrayed CRISPRi screening in microorganisms and highlights representative studies that have employed this method. Additionally, the challenges associated with CRISPRi screening are discussed, and potential solutions for optimizing this strategy are proposed.