Engineering Microbiology最新文献

{"title":"Advances in the dynamic control of metabolic pathways in Saccharomyces cerevisiae","authors":"Chufan Xiao,&nbsp;Yuyang Pan,&nbsp;Mingtao Huang","doi":"10.1016/j.engmic.2023.100103","DOIUrl":"https://doi.org/10.1016/j.engmic.2023.100103","url":null,"abstract":"<div><p>The metabolic engineering of <em>Saccharomyces cerevisiae</em> 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 <em>S. cerevisiae</em> metabolism. Here, we focused on the successful utilization of transcription factor (TF)-based biosensors within the dynamic regulatory network of <em>S. cerevisiae</em>. 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.</p></div>","PeriodicalId":100478,"journal":{"name":"Engineering Microbiology","volume":"3 4","pages":"Article 100103"},"PeriodicalIF":0.0,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50203812","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Investigating the cellular functions of β-Glucosidases for synthesis of lignocellulose-degrading enzymes in Trichoderma reesei","authors":"Ai–Ping Pang ,&nbsp;Haiyan Wang ,&nbsp;Yongsheng Luo ,&nbsp;Funing Zhang ,&nbsp;Fu–Gen Wu ,&nbsp;Zhihua Zhou ,&nbsp;Zuhong Lu ,&nbsp;Fengming Lin","doi":"10.1016/j.engmic.2023.100105","DOIUrl":"https://doi.org/10.1016/j.engmic.2023.100105","url":null,"abstract":"<div><p>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 <em>Trichoderma reesei</em> facilitate cellulase production, with <em>cel3j</em> being the most crucial<em>.</em> Transcriptional analysis revealed that the most affected biological processes in △<em>cel3j</em> strain were cellulase synthesis, ribosome biogenesis, and RNA polymerases. Moreover, CEL3J was unconventionally transported through the endoplasmic reticulum, bypassing the Golgi apparatus, whereas <em>cel3j</em> 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 <em>cel3j</em>. These data give new clues for exploring the roles of β-glucosidases and the molecular mechanisms of their unconventional secretion in fungi.</p></div>","PeriodicalId":100478,"journal":{"name":"Engineering Microbiology","volume":"3 4","pages":"Article 100105"},"PeriodicalIF":0.0,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50203814","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Combinatorial metabolic engineering of Saccharomyces cerevisiae for improved production of 7-dehydrocholesterol","authors":"Yuehao Gu ,&nbsp;Shuhui Chen ,&nbsp;Xue Jiao ,&nbsp;Qi Bian ,&nbsp;Lidan Ye ,&nbsp;Hongwei Yu","doi":"10.1016/j.engmic.2023.100100","DOIUrl":"https://doi.org/10.1016/j.engmic.2023.100100","url":null,"abstract":"<div><p>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 <em>Saccharomyces cerevisiae</em> 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 <em>ERG5</em> and <em>ERG6</em> and introducing <em>DHCR24</em> from <em>Gallus gallus, S. cerevisiae</em> 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 <em>ROX1</em> and overexpressing <em>UPC2-1</em> 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<em>_GAL1</em>-driven <em>ERG11</em> and P<em>_GAL10</em>-driven <em>ERG1</em>, 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.</p></div>","PeriodicalId":100478,"journal":{"name":"Engineering Microbiology","volume":"3 4","pages":"Article 100100"},"PeriodicalIF":0.0,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50203818","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISPR-based gene editing technology and its application in microbial engineering","authors":"Junwei Wei,&nbsp;Yingjun Li","doi":"10.1016/j.engmic.2023.100101","DOIUrl":"https://doi.org/10.1016/j.engmic.2023.100101","url":null,"abstract":"<div><p>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.</p></div>","PeriodicalId":100478,"journal":{"name":"Engineering Microbiology","volume":"3 4","pages":"Article 100101"},"PeriodicalIF":0.0,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50203813","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A strategy to enhance the insecticidal potency of Vip3Aa by introducing additional cleavage sites to increase its proteolytic activation efficiency","authors":"Kun Jiang ,&nbsp;Zhe Chen ,&nbsp;Yiting Shi ,&nbsp;Yuanrong Zang ,&nbsp;Chengbin Shang ,&nbsp;Xi Huang ,&nbsp;Jiahe Zang ,&nbsp;Zhudong Bai ,&nbsp;Xuyao Jiao ,&nbsp;Jun Cai ,&nbsp;Xiang Gao","doi":"10.1016/j.engmic.2023.100083","DOIUrl":"https://doi.org/10.1016/j.engmic.2023.100083","url":null,"abstract":"<div><p>Microbially derived, protein-based biopesticides have become a vital element in pest management strategies. Vip3 family proteins from <em>Bacillus thuringiensis</em> have distinct characteristics from known insecticidal Cry toxins and show efficient insecticidal activity against several detrimental lepidopteran pests. They are considered to be a promising toxic candidate for the management of various detrimental pests. In this study, we found that in addition to the preliminary digestion sites lysine, there are multiple cleavage activation sites in the linker region between domain I (DI) and DII of Vip3Aa. We further demonstrated that by adding more cleavage sites between DI and DII of Vip3Aa, its proteolysis efficiency by midgut proteases can be significantly increased, and correspondingly enhance its insecticidal activity against <em>Spodoptera frugiperda</em> and <em>Helicoverpa armigera</em> larvae. Our study promotes the understanding of the insecticidal mechanism of Vip3 proteins and illustrates an easily implementable strategy to increase the insecticidal potency of Vip3Aa. This facilitates their potential future development and efficient application for sustainable agriculture.</p></div>","PeriodicalId":100478,"journal":{"name":"Engineering Microbiology","volume":"3 4","pages":"Article 100083"},"PeriodicalIF":0.0,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50203841","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The diverse landscape of AB5-type toxins","authors":"Paris I. Brown,&nbsp;Adaobi Ojiakor,&nbsp;Antonio J. Chemello,&nbsp;Casey C. Fowler","doi":"10.1016/j.engmic.2023.100104","DOIUrl":"https://doi.org/10.1016/j.engmic.2023.100104","url":null,"abstract":"<div><p>AB₅-type toxins are a group of secreted protein toxins that are central virulence factors for bacterial pathogens such as <em>Shigella dysenteriae, Vibrio cholerae, Bordetella pertussis,</em> and certain lineages of pathogenic <em>Escherichia coli</em> and <em>Salmonella enterica</em>. 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.</p></div>","PeriodicalId":100478,"journal":{"name":"Engineering Microbiology","volume":"3 4","pages":"Article 100104"},"PeriodicalIF":0.0,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50203811","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Exploring the diversity of microbes and natural products from fungus-growing termite tripartite symbiosis","authors":"Muhammad Shoaib ,&nbsp;Ruining Bai ,&nbsp;Shuai Li ,&nbsp;Yan Xie ,&nbsp;Yulong Shen ,&nbsp;Jinfeng Ni","doi":"10.1016/j.engmic.2023.100124","DOIUrl":"10.1016/j.engmic.2023.100124","url":null,"abstract":"<div><p>The fungus-growing termite is considered a distinct ecological niche because it involves a tripartite symbiosis between the termite host, gut microflora, and the <em>in vitro</em> fungus <em>Termitomyces</em>, 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.</p></div>","PeriodicalId":100478,"journal":{"name":"Engineering Microbiology","volume":"4 1","pages":"Article 100124"},"PeriodicalIF":0.0,"publicationDate":"2023-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2667370323000565/pdfft?md5=626d1e0eddd0b16494e5e73d294b7fe8&pid=1-s2.0-S2667370323000565-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135455640","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Recombineering enables genome mining of novel siderophores in a non-model Burkholderiales strain","authors":"Xingyan Wang,&nbsp;Haibo Zhou,&nbsp;Xiangmei Ren,&nbsp;Hanna Chen,&nbsp;Lin Zhong,&nbsp;Xianping Bai,&nbsp;Xiaoying Bian","doi":"10.1016/j.engmic.2023.100106","DOIUrl":"https://doi.org/10.1016/j.engmic.2023.100106","url":null,"abstract":"<div><p>Iron is essential for bacterial survival, and most bacteria capture iron by producing siderophores. <em>Burkholderiales</em> bacteria produce various types of bioactive secondary metabolites, such as ornibactin and malleobactin siderophores. In this study, the genome analysis of <em>Burkholderiales</em> genomes showed a putative novel siderophore gene cluster <em>crb</em>, which is highly similar to the ornibactin and malleobactin gene clusters but does not have <em>pvdF</em>, a gene encoding a formyltransferase for N-<em>δ</em>‑hydroxy-ornithine formylation. Establishing the bacteriophage recombinase Redγ-Redαβ7029 mediated genome editing system in a non-model <em>Burkholderiales</em> strain <em>Paraburkholderia caribensis</em> CICC 10960 allowed the rapid identification of the products of <em>crb</em> gene cluster, caribactins A-F (<strong>1–6</strong>). Caribactins contain a special amino acid residue N-<em>δ</em>‑hydroxy-N-<em>δ</em>-acetylornithine (haOrn), which differs from the counterpart N-<em>δ</em>‑hydroxy-N-<em>δ</em>-formylornithine (hfOrn) in ornibactin and malleobactin, owing to the absence of <em>pvdF</em>. 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 <em>Burkholderiales</em> species.</p></div>","PeriodicalId":100478,"journal":{"name":"Engineering Microbiology","volume":"3 3","pages":"Article 100106"},"PeriodicalIF":0.0,"publicationDate":"2023-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49891302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Profiling proteomic responses to hexokinase-II depletion in terpene-producing Saccharomyces cerevisiae","authors":"Zeyu Lu ,&nbsp;Qianyi Shen ,&nbsp;Lian Liu ,&nbsp;Gert Talbo ,&nbsp;Robert Speight ,&nbsp;Matt Trau ,&nbsp;Geoff Dumsday ,&nbsp;Christopher B. Howard ,&nbsp;Claudia E. Vickers ,&nbsp;Bingyin Peng","doi":"10.1016/j.engmic.2023.100079","DOIUrl":"https://doi.org/10.1016/j.engmic.2023.100079","url":null,"abstract":"<div><p>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 <em>Saccharomyces cerevisiae</em>. 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 <em>GAL</em> promoters-controlled nerolidol synthetic pathway, at the exponential and ethanol growth phases and in <em>GAL80</em>-wildtype and <em>gal80Δ</em> 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 <em>GAL</em> 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 <em>RIM4</em> 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 <em>HXT1</em> promoter activities are glucose-dependent in <em>hxk2Δ</em> background, but much weaker than those in <em>HXK2</em>-wildtype background. In summary, inactivating <em>HXK2</em> may relieve glucose repression on respiration and <em>GAL</em> promoters for improved bioproduction under aerobic conditions in <em>S. cerevisiae</em>. The proteomics profiles provide a better genetics overview for a better metabolic engineering design in <em>Hxk2-deficient</em> backgrounds.</p></div>","PeriodicalId":100478,"journal":{"name":"Engineering Microbiology","volume":"3 3","pages":"Article 100079"},"PeriodicalIF":0.0,"publicationDate":"2023-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49891366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Recent advances in the direct cloning of large natural product biosynthetic gene clusters","authors":"Jiaying Wan,&nbsp;Nan Ma,&nbsp;Hua Yuan","doi":"10.1016/j.engmic.2023.100085","DOIUrl":"https://doi.org/10.1016/j.engmic.2023.100085","url":null,"abstract":"<div><p>Large-scale genome-mining analyses have revealed that microbes potentially harbor a huge reservoir of uncharacterized natural product (NP) biosynthetic gene clusters (<em>BGCs</em>), and this has spurred a renaissance of novel drug discovery. However, the majority of these <em>BGCs</em> are often poorly or not at all expressed in their native hosts under laboratory conditions, and thus are regarded as silent/orphan <em>BGCs</em>. Currently, connecting silent <em>BGCs</em> 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 <em>BGCs</em>. In the last ten years, a variety of methods have been reported for the direct cloning of <em>BGCs</em> 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 <em>DNA</em>; 2) how to digest the bilateral boundaries for release of the target <em>BGC</em>; and 3) how to assemble the <em>BGC</em> and the capture vector. Here, we summarize recent reports regarding how to directly capture a <em>BGC</em> 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.</p></div>","PeriodicalId":100478,"journal":{"name":"Engineering Microbiology","volume":"3 3","pages":"Article 100085"},"PeriodicalIF":0.0,"publicationDate":"2023-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49891304","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}