Engineering Microbiology最新文献

Type III CRISPR-Cas10 systems employ multiple immune activities to defend their hosts against invasion from mobile genetic elements (MGEs), including DNase and cyclic oligoadenylates (cOA) synthesis both of which are hosted by the type-specific protein Cas10. Extensive investigations conducted for the activation of Cas accessory proteins by cOAs have revealed their functions in the type III immunity, but the function of the Cas10 DNase in the same process remains elusive. Here, Lactobacillus delbrueckii subsp. Bulgaricus type III-A (Ld) Csm system, a type III CRISPR system that solely relies on its Cas10 DNase for providing immunity, was employed as a model to investigate the DNase function. Interference assay was conducted in Escherichia coli using two plasmids: pCas carrying the LdCsm system and pTarget producing target RNAs. The former functioned as a de facto “CRISPR host element” while the latter, mimicking an invading MGE. We found that, upon induction of immune responses, the fate of each genetic element was determined by their copy numbers: plasmid of a low copy number was selectively eliminated from the E. coli cells regardless whether it represents a de facto CRISPR host or an invader. Together, we reveal, for the first time, that the immune mechanisms of Cas10 DNases are of two folds: the DNase activity is capable of removing low-copy invaders from infected cells, but it also leads to abortive infection when the invader copy number is high.

The metabolic engineering of Saccharomyces cerevisiae 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 S. cerevisiae metabolism. Here, we focused on the successful utilization of transcription factor (TF)-based biosensors within the dynamic regulatory network of S. cerevisiae. 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.

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.

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 Saccharomyces cerevisiae 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 ERG5 and ERG6 and introducing DHCR24 from Gallus gallus, S. cerevisiae 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 ROX1 and overexpressing UPC2-1 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_GAL1-driven ERG11 and P_GAL10-driven ERG1, 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.

The methylotrophic yeast Pichia pastoris (also known as Komagataella phaffii) is widely used as a yeast cell factory for producing heterologous proteins. Recently, it has gained attention for its potential in producing chemicals from inexpensive feedstocks, which requires efficient genetic engineering platforms. This review provides an overview of the current advances in developing genetic tools for metabolic engineering of P. pastoris. The topics cover promoters, terminators, plasmids, genome integration sites, and genetic editing systems, with a special focus on the development of CRISPR/Cas systems and their comparison to other genome editing tools. Additionally, this review highlights the prospects of multiplex genome integration, fine-tuning gene expression, and single-base editing systems. Overall, the aim of this review is to provide valuable insights into current genetic engineering and discuss potential directions for future efforts in developing efficient genetic tools in P. pastoris.

Microbial transglutaminase (TGase) is a protein that is secreted in a mature form and finds wide applications in meat products, tissue scaffold crosslinking, and textile engineering. Streptomyces mobaraensis is the only licensed producer of TGase. However, increasing the production of TGase using metabolic engineering and heterologous expression approaches has encountered challenges in meeting industrial demands. Therefore, it is necessary to identify the regulatory networks involved in TGase biosynthesis to establish a stable and highly efficient TGase cell factory. In this study, we employed a DNA-affinity capture assay and mass spectrometry analysis to discover several transcription factors. Among the candidates, eight were selected and found to impact TGase biosynthesis. Notably, SMDS_4150, an AdpA-family regulator, exhibited a significant influence and was hence named AdpA_Sm. Through electrophoretic mobility shift assays, we determined that AdpA_Sm regulates TGase biosynthesis by directly repressing the transcription of tg and indirectly inhibiting the transcription of SMDS_3961. The latter gene encodes a LytR-family positive regulator of TGase biosynthesis. Additionally, AdpA_Sm exhibited negative regulation of its own transcription. To further enhance TGase production, we combined the overexpression of SMDS_3961 with the repression of SMDS_4150, resulting in a remarkable improvement in TGase titer from 28.67 to 52.0 U/mL, representing an 81.37% increase. This study establishes AdpA as a versatile regulator involved in coordinating enzyme biosynthesis in Streptomyces species. Furthermore, we elucidated a cascaded regulatory network governing TGase production.

Biosynthesis of the functional factor γ-aminobutyric acid (GABA) in bacteria involves two key proteins an intracellular glutamate decarboxylase (GadB) and a membrane-bound antiporter (GadC). Efficient co-expression of suitable GadB and GadC candidates is crucial for improving GABA productivity. In this study, gadB_ΔC11 of Lactiplantibacillus plantarum and gadC_ΔC41 of Escherichia coli were inserted into the designed double promoter (P_T7lac and P_BAD) expression system. Then, E. coli Lemo21(DE3) was chosen as the host to minimize the toxic effects of GadC_ΔC41 overexpression. Furthermore, a green and high-efficiency GABA synthesis system using dormant engineered Lemo21(DE3) cells as biocatalysts was developed. The total GABA yield reached 829.08 g/L with a 98.7% conversion ratio within 13 h, when engineered E. coli Lemo21(DE3) cells were concentrated to an OD₆₀₀ of 20 and reused for three cycles in a 3 M L-glutamate solution at 37 °C, which represented the highest GABA productivity ever reported. Overall, expanding the active pH ranges of GadB and GadC toward physiological pH and employing a tunable expression host for membrane-bound GadC production is a promising strategy for high-level GABA biosynthesis in E. coli.

A greener method has been tested to utilize algal biomass as a feedstock to produce bio-oil in addition to acetone, butanol, and ethanol (ABE) products. Various hydrolysis treatments were used prior to fermentation including combination of thermal, chemical, and enzymatic, which resulted in maximum sugar release of 27.78 g/L. Bio-based terpenes was used instead of common toxic chemicals together with Clostridial fustants to produce bio-alcoholic fuels. Protoplast fusion technique were used to produce the novel Clostridia fusants (C. beijernickii + C. thermocellum and C. acetobutylicum + C. thermocellocum). Fused strains were then subjected to UV radiation for strain enhancement. Final fusansts showed clear improvement in thermal stability and resistance to biobutanol toxicity. Fermentation experiments showed maximum biobutanol final production of 7.98 g/L using CbCt versus 7.39 g/L using CaCt. Oil extraction from virgin algae was tested using a green, bio-based approach using terpenes with ultrasonication and green Bligh and Dyer method, separately. In preliminary study on algal biomass, the combinations of ultrasonication followed by the green Bligh and Dyer have resulted in oil yield of 46.27% (d-limonene) and 39.85% (p-cymene). Oil extraction from an algae sample following fermentation using the combined extraction method resulted in significantly higher oil yield of 65.04%.

Formic acid is one of the main weak acids in lignocellulosic hydrolysates that is known to be inhibitory to yeast growth even at low concentrations. In this study, we employed a CRISPR interference (CRISPRi) strain library comprising >9000 strains encompassing >98% of all essential and respiratory growth-essential genes, to study formic acid tolerance in Saccharomyces cerevisiae. To provide quantitative growth estimates on formic acid tolerance, the strains were screened individually on solid medium supplemented with 140 mM formic acid using the Scan-o-Matic platform. Selected resistant and sensitive strains were characterized in liquid medium supplemented with formic acid and in synthetic hydrolysate medium containing a combination of inhibitors. Strains with gRNAs targeting genes associated with chromatin remodeling were significantly enriched for strains showing formic acid tolerance. In line with earlier findings on acetic acid tolerance, we found genes encoding proteins involved in intracellular vesicle transport enriched among formic acid sensitive strains. The growth of the strains in synthetic hydrolysate medium followed the same trend as when screened in medium supplemented with formic acid. Strains sensitive to formic acid had decreased growth in the synthetic hydrolysate and all strains that had improved growth in the presence of formic acid also grew better in the hydrolysate medium. Systematic analysis of CRISPRi strains allowed identification of genes involved in tolerance mechanisms and provided novel engineering targets for bioengineering strains with increased resistance to inhibitors in lignocellulosic hydrolysates.

Toxin-antitoxin (TA) systems are ubiquitous in bacteria and archaea. Most are composed of two neighboring genetic elements, a stable toxin capable of inhibiting crucial cellular processes, including replication, transcription, translation, cell division and membrane integrity, and an unstable antitoxin to counteract the toxicity of the toxin. Many new discoveries regarding the biochemical properties of the toxin and antitoxin components have been made since the first TA system was reported nearly four decades ago. The physiological functions of TA systems have been hotly debated in recent decades, and it is now increasingly clear that TA systems are important immune systems in prokaryotes. In addition to being involved in biofilm formation and persister cell formation, these modules are antiphage defense systems and provide host defenses against various phage infections via abortive infection. In this review, we explore the potential applications of TA systems based on the recent progress made in elucidating TA functions. We first describe the most recent classification of TA systems and then introduce the biochemical functions of toxins and antitoxins, respectively. Finally, we primarily focus on and devote considerable space to the application of TA complexes in synthetic biology.