Engineering Microbiology最新文献

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.

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.

Adenoviruses typically cause mild illnesses, but severe diseases may occur primarily in immunodeficient individuals, particularly children. Recently, adenoviruses have garnered significant interest as a versatile tool in gene therapy, tumor treatment, and vaccine vector development. Over the past two decades, the advent of recombineering, a method based on homologous recombination, has notably enhanced the utility of adenoviral vectors in therapeutic applications. This review summarizes recent advancements in the use of human adenoviral vectors in medicine and discusses the pivotal role of recombineering in the development of these vectors. Additionally, it highlights the current achievements and potential future impact of therapeutic adenoviral vectors.

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.

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 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.

Leech hyaluronidase (LHyal) is a hyperactive hyaluronic acid (HA) hydrolase that belongs to the hyaluronoglucuronidase family. Traditionally, LHyal is extracted from the heads of leeches, but the recent development of the Pichia pastoris recombinant LHyal expression method permitted the industrial production of size-specific HA oligosaccharides. However, at present LHyal expressed by recombinant yeast strains requires laborious protein purification steps. Moreover, the enzyme is deactivated and removed after single use. To solve this problem, we developed a recyclable LHyal biocatalyst using a yeast surface display (YSD) system. After screening and characterization, we found that the cell wall protein Sed1p displayed stronger anchoring to the P. pastoris cell wall than other cell wall proteins. By optimizing the type and length of the linkers between LHyal and Sed1p, we increased the activity of enzymes displayed on the P. pastoris cell wall by 50.34% in flask cultures. LHyal-(GGGS)₆-Sed1p activity further increased to 3.58 × 10⁵ U mL⁻¹ in fed-batch cultivation in a 5 L bioreactor. Enzymatic property analysis results revealed that the displayed LHyal-(GGGS)₆-Sed1p generated the same oligosaccharides but exhibited higher thermal stability than free LHyal enzyme. Moreover, displayed LHyal-(GGGS)₆-Sed1p could be recovered easily from HA hydrolysis solutions via low-speed centrifugation and could be reused at least 5 times. YSD of LHyal not only increased the utilization efficiency of the enzyme but also simplified the purification process for HA oligosaccharides. Thus, this study provides an alternative approach for the industrial preparation of LHyal and HA oligosaccharides.

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.