Search

Sinorhizobium meliloti is a free-living soil Gram-negative bacterium that participates in nitrogen-fixation symbiosis with several legumes. S. meliloti has the potential to be utilized for the production of high-value nutritional compounds, such as vitamin B 12 . Advances in gene editing tools play a vital role in the development of S. meliloti strains with enhanced characteristics for biotechnological applications. Several novel genetic engineering strategies have emerged in recent years to investigate genetic modifications in S. meliloti . This review provides a comprehensive overview of the mechanism and application of the extensively used Tn5-mediated genetic engineering strategies. Strategies based on homologous recombination and site-specific recombination were also discussed. Subsequently, the development and application of the genetic engineering strategies utilizing various CRISPR/Cas systems in S. meliloti are summarized. This review may stimulate research interest among scientists, foster studies in the application areas of S. meliloti , and serve as a reference for the utilization of genome editing tools for other Rhizobium species.

Rhizobia interact with leguminous plants in the soil to form nitrogen fixing nodules in which rhizobia and plant cells coexist. Although there are emerging studies on rhizobium-associated nitrogen fixation in cereals, the legume-rhizobium interaction is more well-studied and usually serves as the model to study rhizobium-mediated nitrogen fixation in plants. Rhizobia play a crucial role in the nitrogen cycle in many ecosystems. However, rhizobia are highly sensitive to variations in soil conditions and physicochemical properties (i.e. moisture, temperature, salinity, pH, and oxygen availability). Such variations directly caused by global climate change are challenging the adaptive capabilities of rhizobia in both natural and agricultural environments. Although a few studies have identified rhizobial genes that confer adaptation to different environmental conditions, the genetic basis of rhizobial stress tolerance remains poorly understood. In this review, we highlight the importance of improving the survival of rhizobia in soil to enhance their symbiosis with plants, which can increase crop yields and facilitate the establishment of sustainable agricultural systems. To achieve this goal, we summarize the key challenges imposed by global climate change on rhizobium-plant symbiosis and collate current knowledge of stress tolerance-related genes and pathways in rhizobia. And finally, we present the latest genetic engineering approaches, such as synthetic biology, implemented to improve the adaptability of rhizobia to changing environmental conditions., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Chlamydomonas reinhardtii is gaining recognition as a promising expression system for the production of recombinant proteins. However, its performance as a cellular biofactory remains suboptimal, especially with respect to consistent expression of heterologous genes. Gene silencing mechanisms, position effect, and low nuclear transgene expression are major drawbacks for recombinant protein production in this model system. To unveil the molecular changes following transgene insertion, retention, and expression in this species, we genetically engineered C. reinhardtii wild type strain 137c (strain cc-125 mt+) to express the fluorescent protein mVenus and subsequently analysed its intracellular proteome., Results: The obtained transgenic cell lines showed differences in abundance in more than 400 proteins, with multiple pathways altered post-transformation. Proteins involved in chromatin remodelling, translation initiation and elongation, and protein quality control and transport were found in lower abundance. On the other hand, ribosomal proteins showed higher abundance, a signal of ribosomal stress response., Conclusions: These results provide new insights into the modifications of C. reinhardtii proteome after transformation, highlighting possible pathways involved in gene silencing. Moreover, this study identifies multiple protein targets for future genetic engineering approaches to improve the prospective use of C. reinhardtii as cell biofactory for industrial applications., (© 2024. The Author(s).)

The combination of nanoparticles and tumor-targeting bacteria for cancer immunotherapy can overcome the shortcomings of poor nanoparticle accumulation, limited penetration, and restricted distribution. However, it remains a great challenge for the hybrid system to improve therapeutic efficacy through the simultaneous and controllable regulation of immune cells and tumor cells. Herein, a hybrid therapeutic platform is rationally designed to achieve immune cascade-augmented cancer immunotherapy. To construct the hybrids, photothermal nanoparticles responsive to light in the second near-infrared (NIR-II) region are conjugated onto the surface of engineered bacteria through pH-responsive Schiff base bonds. Taking advantage of the hypoxia targeting and deep penetration characteristics of the bacteria, the hybrids can accumulate at tumor sites. Then nanoparticles detach from the bacteria to realize genetic engineering of tumor cells, which induces tumor cell apoptosis and down-regulate the expression of programmed cell death ligand 1 to alleviate immunosuppressive tumor microenvironment. The mild photothermal heating can not only induce tumor-associated antigen release, but also trigger sustainable expression of cytokine interleukin-2. Notably, a synergistic antitumor effect is achieved between the process of p53 transfection and NIR-II light-activated genetic engineering of bacteria. This work proposes a facile strategy for the construction of hybrid system to achieve cascade-augmented cancer immunotherapy., (© 2024 Wiley‐VCH GmbH.)

With the depletion of non-renewable fossil fuels, there has been an increasing emphasis on renewable biomass. Penicillium oxalicum is notable for its exceptional capacity to secrete a diverse array of enzymes that degrade plant polysaccharides into monosaccharides. These valuable monosaccharides can be harnessed in the production of bioethanol and other sustainable forms of energy. By enhancing the production of plant-polysaccharide-degrading enzymes (PPDEs) in P. oxalicum, we can optimize the utilization of plant biomass. This paper presents recent advances in augmenting PPDE expression in P. oxalicum through genetic engineering strategies involving protoplast preparation, transformation, and factors influencing PPDE gene expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bacterial nanocellulose (BNC) is a biopolymer that is drawing significant attention for a wide range of applications thanks to its unique structure and excellent properties, such as high purity, mechanical strength, high water holding capacity and biocompatibility. Nevertheless, the biomanufacturing of BNC is hindered due to its low yield, the instability of microbial strains and cost limitations that prevent it from being mass-produced on a large scale. Various approaches have been developed to address these problems by genetically modifying strains and to produce BNC-based biomaterials with added value. These works are summarized and discussed in the present article, which include the overexpression and knockout of genes related and not related with the nanocellulose biosynthetic operon, the application of synthetic biology approaches and CRISPR/Cas techniques to modulate BNC biosynthesis. Further discussion is provided on functionalized BNC-based biomaterials with tailored properties that are incorporated in-vivo during its biosynthesis using genetically modified strains either in single or co-culture systems (in-vivo manufacturing). This novel strategy holds potential to open the road toward cost-effective production processes and to find novel applications in a variety of technology and industrial fields., Competing Interests: Declaration of competing interest The authors for the manuscript titled Toward biomanufacturing of next-generation bacterial nanocellulose (BNC)-based materials with tailored properties: A review on metabolic engineering approaches declare no conflicting interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts that have the potential to contribute to the sustainable bioeconomy. Among the Alphaproteobacteria, Novosphingobium aromaticivorans , Rhodobacter sphaeroides , and Zymomonas mobilis show promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for Z. mobilis , N. aromaticivorans and R. sphaeroides still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for N. aromaticivorans and R. sphaeroides to address these issues. We demonstrate that Tn 7 transposition is an effective approach for introducing engineered DNA into the chromosome of N. aromaticivorans and R. sphaeroides . We screen a synthetic promoter library to identify isopropyl β-D-1-thiogalactopyranoside-inducible promoters with regulated activity in both organisms (up to ~15-fold induction in N. aromaticivorans and ~5-fold induction in R. sphaeroides ). Combining Tn 7 integration with promoters from our library, we establish CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference systems for N. aromaticivorans and R. sphaeroides (up to ~10-fold knockdown in N. aromaticivorans and R. sphaeroides ) that can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these species and other Alphaproteobacteria.IMPORTANCEIt is important to increase our understanding of the microbial world to improve health, agriculture, the environment, and biotechnology. For example, building a sustainable bioeconomy depends on the efficient conversion of plant material to valuable biofuels and bioproducts by microbes. One limitation in this conversion process is that microbes with otherwise promising properties for conversion are challenging to genetically engineer. Here we report genetic tools for Novosphingobium aromaticivorans and Rhodobacter sphaeroides that add to the burgeoning set of tools available for genome engineering and gene expression in Alphaproteobacteria. Our approaches allow straightforward insertion of engineered pathways into the N. aromaticivorans or R. sphaeroides genome and control of gene expression by inducing genes with synthetic promoters or repressing genes using CRISPR interference. These tools can be used in future work to gain additional insight into these and other Alphaproteobacteria and to aid in optimizing yield of biofuels and bioproducts., Competing Interests: J.M.P. and A.B.B. have filed for patents related to Mobile-CRISPRi technology and bacterial promoters.

One objection to xenotransplantation is that it will require the large-scale breeding, raising and killing of genetically modified pigs. The pigs will need to be raised in designated pathogen-free facilities and undergo a range of medical tests before having their organs removed and being euthanised. As a result, they will have significantly shortened life expectancies, will experience pain and suffering and be subject to a degree of social and environmental deprivation. To minimise the impact of these factors, we propose the following option for consideration-ethically defensible xenotransplantation should entail the use of genetic disenhancement if it becomes possible to do so and if that pain and suffering cannot be eliminated by other means. Despite not being a morally ideal 'solution', it is morally better to prevent unavoidable pain until a viable non-animal alternative becomes available., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Chimeric antigen receptor (CAR) T-cell therapy has achieved notable success in treating hematological cancers but faces significant challenges in solid-tumor treatment and overall efficacy. Key limitations include T-cell exhaustion, tumor relapse, immunosuppressive tumor microenvironments (TME), immunogenicity, and antigen heterogeneity. To address these issues, various genetic engineering strategies have been proposed. Approaches such as overexpression of transcription factors or metabolic armoring and dynamic CAR regulation are being explored to improve CAR T-cell function and safety. Other efforts to improve CAR T-cell efficacy in solid tumors include targeting novel antigens or developing alternative strategies to address antigen diversity. Despite the promising preclinical results of these solutions, challenges remain in translating CAR T-cell therapies to the clinic to enable economically viable access to these transformative medicines. The efficiency and scalability of autologous CAR T-cell therapy production are hindered by traditional, manual processes which are costly, time-consuming, and prone to variability and contamination. These high-cost, time-intensive processes have complex quality-control requirements. Recent advancements suggest that smaller, decentralized solutions such as microbioreactors and automated point-of-care systems could improve production efficiency, reduce costs, and shorten manufacturing timelines, especially when coupled with innovative manufacturing methods such as transposons and lipid nanoparticles. Future advancements may include harmonized consumables and AI-enabled technologies, which promise to streamline manufacturing, reduce costs, and enhance production quality.

The spotted wing Drosophila (Drosophila suzukii Matsumura) is recognized globally as a significant economic pest. Here, we present a protocol for genetic engineering in D. suzukii using microinjection. We describe steps for genetic engineering techniques, including transposon-mediated germline transformation, recombinase-mediated genome targeting, and CRISPR-mediated gene editing. This protocol can significantly expand the toolkit for functional genomics and genetic control studies of this pest. For complete details on the use and execution of this protocol, please refer to Schetelig and Handler, 1 Schetelig et al. 2 Yan et al., 3 and Yan et al. 4 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Collaboration between cancer treatment and inflammation management has emerged as an integral facet of comprehensive cancer care. Nevertheless, the development of interventions concurrently targeting both inflammation and cancer has encountered significant challenges stemming from various external factors. Herein, a bioactive agent synthesized by genetically engineering melanin-producing Bacillus thuringiensis (B. thuringiensis) bacteria, simultaneously achieves eco-friendly photothermal agent and efficient reactive oxygen/nitrogen species (RONS) scavenger benefits, perfectly tackling present toughies from inflammation to cancer therapies. The biologically derived melanin exhibits exceptional photothermal-conversion performance, facilitating potent photonic hyperthermia that effectively eradicates tumor cells and tissues, thereby impeding tumor growth. Additionally, the RONS-scavenging properties of melanin produced by B. thuringiensis bacteria contribute to inflammation reduction, augmenting the efficacy of photothermal tumor repression. This study presents a representative paradigm of genetic engineering in B. thuringiensis bacteria to produce functional agents tailored for diverse biomedical applications, encompassing inflammation and cancer therapy., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Addressing the threat of harmful cyanobacterial blooms (CyanoHABs) and their associated microcystins (MCs) is crucial for global drinking water safety. In this review, we comprehensively analyze and compares the physical, chemical, and biological methods and genetic engineering for MCs degradation in aquatic environments. Physical methods, such as UV treatments and photocatalytic reactions, have a high efficiency in breaking down MCs, with the potential for further enhancement in performance and reduction of hazardous byproducts. Chemical treatments using chlorine dioxide and potassium permanganate can reduce MC levels but require careful dosage management to avoid toxic by-products and protect aquatic ecosystems. Biological methods, including microbial degradation and phytoremediation techniques, show promise for the biodegradation of MCs, offering reduced environmental impact and increased sustainability. Genetic engineering, such as immobilization of microcystinase A (MlrA) in Escherichia coli and its expression in Synechocystis sp., has proven effective in decomposing MCs such as MC-LR. However, challenges related to specific environmental conditions such as temperature variations, pH levels, presence of other contaminants, nutrient availability, oxygen levels, and light exposure, as well as scalability of biological systems, necessitate further exploration. We provide a comprehensive evaluation of MCs degradation techniques, delving into their practicality, assessing the environmental impacts, and scrutinizing their efficiency to offer crucial insights into the multifaceted nature of these methods in various environmental contexts. The integration of various methodologies to enhance degradation efficiency is vital in the field of water safety, underscoring the need for ongoing innovation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Carotenoids, with their diverse biological activities and potential pharmaceutical applications, have garnered significant attention as essential nutraceuticals. Microalgae, as natural producers of these bioactive compounds, offer a promising avenue for sustainable and cost-effective carotenoid production. Despite the ability to cultivate microalgae for its high-value carotenoids with health benefits, only astaxanthin and β-carotene are produced on a commercial scale by Haematococcus pluvialis and Dunaliella salina, respectively. This review explores recent advancements in genetic engineering and cultivation strategies to enhance the production of lutein by microalgae. Techniques such as random mutagenesis, genetic engineering, including CRISPR technology and multi-omics approaches, are discussed in detail for their impact on improving lutein production. Innovative cultivation strategies are compared, highlighting their advantages and challenges. The paper concludes by identifying future research directions, challenges, and proposing strategies for the continued advancement of cost-effective and genetically engineered microalgal carotenoids for pharmaceutical applications.

The interface between electrodes and neural tissues plays a pivotal role in determining the efficacy and fidelity of neural activity recording and modulation. While considerable efforts have been made to improve the electrode-tissue interface, the majority of studies have primarily concentrated on the development of biocompatible neural electrodes through abiotic materials and structural engineering. In this study, an approach is presented that seamlessly integrates abiotic and biotic engineering principles into the electrode-tissue interface. Specifically, ultraflexible neural electrodes with short hairpin RNAs (shRNAs) designed to silence the expression of endogenous genes within neural tissues are combined. The system facilitates shRNA-mediated knockdown of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and polypyrimidine tract-binding protein 1 (PTBP1), two essential genes associated in neural survival/growth and neurogenesis, within specific cell populations located at the electrode-tissue interface. Additionally, it is demonstrated that the downregulation of PTEN in neurons can result in an enlargement of neuronal cell bodies at the electrode-tissue interface. Furthermore, the system enables long-term monitoring of neuronal activities following PTEN knockdown in a mouse model of Parkinson's disease and traumatic brain injury. The system provides a versatile approach for genetically engineering the electrode-tissue interface with unparalleled precision, paving the way for the development of regenerative electronics and next-generation brain-machine interfaces., (© 2024 Wiley‐VCH GmbH.)

Background: Betalains are reddish and yellow pigments that accumulate in a few plant species of the order Caryophyllales. These pigments have antioxidant and medicinal properties and can be used as functional foods. They also enhance resistance to stress or disease in crops. Several plant species belonging to other orders have been genetically engineered to express betalain pigments. Betalains can also be used for flower color modification in ornamental plants, as they confer vivid colors, like red and yellow. To date, betalain engineering to modify the color of Torenia fournieri-or wishbone flower-a popular ornamental plant, has not been attempted., Results: We report the production of purple-reddish-flowered torenia plants from the purple torenia cultivar "Crown Violet."  Three betalain-biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were constitutively ectopically expressed under the cauliflower mosaic virus (CaMV) 35S promoter, and their expression was confirmed by quantitative real-time PCR (qRT-PCR) analysis. The color traits, measured by spectrophotometric colorimeter and spectral absorbance of fresh petal extracts, revealed a successful flower color modification from purple to reddish. Red pigmentation was also observed in whole plants. LC-DAD-MS and HPLC analyses confirmed that the additional accumulated pigments were betacyanins-mainly betanin (betanidin 5-O-glucoside) and, to a lesser extent, isobetanin (isobetanidin 5-O-glucoside). The five endogenous anthocyanins in torenia flower petals were also detected., Conclusions: This study demonstrates the possibility of foreign betacyanin accumulation in addition to native pigments in torenia, a popular garden bedding plant. To our knowledge, this is the first report presenting engineered expression of betalain pigments in the family Linderniaceae. Genetic engineering of betalains would be valuable in increasing the flower color variation in future breeding programs for torenia., (© 2024. The Author(s).)

The fastest replicating bacterium Vibrio natriegens is a rising workhorse for molecular and biotechnological research with established tools for efficient genetic manipulation. Here, we expand on the capabilities of multiplex genome editing by natural transformation (MuGENT) by identifying a neutral insertion site and showing how two selectable markers can be swapped at this site for sequential rounds of natural transformation. Second, we demonstrated that MuGENT can be used for complementation by gene insertion at an ectopic chromosomal locus. Additionally, we developed a robust method to cure the competence plasmid required to induce natural transformation. Finally, we demonstrated the ability of MuGENT to create massive deletions; the 280 kb deletion created in this study is one of the largest artificial deletions constructed in a single round of targeted mutagenesis of a bacterium. These methods each advance the genetic potential of V. natriegens and collectively expand upon its utility as an emerging model organism for synthetic biology., Importance: Vibrio natriegens is an emerging model organism for molecular and biotechnological applications. Its fast growth, metabolic versatility, and ease of genetic manipulation provide an ideal platform for synthetic biology. Here, we develop and apply novel methods that expand the genetic capabilities of the V. natriegens model system. Prior studies developed a method to manipulate multiple regions of the chromosome in a single step. Here, we provide new resources that diversify the utility of this method. We also provide a technique to remove the required genetic tools from the cell once the manipulation is performed, thus establishing "clean" derivative cells. Finally, we show the full extent of this technique's capability by generating one of the largest chromosomal deletions reported in the literature. Collectively, these new tools will be beneficial broadly to the Vibrio community and specifically to the advancement of V. natriegens as a model system., Competing Interests: The authors declare no conflict of interest.

Hyaluronic acid (HA), which is a highly versatile glycosaminoglycan, is widely applied across the fields of food, cosmetics, and pharmaceuticals. It is primary produced through Streptococcus fermentation, but the product presents inherent challenges concerning consistency and potential pathogenicity. However, recent strides in molecular biology have paved the way for genetic engineering, which facilitates the creation of high-yield, nonpathogenic strains adept at synthesizing HA with specific molecular weights. This comprehensive review extensively explores the molecular biology underpinning pivotal HA synthase genes, which elucidates the intricate mechanisms governing HA synthesis. Moreover, it delineates various strategies employed in engineering HA-producing strains., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Methanogenic archaea, which are integral to global carbon and nitrogen cycling, currently face challenges in genetic manipulation due to unique physiology and limited genetic tools. This review provides a survey of current and past developments in the genetic engineering of methanogens, including selection and counterselection markers, reporter systems, shuttle vectors, mutagenesis methods, markerless genetic exchange, and gene expression control. This review discusses genetic tools and emphasizes challenges tied to tool scarcity for specific methanogenic species. Mutagenesis techniques for methanogens, including physicochemical, transposon-mediated, liposome-mediated mutagenesis, and natural transformation, are outlined, along with achievements and challenges. Markerless genetic exchange strategies, such as homologous recombination and CRISPR/Cas-mediated genome editing, are also detailed. Finally, the review concludes by examining the control of gene expression in methanogens. The information presented underscores the urgent need for refined genetic tools in archaeal research. Despite historical challenges, recent advancements, notably CRISPR-based systems, hold promise for overcoming obstacles, with implications for global health, agriculture, climate change, and environmental engineering. This comprehensive review aims to bridge existing gaps in the literature, guiding future research in the expanding field of archaeal genetic engineering., Competing Interests: The authors declare no conflict of interest.

Despite the development of a vaccine against cutaneous leishmaniasis in preclinical and clinical studies, we still do not have a safe and effective vaccine for human use. Given this situation, the search for a new prophylactic alternative to control leishmaniasis should be a global priority. A first-generation vaccine strategy-leishmanization, in which live Leishmania major parasites are inoculated into the skin to protect against reinfection, is taking advantage of this situation. Live attenuated Leishmania vaccine candidates are promising alternatives due to their robust protective immune responses. Importantly, they do not cause disease and could provide long-term protection following challenges with a virulent strain. In addition to physical and chemical methods, genetic tools, including the Cre- loxP system, have enabled the selection of safer null mutant live attenuated Leishmania parasites obtained by gene disruption. This was followed by the discovery and introduction of CRISPR/Cas-based gene editing tools, which can be easily and precisely used to modify genes. Here, we briefly review the immunopathology of L. major parasites and then present the classical methods and their limitations for the production of live attenuated vaccines. We then discuss the potential of current genetic engineering tools to generate live attenuated vaccine strains by targeting key genes involved in L. major pathogenesis and then discuss their discovery and implications for immune responses to control leishmaniasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rooholamini, Dianat-Moghadam, Esmaeilifallah and Khanahmad.)

Bacteriophages (phages) are viruses specific to bacteria that target them with great efficiency and specificity. Phages were first studied for their antibacterial potential in the early twentieth century; however, their use was largely eclipsed by the popularity of antibiotics. Given the surge of antimicrobial-resistant strains worldwide, there has been a renaissance in harnessing phages as therapeutics once more. One of the key advantages of phages is their amenability to modification, allowing the generation of numerous derivatives optimised for specific functions depending on the modification. These enhanced derivatives could display higher infectivity, expanded host range or greater affinity to human tissues, where some bacterial species exert their pathogenesis. Despite this, there has been a noticeable discrepancy between the generation of derivatives in vitro and their clinical application in vivo. In most instances, phage therapy is only used on a compassionate-use basis, where all other treatment options have been exhausted. A lack of clinical trials and numerous regulatory hurdles hamper the progress of phage therapy and in turn, the engineered variants, in becoming widely used in the clinic. In this review, we outline the various types of modifications enacted upon phages and how these modifications contribute to their enhanced bactericidal function compared with wild-type phages. We also discuss the nascent progress of genetically modified phages in clinical trials along with the current issues these are confronted with, to validate it as a therapy in the clinic., (© 2024 The Author(s).)

Compared with the widely used rodents, pigs are anatomically, physiologically, and genetically more similar to humans, making them high-quality models for the study of liver diseases. Here, we review the latest research progress on pigs as a model of human liver disease, including methods for establishing them and their advantages in studying cystic fibrosis liver disease, acute liver failure, liver regeneration, non-alcoholic fatty liver disease, liver tumors, and xenotransplantation. We also emphasize the importance of genetic engineering techniques, mainly the CRISPR/Cas9 system, which has greatly enhanced the utility of porcine models as a tool for substantially advancing liver disease research. Genetic engineering is expected to propel the pig as one of the irreplaceable animal models for future biomedical research.

Abstract Background Chlamydomonas reinhardtii is gaining recognition as a promising expression system for the production of recombinant proteins. However, its performance as a cellular biofactory remains suboptimal, especially with respect to consistent expression of heterologous genes. Gene silencing mechanisms, position effect, and low nuclear transgene expression are major drawbacks for recombinant protein production in this model system. To unveil the molecular changes following transgene insertion, retention, and expression in this species, we genetically engineered C. reinhardtii wild type strain 137c (strain cc-125 mt+) to express the fluorescent protein mVenus and subsequently analysed its intracellular proteome. Results The obtained transgenic cell lines showed differences in abundance in more than 400 proteins, with multiple pathways altered post-transformation. Proteins involved in chromatin remodelling, translation initiation and elongation, and protein quality control and transport were found in lower abundance. On the other hand, ribosomal proteins showed higher abundance, a signal of ribosomal stress response. Conclusions These results provide new insights into the modifications of C. reinhardtii proteome after transformation, highlighting possible pathways involved in gene silencing. Moreover, this study identifies multiple protein targets for future genetic engineering approaches to improve the prospective use of C. reinhardtii as cell biofactory for industrial applications.

Our reliance on agriculture for sustenance, healthcare, and resources has been essential since the dawn of civilization. However, traditional agricultural practices are no longer adequate to meet the demands of a burgeoning population amidst climate-driven agricultural challenges. Microalgae emerge as a beacon of hope, offering a sustainable and renewable source of food, animal feed, and energy. Their rapid growth rates, adaptability to non-arable land and non-potable water, and diverse bioproduct range, encompassing biofuels and nutraceuticals, position them as a cornerstone of future resource management. Furthermore, microalgaes ability to capture carbon aligns with environmental conservation goals. While microalgae offers significant benefits, obstacles in cost-effective biomass production persist, which curtails broader application. This review examines microalgae compared to other host platforms, highlighting current innovative approaches aimed at overcoming existing barriers. These approaches include a range of techniques, from gene editing, synthetic promoters, and mutagenesis to selective breeding and metabolic engineering through transcription factors.

Background: Lovastatin has widespread applications thanks to its multiple pharmacological effects. Fermentation by filamentous fungi represents the major way of lovastatin production. However, the current lovastatin productivity by fungal fermentation is limited and needs to be improved., Results: In this study, the lovastatin-producing strains of Aspergillus terreus from marine environment were screened, and their lovastatin productions were further improved by genetic engineering. Five strains of A. terreus were isolated from various marine environments. Their secondary metabolites were profiled by metabolomics analysis using Ultra Performance Liquid Chromatography-Mass spectrometry (UPLC-MS) with Global Natural Products Social Molecular Networking (GNPS), revealing that the production of secondary metabolites was variable among different strains. Remarkably, the strain of A. terreus MJ106 could principally biosynthesize the target drug lovastatin, which was confirmed by High Performance Liquid Chromatography (HPLC) and gene expression analysis. By one-factor experiment, lactose was found to be the best carbon source for A. terreus MJ106 to produce lovastatin. To improve the lovastatin titer in A. terreus MJ106, genetic engineering was applied to this strain. Firstly, a series of strong promoters was identified by transcriptomic and green fluorescent protein reporter analysis. Then, three selected strong promoters were used to overexpress the transcription factor gene lovE encoding the major transactivator for lov gene cluster expression. The results revealed that compared to A. terreus MJ106, all lovE over-expression mutants exhibited significantly more production of lovastatin and higher gene expression. One of them, LovE-b19, showed the highest lovastatin productivity at a titer of 1512 mg/L, which represents the highest production level reported in A. terreus., Conclusion: Our data suggested that combination of strain screen and genetic engineering represents a powerful tool for improving the productivity of fungal secondary metabolites, which could be adopted for large-scale production of lovastatin in marine-derived A. terreus., (© 2024. The Author(s).)

Microbial products are essential for developing various therapeutic agents, including antibiotics, anticancer drugs, vaccines, and therapeutic enzymes. Genetic engineering techniques, functional genomics, and synthetic biology unlock previously uncharacterized natural products. This review highlights major advances in microbial biotechnology, focusing on gene-based technologies for medical applications., (© 2024 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

While diatoms are promising synthetic biology platforms, there currently exists a limited number of validated genetic regulatory parts available for genetic engineering. The standard method for diatom transformation, nonspecific introduction of DNA into chromosomes via biolistic particle bombardment, is low throughput and suffers from clonal variability and epigenetic effects. Recent developments in diatom engineering have demonstrated that autonomously replicating episomal plasmids serve as stable expression platforms for diverse gene expression technologies. These plasmids are delivered via bacterial conjugation and, when combined with modular DNA assembly technologies, provide a flexibility and speed not possible with biolistic-mediated strain generation. In order to expand the current toolbox for plasmid-based engineering in the diatom Phaeodactylum tricornutum, a conjugation-based forward genetics screen for promoter discovery was developed, and application to a diatom genomic DNA library defined 252 P. tricornutum promoter elements. From this library, 40 promoter/terminator pairs were delivered via conjugation on episomal plasmids, characterized in vivo, and ranked across 4 orders of magnitude difference in reporter gene expression levels.

D-xylose is the second most abundant monosaccharide found in lignocellulose and is of biotechnological importance for producing second-generation ethanol and other high-value chemical compounds. D-xylose conversion to ethanol is promoted by microbial fermentation, mainly by bacteria, yeasts, or filamentous fungi. Among yeasts, species belonging to the CTG(Ser1) or CTG(Ala) clade display a remarkable ability to ferment D-xylose to ethanol and other compounds; however, these yeasts are not employed on an industrial scale given their poor fermentative performance compared to that of conventional yeasts, such as Saccharomyces cerevisiae, and because of the lack of a molecular toolbox for the development of new strains tailored to fermentation stress tolerance and performance. Thus, the purpose of this review was to evaluate the major genetic engineering tools (e.g., transformation markers and techniques, vectors, regulatory sequences, and gene editing techniques) available for the most studied yeasts of the CTG(Ser1) clade, such as Scheffersomyces , Spathaspora , Candida , and Yamadazyma species, and the CTG(Ala) clade, representative Pachysolen tannophilus. Furthermore, we systematized state-of-the-art molecular developments and perspectives to design D-xylose-fermenting yeast strains. • Review of genetic tools in CTG(Ser1) and CTG(Ala) yeasts for bioethanol production. • CTG codon reassignment impairs the use of conventional genetic engineering tools. • CTG(Ser1) yeasts can use genetic tools from clinically important Candida strains for research. [ABSTRACT FROM AUTHOR]

Drought and soil salinization substantially impact agriculture. While proline's role in enhancing stress tolerance is known, the exact molecular mechanism by which plants process stress signals and control proline synthesis under stress is still not fully understood. In tomato (Solanum lycopersicum L.), drought and salt stress stimulate nitric oxide (NO) production, which boosts proline synthesis by activating Δ1-pyrroline-5-carboxylate synthetase (SlP5CS) and Δ1-pyrroline-5-carboxylate reductase (SlP5CR) genes and the P5CR enzyme. The crucial factor is stress-triggered NO production, which regulates the S-nitrosylation of SlP5CR at Cys-5, thereby increasing its NAD(P)H affinity and enzymatic activity. S-nitrosylation of SlP5CR enables tomato plants to better adapt to changing NAD(P)H levels, boosting both SlP5CR activity and proline synthesis during stress. By comparing tomato lines genetically modified to express different forms of SlP5CR, including a variant mimicking S-nitrosylation (SlP5CRC5W), we found that SlP5CRC5W plants show superior growth and stress tolerance. This is attributed to better P5CR activity, proline production, water use efficiency, reactive oxygen species scavenging, and sodium excretion. Overall, this study demonstrates that tomato engineered to mimic S-nitrosylated SlP5CR exhibits enhanced growth and yield under drought and salt stress conditions, highlighting a promising approach for stress-tolerant tomato cultivation., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Although bioactive peptides enhancing bone healing have demonstrated effectiveness in treating bone defects, in vivo instability poses a challenge to their clinical application. Currently reported peptide delivery systems do not meet the demands of bone tissue repair regarding stability and peptide release efficacy. Herein, the self-assembling recombinant chimeric protein (Sbp5-2 RGD ) is developed by genetic engineering with cell adhesion peptide RGD as the targeted peptide and a newly discovered scallop byssal-derived protein Sbp5-2 that can assemble into wet stable films as the structural domain. In vitro studies show that the Sbp5-2 RGD film exhibits excellent extensibility and biocompatibility. In vitro and in vivo degradation experiments demonstrate that the film remains stable due to the layer-by-layer degradation mode, resulting in sustained delivery of RGD in situ for up to 4 weeks. Consequently, the film can effectively promote osteogenesis, which accelerates bone defect healing and the implants osseointegration. Cell-level studies further show that the film up-regulates the expression of genes and proteins (ALP, OCN, OSX, OPN, RUNX2, VEGF) associated with osteogenesis and angiogenesis. Overall, this novel protein film represents an intelligent platform for peptide immobilization, protection, and release through its self-assembly, dense structure, and degradation mode, providing a therapeutic strategy for bone repair., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Since their initial development, cell membrane-coated nanoparticles (CNPs) have become increasingly popular in the biomedical field. Despite their inherent versatility and ability to enable complex biological applications, there is considerable interest in augmenting the performance of CNPs through the introduction of additional functionalities. Here we demonstrate a genetic-engineering-based modular approach to CNP functionalization that can encompass a wide range of ligands onto the nanoparticle surface. The cell membrane coating is engineered to express a SpyCatcher membrane anchor that can readily form a covalent bond with any moiety modified with SpyTag. To demonstrate the broad utility of this technique, three unique targeted CNP formulations are generated using different classes of targeting ligands, including a designed ankyrin repeat protein, an affibody and a single-chain variable fragment. In vitro, the modified nanoparticles exhibit enhanced affinity towards cell lines overexpressing the cognate receptors for each ligand. When formulated with a chemotherapeutic payload, the modularly functionalized nanoparticles display strong targeting ability and growth suppression in a murine tumour xenograft model of ovarian cancer. Our data suggest genetic engineering offers a feasible approach for accelerating the development of multifunctional CNPs for a broad range of biomedical applications., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)