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Although gene delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective for numerous clinical gene therapy applications, many challenges remain. Recent advances in AAV vector development through rational design and directed evolution, as well as in the design of novel genetic cargoes, promise to extend clinical successes of AAV-mediated gene therapy. Clinical gene therapy has been increasingly successful owing both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among these technologies, delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective and, in one recent case, have led to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers.

A 15-year-old patient with cystic fibrosis with a disseminated Mycobacterium abscessus infection was treated with a three-phage cocktail following bilateral lung transplantation. Effective lytic phage derivatives that efficiently kill the infectious M. abscessus strain were developed by genome engineering and forward genetics. Intravenous phage treatment was well tolerated and associated with objective clinical improvement, including sternal wound closure, improved liver function, and substantial resolution of infected skin nodules.Clinical use of engineered bacteriophages for the treatment of disseminated mycobacterial infection.

Lipid-based biofuels, such as biodiesel and hydroprocessed esters, are a central part of the global initiative to reduce the environmental impact of the transport sector. The vast majority of production is currently from first-generation feedstocks, such as rapeseed oil, and waste cooking oils. However, the increased exploitation of soybean oil and palm oil has led to vast deforestation, smog emissions and heavily impacted on biodiversity in tropical regions. One promising alternative, potentially capable of meeting future demand sustainably, are oleaginous yeasts. Despite being known about for 143 years, there has been an increasing effort in the last decade to develop a viable industrial system, with currently around 100 research papers published annually. In the academic literature, approximately 160 native yeasts have been reported to produce over 20% of their dry weight in a glyceride-rich oil. The most intensively studied oleaginous yeast have been Cutaneotrichosporon oleaginosus (20% of publications), Rhodotorula toruloides (19%) and Yarrowia lipolytica (19%). Oleaginous yeasts have been primarily grown on single saccharides (60%), hydrolysates (26%) or glycerol (19%), and mainly on the mL scale (66%). Process development and genetic modification (7%) have been applied to alter yeast performance and the lipids, towards the production of biofuels (77%), food/supplements (24%), oleochemicals (19%) or animal feed (3%). Despite over a century of research and the recent application of advanced genetic engineering techniques, the industrial production of an economically viable commodity oil substitute remains elusive. This is mainly due to the estimated high production cost, however, over the course of the twenty-first century where climate change will drastically change global food supply networks and direct governmental action will likely be levied at more destructive crops, yeast lipids offer a flexible platform for localised, sustainable lipid production. Based on data from the large majority of oleaginous yeast academic publications, this review is a guide through the history of oleaginous yeast research, an assessment of the best growth and lipid production achieved to date, the various strategies employed towards industrial production and importantly, a critical discussion about what needs to be built on this huge body of work to make producing a yeast-derived, more sustainable, glyceride oil a commercial reality.

The manufacture of recombinant therapeutics is a fastest-developing section of therapeutic pharmaceuticals and presently plays a significant role in disease management. Yeasts are established eukaryotic host for heterologous protein production and offer distinctive benefits in synthesising pharmaceutical recombinants. Yeasts are proficient of vigorous growth on inexpensive media, easy for gene manipulations, and are capable of adding post translational changes of eukaryotes. Saccharomyces cerevisiae is model yeast that has been applied as a main host for the manufacture of pharmaceuticals and is the major tool box for genetic studies; nevertheless, numerous other yeasts comprising Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Yarrowia lipolytica have attained huge attention as non-conventional partners intended for the industrial manufacture of heterologous proteins. Here we review the advances in yeast gene manipulation tools and techniques for heterologous pharmaceutical protein synthesis. Application of secretory pathway engineering, glycosylation engineering strategies and fermentation scale-up strategies in customizing yeast cells for the synthesis of therapeutic proteins has been meticulously described.

Soil microbiomes are extremely complex, with dense networks of interconnected microbial species underpinning vital functions for the ecosystem. In advanced agricultural research, rhizosphere microbiome engineering is gaining much attention, as the microbial community has been acknowledged to be a crucial partner of associated plants for their health fitness and yield. However, single or combined effects of a wide range of soil biotic and abiotic factors impact the success of engineered microbiomes, as these microbial communities exhibit uneven structural and functional networks in diverse soil conditions. Therefore, once a deep understanding of major influential factors and corresponding microbial responses is developed, the microbiome can be more effectively manipulated and optimized for cropping benefits. In this mini-review, we propose the concept of a microbiome-mediated smart agriculture system (MiMSAS). We summarize some of the advanced strategies for engineering the rhizosphere microbiome to withstand the stresses imposed by dominant abiotic and biotic factors. This work will help the scientific community gain more clarity about engineered microbiome technologies for increasing crop productivity and environmental sustainability.Key points• Individual or combined effects of soil biotic and abiotic variables hamper the implementation of engineered microbiome technologies in the field.• As a traditional approach, reduced-tillage practices coinciding with biofertilization can promote a relatively stable functional microbiome.• Increasing the complexity and efficiency of the synthetic microbiome is one way to improve its field-application success rate.• Plant genome editing/engineering is a promising approach for recruiting desired microbiomes for agricultural benefit.

Adaptive laboratory evolution is a frequent method in biological studies to gain insights into the basic mechanisms of molecular evolution and adaptive changes that accumulate in microbial populations during long term selection under specified growth conditions. Although regularly performed for more than 25 years, the advent of transcript and cheap next-generation sequencing technologies has resulted in many recent studies, which successfully applied this technique in order to engineer microbial cells for biotechnological applications. Adaptive laboratory evolution has some major benefits as compared with classical genetic engineering but also some inherent limitations. However, recent studies show how some of the limitations may be overcome in order to successfully incorporate adaptive laboratory evolution in microbial cell factory design. Over the last two decades important insights into nutrient and stress metabolism of relevant model species were acquired, whereas some other aspects such as niche-specific differences of non-conventional cell factories are not completely understood. Altogether the current status and its future perspectives highlight the importance and potential of adaptive laboratory evolution as approach in biotechnological engineering.

In this study, we successfully applied the strategy of combining tandem promoters and tandem signal peptides with overexpressing signal peptidase to efficiently express and produce [gamma]-glutamyl peptidase (GGT) enzymes (BsGGT, BaGGT, and BlGGT) from Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis in Bacillus subtilis ATCC6051[DELTA]5. In order to avoid the problem of instability caused by duplicated strong promoters, we assembled tandem promoters of different homologous genes from different species. To achieve resistance marker-free enzyme in the food industry, we first removed the replication origin and corresponding resistance marker of Escherichia coli from the expression vector. The plasmid was then transformed into the B. subtilis host, and the Kan resistance gene in the expression plasmid was directly edited and silenced using the CRISPR/Cas9n-AID base editing system. As a result, a recombinant protein expression carrier without resistance markers was constructed, and the enzyme activity of the BlGGT strain during shake flask fermentation can reach 53.65 U/mL. The recombinant BlGGT was immobilized with epoxy resin and maintained 82.8% enzyme activity after repeated use for 10 times and 87.36% enzyme activity after storage at 4 °C for 2 months. The immobilized BlGGT enzyme was used for the continuous synthesis of theanine with a conversion rate of 65.38%. These results indicated that our approach was a promising solution for improving enzyme production efficiency and achieving safe production of enzyme preparations in the food industry.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/ , constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

Cryptosporidium is an important cause of diarrhoeal disease in young children but until now it has been difficult to study; here, the parasite is genetically modified, paving the way for in-depth investigation and the development of effective treatments. Cryptosporidium engineered for drug screening The protozoan parasite Cryptosporidium is a major cause of diarrhoeal disease in young children but until now it has been difficult to study and there is currently no vaccine and only a single drug (nitazoxanide) available to counter the infection. Here Boris Striepen and colleagues describe a robust genetic system for cryptosporidiosis. They genetically modify Cryptosporidium parvum by optimizing transfection of sporozoites using a CRISPR/Cas9 system, to generate stable transgenic lines suitable for in vitro and in vivo drug screening. Using this system they knockout the gene encoding thymidine kinase which increases susceptibility to trimethoprim, an antimalarial drug to which wild-type Cryptosporidium is resistant. Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus 1 , 2 , 3 . Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality 4 . Cryptosporidium is also an opportunistic pathogen in the contexts of human immunodeficiency virus (HIV)-caused AIDS and organ transplantation 5 , 6 . There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger: malnourished children and immunocompromised patients 7 , 8 . Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes a lack of systems for continuous culture, facile animal models, and molecular genetic tools 3 , 9 . Here we describe an experimental framework to genetically modify this important human pathogen. We established and optimized transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we developed a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and in vivo selection for aminoglycoside resistance. We derived reporter parasites suitable for in vitro and in vivo drug screening, and we evaluated the basis of drug susceptibility by gene knockout. We anticipate that the ability to genetically engineer this parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection, and the role of parasite genes in these processes is now open to rigorous investigation.

Background Iturins, which belong to antibiotic cyclic lipopeptides mainly produced by Bacillus sp., have the potential for application in biomedicine and biocontrol because of their hemolytic and antifungal properties. Bacillus amyloliquefaciens LL3, isolated previously by our lab, possesses a complete iturin A biosynthetic pathway as shown by genomic analysis. Nevertheless, iturin A could not be synthesized by strain LL3, possibly resulting from low transcription level of the itu operon. Results In this work, enhanced transcription of the iturin A biosynthetic genes was implemented by inserting a strong constitutive promoter C2up into upstream of the itu operon, leading to the production of iturin A with a titer of 37.35 mg l −1 . Liquid chromatography-mass spectrometry analyses demonstrated that the strain produced four iturin A homologs with molecular ion peaks at m / z 1044, 1058, 1072 and 1086 corresponding to [C 14  + 2H] 2+ , [C 15  + 2H] 2+ , [C 16  + 2H] 2+ and [C 17  + 2H] 2+ . The iturin A extract exhibited strong inhibitory activity against several common plant pathogens. The yield of iturin A was improved to 99.73 mg l −1 by the optimization of the fermentation conditions using a response surface methodology. Furthermore, the yield of iturin A was increased to 113.1 mg l −1 by overexpression of a pleiotropic regulator DegQ. Conclusions To our knowledge, this is the first report on simultaneous production of four iturin A homologs (C 14 –C 17 ) by a Bacillus strain. In addition, this study suggests that metabolic engineering in combination with culture conditions optimization may be a feasible method for enhanced production of bacterial secondary metabolites.