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The SubtiToolKit (STK) is a high-efficiency Golden Gate (GG) toolkit for Bacillus subtilis and other Gram-positive bacteria, addressing a key gap in synthetic biology tools. It includes a GeoBox for Geobacillus spp. engineering, which demonstrates versatility for other bacteria.The GG method allows a rapid modular construction of complex genetic circuits by exploiting Type IIS restriction enzymes and standardized overhangs.The STK is designed with high-efficiency overhangs that enables precise assembly of transcriptional units (TU), operons, and constructs for genomic integration, reducing time, cost, and experimental complexity.Includes libraries of promoters, ribosome-binding site (RBSs), terminators, protein tags, fluorescent proteins, antibiotic cassettes, and tools to deal with toxic constructs during the assembly process.The STK unlocks the cutting-edge methods for bioengineering in Gram-positive bacteria. Building DNA constructs of increasing complexity is key to synthetic biology. Golden Gate (GG) methods led to the creation of cloning toolkits – collections of modular standardized DNA parts hosted on hierarchic plasmids, developed for yeast, plants, Gram-negative bacteria, and human cells. However, Gram-positive bacteria have been neglected. Bacillus subtilis is a Gram-positive model organism and a workhorse in the bioindustry. Here, we present the SubtiToolKit (STK), a high-efficiency cloning toolkit for B. subtilis and Gram-positive bacteria. Its design permits DNA constructs for transcriptional units (TUs), operons, and knockin and knockout applications. The STK contains libraries of promoters, ribosome-binding site (RBSs), fluorescent proteins, protein tags, terminators, genome integration parts, a no-leakage genetic device to control the expression of toxic products during Escherichia coli assembly, and a toolbox for industrially relevant strains of Geobacillus and Parageobacillus as an example of the STK versatility for other Gram-positive bacteria and its future perspective as a reference toolkit. [Display omitted] The SubtiToolKit (STK) is a high-efficiency Golden Gate (GG) cloning toolkit for Bacillus subtilis and Gram-positive bacteria. It contains pilot libraries of characterized parts, genetic devices for species-specific gene expression control, and a GeoBox with characterized plasmids and parts for Geobacillus spp., broadening synthetic biology applications across Gram-positive bacteria. The SubtiToolKit (STK) technology standardizes the assembly of genetic constructs in Gram-positive bacteria, with the potential for extension to Gram-negative species by building part libraries using the STK syntax. This technology is ready to be implemented as a tool to accelerate genetic modification in bacteria, thereby fostering advancements in synthetic biology and its biotechnological applications. The STK is designed for use in both basic and applied research projects, including upstream bioproduction to modify or adapt bacteria to industrial bioprocesses. We anticipate that it will be primarily used in projects at Technology Readiness Levels (TRLs) 1–4. In this work, we have validated the STK in a relevant laboratory environment, which corresponds to TRL 5. Advanced users may encounter challenges with the size of genetic constructs that can be assembled using STK, due to limitations in the stability of large plasmids. These challenges can be addressed by fragmenting constructs or by using cosmids specifically designed with the STK syntax.

Genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner. The library consists currently of more than 2600 plasmids and 1700 fly lines with a focus on targeting kinases, phosphatases and transcription factors, each expressing two sgRNAs under control of the Gal4/UAS system. We show that conditional CRISPR mutagenesis is robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. In order to prevent artefacts commonly associated with excessive amounts of Cas9 protein, we have developed a series of novel UAS-Cas9 transgenes, which allow fine tuning of Cas9 expression to achieve high gene editing activity without detectable toxicity. Functional assays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption. Furthermore, we conducted the so far largest fully transgenic CRISPR screen in any metazoan organism, which further supported the high efficiency and accuracy of our library and revealed many so far uncharacterized genes essential for development. Twenty years after the release of the sequence of the human genome, the role of many genes is still unknown. This is partly because some of these genes may only be active in specific types of cells or for short periods of time, which makes them difficult to study. A powerful way to gather information about human genes is to examine their equivalents in ‘model’ animals such as fruit flies. Researchers can use genetic methods to create strains of insects where genes are deactivated; evaluating the impact of these manipulations on the animals helps to understand the roles of the defunct genes. However, the current methods struggle to easily delete target genes, especially only in certain cells, or at precise times. Here, Port et al. genetically engineered flies that carry CRISPR-Cas9, a biological system that can be programmed to ‘cut’ and mutate precise genetic sequences. The insects were also manipulated in such a way that the CRISPR elements could be switched on at will, and their quantity finely tuned. This work resulted in a collection of more than 1,700 fruit fly strains in which specific genes could be deactivated on demand in precise cells. Further experiments confirmed that this CRISPR system could mutate target genes in different parts of the fly, including in the eyes, gut and wings. Port et al. have made their collection of genetically engineered fruit flies publically available, so that other researchers can use the strains in their experiments. The CRISPR technology they refined and developed may also lay the foundation for similar collections in other model organisms.

Abstract This mini-review provides a perspective of traditional, emerging and future applications of lactic acid bacteria (LAB) and how genome editing tools can be used to overcome current challenges in all these applications. It also describes available tools and how these can be further developed, and takes current legislation into account. Genome editing tools are necessary for the construction of strains for new applications and products, but can also play a crucial role in traditional ones, such as food and probiotics, as a research tool for gaining mechanistic insights and discovering new properties. Traditionally, recombinant DNA techniques for LAB have strongly focused on being food-grade, but they lack speed and the number of genetically tractable strains is still rather limited. Further tool development will enable rapid construction of multiple mutants or mutant libraries on a genomic level in a wide variety of LAB strains. We also propose an iterative Design–Build–Test–Learn workflow cycle for LAB cell factory development based on systems biology, with ‘cell factory’ expanding beyond its traditional meaning of production strains and making use of genome editing tools to advance LAB understanding, applications and strain development. Traditional, emerging and future applications of lactic acid bacteria can all benefit from genome editing and a proposed Design–Build–Test–Learn workflow cycle for advancement of strain development.

Phages are a valuable resource for the genetic engineering of Streptomyces antibiotic-producing bacteria. Indeed, a few integrative vectors based on phage integrase are available to insert transgenes at specific genomic loci. Chromosome conformation captures previously demonstrated that the Streptomyces linear chromosome is organized in two spatial compartments: the central compartment encompassing the most conserved and highly expressed genes in exponential phase, and the terminal compartments enriched in poorly conserved sequences including specialized metabolite biosynthetic gene clusters. This study introduces a new integrative tool based on a recently described phage, Samy, which specifically targets the terminal compartment of its native host chromosome. Samy is related to PhiC31 phage and, like the latter, encodes a serine integrase. Whereas PhiC31 targets a site generally located near the origin of replication, the Samy integration site is one of the farthest known attB sites from it. We demonstrated that the Samy integrase efficiently mediates the specific integration of a non-replicating plasmid in six Streptomyces strains from distinct clades. Bioinformatic analyses revealed that the Samy- att B site is rather conserved and located in the terminal compartment of most Streptomyces chromosomes. Finally, heterologous expression of the albonoursin biosynthetic gene cluster from the Samy-, PhiC31-, and R4- attB sites yields quantitatively equivalent levels of production, though qualitative differences were observed. Altogether, these results demonstrate that the att-int Samy system expands Streptomyces genetic engineering tools by enabling targeted integration in the terminal chromosomal compartment. Key points •  Samy-based integrative vectors are new tools for engineering Streptomyces strains. • They target the terminal compartment, farthest from the origin in most strains. •  They facilitate efficient heterologous production of the albonoursin antibiotic.

Developing inducible and titratable gene regulation toolkits expands the ability to control and characterize gene expression for metabolic engineering in Rhodococcus jostii RPET.Serine integrase-based recombinational tools (SIRT) allow precise genome editing for RPET.Multiple chemicals were produced from postconsumer PET through systems metabolic engineering.The practicality of fed-batch fermentation was validated for the simultaneous production of lycopene, lipids, and succinate from postconsumer PET. Poly(ethylene terephthalate) (PET) waste is of low degradability in nature, and its mismanagement threatens numerous ecosystems. To combat the accumulation of waste PET in the biosphere, PET bio-upcycling, which integrates chemical pretreatment to produce PET-derived monomers with their microbial conversion into value-added products, has shown promise. The recently discovered Rhodococcus jostii RPET strain can metabolically degrade terephthalic acid (TPA) and ethylene glycol (EG) as sole carbon sources, and it has been developed into a microbial chassis for PET upcycling. However, the scarcity of synthetic biology tools, specifically designed for this non-model microbe, limits the development of a microbial cell factory for expanding the repertoire of bioproducts from postconsumer PET. Herein, we describe the development of potent genetic tools for RPET, including two inducible and titratable expression systems for tunable gene expression, along with serine integrase-based recombinational tools (SIRT) for genome editing. Using these tools, we systematically engineered the RPET strain to ultimately establish microbial supply chains for producing multiple chemicals, including lycopene, lipids, and succinate, from postconsumer PET waste bottles, achieving the highest titer of lycopene ever reported thus far in RPET [i.e., 22.6 mg/l of lycopene, ~10 000-fold higher than that of the wild-type (WT) strain]. This work highlights the great potential of plastic upcycling as a generalizable means of sustainable production of diverse chemicals. [Display omitted] Genetic tools were developed to engineer a promising Rhodococcus jostii RPET chassis for the generalizable production of various chemicals from postconsumer PET, advancing the sustainability of PET plastic upcycling. Our results show a paradigm shift from traditional carbohydrate-based bioconversion to utilizing plastic-derived carbon sources as next-generation feedstocks for biomanufacturing. Plastic upcycling offers a promising, sustainable avenue to combat the environmental crisis caused by the accumulation of post-consumer plastic waste in terrestrial ecosystems. This study highlights the development of powerful synthetic biology tools for the non-model microorganism Rhodococcus jostii RPET, advancing its potential as a promising microbial chassis capable of using PET-derived carbon sources as feedstocks for sustainable biomanufacturing. Moreover, the practicality of this process has been validated through the implementation of fed-batch fermentation for the simultaneous production of lycopene, lipids, and succinate from postconsumer PET, reaching the Technology Readiness Level (TRL) 4. However, several major challenges still need to be effectively addressed. First, although serine integrase-based recombinational tools (SIRT) can efficiently integrate exogeneous plasmid DNA into the RPET genome, the selection of positive colonies after transformation still relies on antibiotic resistance markers, limiting its repeated applications when extensive genetic modifications are needed for strain engineering. Second, the catabolic pathway of ethylene glycol (EG) in RPET is not yet fully mapped, which restricts understanding of the potential interaction between the catabolism of terephthalic acid (TPA) and EG in PET waste upcycling, as well as their respective contributions to biomass accumulation and chemical production. Third, although alkaline hydrolysis of PET is highly efficient, the resulting effluent has a high salt concentration; the continuous supplementation of undiluted PET alkaline hydrolysate in large-scale fed-batch fermentation could elicit a continuous build-up of salts in the medium, eventually hindering the upcycling efficiency by severely disrupting the cellular equilibrium. With the growing global research interests in plastic upcycling, there is a significant opportunity to address these challenges and ultimately achieve a fully circular plastic economy.

Non-model microorganisms often possess complex phenotypes that could be important for the future of biofuel and chemical production. They have received significant interest the last several years, but advancement is still slow due to the lack of a robust genetic toolbox in most organisms. Typically, “domestication” of a new non-model microorganism has been done on an ad hoc basis, and historically, it can take years to develop transformation and basic genetic tools. Here, we review the barriers and solutions to rapid development of genetic transformation tools in new hosts, with a major focus on Restriction-Modification systems, which are a well-known and significant barrier to efficient transformation. We further explore the tools and approaches used for efficient gene deletion, DNA insertion, and heterologous gene expression. Finally, more advanced and high-throughput tools are now being developed in diverse non-model microbes, paving the way for rapid and multiplexed genome engineering for biotechnology.

Abstract Conservation genetic analyses of many endangered species have been based on genotyping of microsatellite loci and sequencing of short fragments of mtDNA. The increase in power and resolution afforded by whole genome approaches may challenge conclusions made on limited numbers of loci and maternally inherited haploid markers. Here, we provide a matched comparison of whole genome sequencing versus microsatellite and control region (CR) genotyping for Eurasian otters (Lutra lutra). Previous work identified four genetically differentiated “stronghold” populations of otter in Britain, derived from regional populations that survived the population crash of the 1950s–1980s. Using whole genome resequencing data from 45 samples from across the British stronghold populations, we confirmed some aspects of population structure derived from previous marker-driven studies. Importantly, we showed that genomic signals of the population crash bottlenecks matched evidence from otter population surveys. Unexpectedly, two strongly divergent mitochondrial lineages were identified that were undetectable using CR fragments, and otters in the east of England were genetically distinct and surprisingly variable. We hypothesize that this previously unsuspected variability may derive from past releases of Eurasian otters from other, non-British source populations in England around the time of the population bottleneck. Our work highlights that even reasonably well-studied species may harbor genetic surprises, if studied using modern high-throughput sequencing methods.

In nature, different bacteria have evolved strategies to transfer electrons far beyond the cell surface. This electron transfer enables the use of these bacteria in bioelectrochemical systems (BES), such as microbial fuel cells (MFCs) and microbial electrosynthesis (MES). The main feature of electroactive bacteria (EAB) in these applications is the ability to transfer electrons from the microbial cell to an electrode or vice versa instead of the natural redox partner. In general, the application of electroactive organisms in BES offers the opportunity to develop efficient and sustainable processes for the production of energy as well as bulk and fine chemicals, respectively. This review describes and compares key microbiological features of different EAB. Furthermore, it focuses on achievements and future prospects of genetic manipulation for efficient strain development.

The low frequency of homologous recombination together with poor efficiency in introducing DNA into the cell are the main factors hampering genetic manipulation of some bacterial strains. We faced this problem when trying to construct mutants of Streptomyces iranensis DSM 41954, a strain in which conjugation is particularly inefficient, and suicidal vectors had failed to yield any exconjugants. In this work, we report the construction and application of a conjugative replicative vector, pDS0007, which allows selection of exconjugants even with poor conjugation efficiency. The persistence of the construct inside the cell for as long as required facilitates the homologous recombination events leading to single and double crossovers. While it was confirmed that the vector is frequently lost without selection, the recognition sequence for the I-SceI endonuclease was included in the backbone of pDS0007. The presence of a I-SceI recognition sequence would allow to force the loss of the vector and the appearance of double crossover recombinants by introducing a second construct (e.g. pIJ12742) that expresses a Streptomyces codon–optimised gene encoding the I-SceI endonuclease. To facilitate screening for vector-free clones, the construct also carries a Streptomyces codon–optimised gusA gene encoding the β-glucuronidase expressed from a constitutive promoter. We prove the usefulness of this vector and strategy with the strain S. iranensis DSM 41954, in which we could readily delete an essential gene of a newly discovered biosynthetic pathway for a phosphonate-containing natural product, which led to loss of phosphonate production according to 31 P NMR spectroscopy. Key points • pDS0007 is a new vector for gene-targeting in difficult-to-manipulate streptomycetes. • pDS0007 is self-replicative but easy to cure, targetable and allows visual screening. • pDS0007 was used to prove the discovery of a novel phosphonate biosynthetic pathway. Graphical Abstract

Significant advances in circuit-level analyses of the brain require tools that allow for labeling, modulation of gene expression, and monitoring and manipulation of cellular activity in specific cell types and/or anatomical regions. Large-scale projects and individual laboratories have produced hundreds of gene-specific promoter-driven Cre mouse lines invaluable for enabling genetic access to subpopulations of cells in the brain. However, the potential utility of each line may not be fully realized without systematic whole brain characterization of transgene expression patterns. We established a high-throughput in situ hybridization (ISH), imaging and data processing pipeline to describe whole brain gene expression patterns in Cre driver mice. Currently, anatomical data from over 100 Cre driver lines are publicly available via the Allen Institute's Transgenic Characterization database, which can be used to assist researchers in choosing the appropriate Cre drivers for functional, molecular, or connectional studies of different regions and/or cell types in the brain.