Efficient gene activation in plants by the MoonTag programmable transcriptional activator.

CRISPR/Cas-based transcriptional activators have been developed to induce gene expression in eukaryotic and prokaryotic organisms. The main advantages of CRISPR/Cas-based systems is that they can achieve high levels of transcriptional activation and are very easy to program via pairing between the guide RNA and the DNA target strand. SunTag is a second-generation system that activates transcription by recruiting multiple copies of an activation domain (AD) to its target promoters. SunTag is a strong activator; however, in some species it is difficult to stably express. To overcome this problem, we designed MoonTag, a new activator that works on the same basic principle as SunTag, but whose components are better tolerated when stably expressed in transgenic plants. We demonstrate that MoonTag is capable of inducing high levels of transcription in all plants tested. In Setaria, MoonTag is capable of inducing high levels of transcription of reporter genes as well as of endogenous genes. More important, MoonTag components are expressed in transgenic plants to high levels without any deleterious effects. MoonTag is also able to efficiently activate genes in eudicotyledonous species such as Arabidopsis and tomato. Finally, we show that MoonTag activation is functional across a range of temperatures, which is promising for potential field applications.

Predicting thresholds for population replacement gene drives.

BACKGROUND: Threshold-dependent gene drives (TDGDs) could be used to spread desirable traits through a population, and are likely to be less invasive and easier to control than threshold-independent gene drives. Engineered Genetic Incompatibility (EGI) is an extreme underdominance system previously demonstrated in Drosophila melanogaster that can function as a TDGD when EGI agents of both sexes are released into a wild-type population. RESULTS: Here we use a single generation fitness assay to compare the fecundity, mating preferences, and temperature-dependent relative fitness to wild-type of two distinct genotypes of EGI agents. We find significant differences in the behavior/performance of these EGI agents that would not be predicted a priori based on their genetic design. We report a surprising temperature-dependent change in the predicted threshold for population replacement in an EGI agent that drives ectopic expression of the developmental morphogen pyramus. CONCLUSIONS: The single-generation fitness assay presented here could reduce the amount of time required to estimate the threshold for TDGD strategies for which hybrid genotypes are inviable. Additionally, this work underscores the importance of empirical characterization of multiple engineered lines, as behavioral differences can arise in unique genotypes for unknown reasons.

Using design of experiments to guide genetic optimization of engineered metabolic pathways.

Design of experiments (DoE) is a term used to describe the application of statistical approaches to interrogate the impact of many variables on the performance of a multivariate system. It is commonly used for process optimization in fields such as chemical engineering and material science. Recent advances in the ability to quantitatively control the expression of genes in biological systems open up the possibility to apply DoE for genetic optimization. In this review targeted to genetic and metabolic engineers, we introduce several approaches in DoE at a high level and describe instances wherein these were applied to interrogate or optimize engineered genetic systems. We discuss the challenges of applying DoE and propose strategies to mitigate these challenges. ONE-SENTENCE SUMMARY: This is a review of literature related to applying Design of Experiments for genetic optimization.

Rational search of genetic design space for a heterologous terpene metabolic pathway in Streptomyces.

Modern tools in DNA synthesis and assembly give genetic engineers control over the nucleotide-level design of complex, multi-gene systems. Systematic approaches to explore genetic design space and optimize the performance of genetic constructs are lacking. Here we explore the application of a five-level Plackett-Burman fractional factorial design to improve the titer of a heterologous terpene biosynthetic pathway in Streptomyces. A library of 125 engineered gene clusters encoding the production of diterpenoid ent-atiserenoic acid (eAA) via the methylerythritol phosphate pathway was constructed and introduced into Streptomyces albidoflavus J1047 for heterologous expression. The eAA production titer varied within the library by over two orders of magnitude and host strains showed unexpected and reproducible colony morphology phenotypes. Analysis of Plackett-Burman design identified expression of dxs, the gene encoding the first and the flux-controlling enzyme, having the strongest impact on eAA titer, but with a counter-intuitive negative correlation between dxs expression and eAA production. Finally, simulation modeling was performed to determine how several plausible sources of experimental error/noise and non-linearity impact the utility of Plackett-Burman analyses.

Genetic engineering of sex chromosomes for batch cultivation of non-transgenic, sex-sorted males.

The field performance of Sterile Insect Technique (SIT) is improved by sex-sorting and releasing only sterile males. This can be accomplished by resource-intensive separation of males from females by morphology. Alternatively, sex-ratio biasing genetic constructs can be used to selectively remove one sex without the need for manual or automated sorting, but the resulting genetically engineered (GE) control agents would be subject to additional governmental regulation. Here we describe and demonstrate a genetic method for the batch production of non-GE males. This method could be applied to generate the heterogametic sex (XY, or WZ) in any organism with chromosomal sex determination. We observed up to 100% sex-selection with batch cultures of more than 103 individuals. Using a stringent transgene detection assay, we demonstrate the potential of mass production of transgene free males.

Harnessing intercellular signals to engineer the soil microbiome.

Covering: Focus on 2015 to 2020Plant and soil microbiomes consist of diverse communities of organisms from across kingdoms and can profoundly affect plant growth and health. Natural product-based intercellular signals govern important interactions between microbiome members that ultimately regulate their beneficial or harmful impacts on the plant. Exploiting these evolved signalling circuits to engineer microbiomes towards beneficial interactions with crops is an attractive goal. There are few reports thus far of engineering the intercellular signalling of microbiomes, but this article argues that it represents a tremendous opportunity for advancing the field of microbiome engineering. This could be achieved through the selection of synergistic consortia in combination with genetic engineering of signal pathways to realise an optimised microbiome.

Bioindustrial manufacturing readiness levels (BioMRLs) as a shared framework for measuring and communicating the maturity of bioproduct manufacturing processes.

Readiness level (RL) frameworks such as technology readiness levels and manufacturing readiness levels describe the status of a technology/manufacturing process on its journey from initial conception to commercial deployment. More importantly, they provide a roadmap to guide technology development and scale-up from a ''totality of system'' approach. Commercialization risks associated with too narrowly focused R&D efforts are mitigated. RLs are defined abstractly so that they can apply to diverse industries and technology sectors. However, differences between technology sectors make necessary the definition of sector specific RL frameworks. Here, we describe bioindustrial manufacturing readiness levels (BioMRLs), a classification system specific to bioindustrial manufacturing. BioMRLs will give program managers, investors, scientists, and engineers a shared vocabulary for prioritizing goals and assessing risks in the development and commercialization of a bioindustrial manufacturing process.

Development of a single culture E. coli expression system for the enzymatic synthesis of fluorinated tyrosine and its incorporation into proteins.

Current experiments that rely on biosynthetic metabolic protein labeling with 19F often require fluorinated amino acids, which in the case of 2- and 3-fluorotyrosine can be expensive. However, using these amino acids has provided valuable insight into protein dynamics, structure, and function. Here, we develop a new in-cell method for fluorinated tyrosine generation from readily available substituted phenols and subsequent metabolic labeling of proteins in a single bacterial expression culture. This approach uses a dual-gene plasmid encoding for a model protein BRD4(D1) and a tyrosine phenol lyase from Citrobacter freundii, which catalyzes the formation of tyrosine from phenol, pyruvate, and ammonium. Our system demonstrated both enzymatic fluorotyrosine production and expression of 19F-labeled proteins as analyzed by 19F NMR and LC-MS methods. Further optimization of our system should provide a cost-effective alternative to a variety of traditional protein-labeling strategies.

Genetic manipulation of sex ratio in mammals: the Reaper comes for Mickey.

In most animals, sexual reproduction results in a 1:1 ratio of females to males. For several sectors of agriculture, for example, milk or egg production, only a single sex is needed. Biasing the sex ratio so that only offspring of the desired sex are produced has the potential to increase breeding efficiency. In this issue of EMBO Reports, Yosef et al [1] demonstrate a genetic approach to bias the sex ratio in mice by specifically disrupting essential genes in male embryos. Their approach is an important first step toward generating sex-ratio biasing applications for agriculture.

Complete genome sequences of Streptomyces spp. isolated from disease-suppressive soils.

BACKGROUND: Bacteria within the genus Streptomyces remain a major source of new natural product discovery and as soil inoculants in agriculture where they promote plant growth and protect from disease. Recently, Streptomyces spp. have been implicated as important members of naturally disease-suppressive soils. To shine more light on the ecology and evolution of disease-suppressive microbial communities, we have sequenced the genome of three Streptomyces strains isolated from disease-suppressive soils and compared them to previously sequenced isolates. Strains selected for sequencing had previously showed strong phenotypes in competition or signaling assays. RESULTS: Here we present the de novo sequencing of three strains of the genus Streptomyces isolated from disease-suppressive soils to produce high-quality complete genomes. Streptomyces sp. GS93-23, Streptomyces sp. 3211-3, and Streptomyces sp. S3-4 were found to have linear chromosomes of 8.24 Mb, 8.23 Mb, and greater than 7.5 Mb, respectively. In addition, two of the strains were found to have large, linear plasmids. Each strain harbors between 26 and 38 natural product biosynthetic gene clusters, on par with previously sequenced Streptomyces spp. We compared these newly sequenced genomes with those of previously sequenced organisms. We see substantial natural product biosynthetic diversity between closely related strains, with the gain/loss of episomal DNA elements being a primary driver of genome evolution. CONCLUSIONS: Long read sequencing data facilitates large contig assembly for high-GC Streptomyces genomes. While the sample number is too small for a definitive conclusion, we do not see evidence that disease suppressive soil isolates are particularly privileged in terms of numbers of biosynthetic gene clusters. The strong sequence similarity between GS93-23 and previously isolated Streptomyces lydicus suggests that species recruitment may contribute to the evolution of disease-suppressive microbial communities.

Leveraging ecological theory to guide natural product discovery.

Technological improvements have accelerated natural product (NP) discovery and engineering to the point that systematic genome mining for new molecules is on the horizon. NP biosynthetic potential is not equally distributed across organisms, environments, or microbial life histories, but instead is enriched in a number of prolific clades. Also, NPs are not equally abundant in nature; some are quite common and others markedly rare. Armed with this knowledge, random 'fishing expeditions' for new NPs are increasingly harder to justify. Understanding the ecological and evolutionary pressures that drive the non-uniform distribution of NP biosynthesis provides a rational framework for the targeted isolation of strains enriched in new NP potential. Additionally, ecological theory leads to testable hypotheses regarding the roles of NPs in shaping ecosystems. Here we review several recent strain prioritization practices and discuss the ecological and evolutionary underpinnings for each. Finally, we offer perspectives on leveraging microbial ecology and evolutionary biology for future NP discovery.

Dedicated ent-kaurene and ent-atiserene synthases for platensimycin and platencin biosynthesis.

Platensimycin (PTM) and platencin (PTN) are potent and selective inhibitors of bacterial and mammalian fatty acid synthases and have emerged as promising drug leads for both antibacterial and antidiabetic therapies. Comparative analysis of the PTM and PTN biosynthetic machineries in Streptomyces platensis MA7327 and MA7339 revealed that the divergence of PTM and PTN biosynthesis is controlled by dedicated ent-kaurene and ent-atiserene synthases, the latter of which represents a new pathway for diterpenoid biosynthesis. The PTM and PTN biosynthetic machineries provide a rare glimpse at how secondary metabolic pathway evolution increases natural product structural diversity and support the wisdom of applying combinatorial biosynthesis methods for the generation of novel PTM and/or PTN analogues, thereby facilitating drug development efforts based on these privileged natural product scaffolds.

High-throughput investigation of genetic design constraints in domesticated Influenza A Virus for transient gene delivery.

Replication-incompetent single cycle infectious Influenza A Virus (sciIAV) has demonstrated utility as a research and vaccination platform. Protein-based therapeutics are increasingly attractive due to their high selectivity and potent efficacy but still suffer from low bioavailability and high manufacturing cost. Transient RNA-mediated delivery is a safe alternative that allows for expression of protein-based therapeutics within the target cells or tissues but is limited by delivery efficiency. Here, we develop recombinant sciIAV as a platform for transient gene delivery in vivo and in vitro for therapeutic, research, and manufacturing applications (in vivo antimicrobial production, cell culture contamination clearance, and production of antiviral proteins in vitro). While adapting the system to deliver new protein cargo we discovered expression differences presumably resulting from genetic context effects. We applied a high-throughput screen to map these within the 3'-untranslated and coding regions of the hemagglutinin-encoding segment 4. This screen revealed permissible mutations in the 3'-UTR and depletion of RNA level motifs in the N-terminal coding region.

The Natural Products Discovery Center: Release of the First 8490 Sequenced Strains for Exploring Actinobacteria Biosynthetic Diversity.

Actinobacteria, the bacterial phylum most renowned for natural product discovery, has been established as a valuable source for drug discovery and biotechnology but is underrepresented within accessible genome and strain collections. Herein, we introduce the Natural Products Discovery Center (NPDC), featuring 122,449 strains assembled over eight decades, the genomes of the first 8490 NPDC strains (7142 Actinobacteria), and the online NPDC Portal making both strains and genomes publicly available. A comparative survey of RefSeq and NPDC Actinobacteria highlights the taxonomic and biosynthetic diversity within the NPDC collection, including three new genera, hundreds of new species, and ~7000 new gene cluster families. Selected examples demonstrate how the NPDC Portal's strain metadata, genomes, and biosynthetic gene clusters can be leveraged using genome mining approaches. Our findings underscore the ongoing significance of Actinobacteria in natural product discovery, and the NPDC serves as an unparalleled resource for both Actinobacteria strains and genomes.

Efficient gene activation in plants by the MoonTag programmable transcriptional activator.

CRISPR/Cas-based transcriptional activators have been developed to induce gene expression in eukaryotic and prokaryotic organisms. The main advantages of CRISPR-Cas based systems is that they can achieve high levels of transcriptional activation and are very easy to program via pairing between the guide RNA and the DNA target strand. SunTag is a second-generation system that activates transcription by recruiting multiple copies of an activation domain (AD) to its target promoters. SunTag is a strong activator; however, in some species it is difficult to stably express. To overcome this problem, we designed MoonTag, a new activator that worked on the same basic principle as SunTag, but whose components are better tolerated when stably expressed in transgenic plants. We demonstrate that MoonTag is capable of inducing high levels of transcription in all plants tested. In Setaria, MoonTag is capable of inducing high levels of transcription of reporter genes as well as of endogenous genes. More important, MoonTag components are expressed in transgenic plants to high levels without any deleterious effects. MoonTag is also able to efficiently activate genes in eudicotyledonous species such as Arabidopsis and tomato. Finally, we show that MoonTag activation is functional across a range of temperatures, which is promising for potential field applications.

Corrigendum: Modeling-informed Engineered Genetic Incompatibility strategies to overcome resistance in the invasive Drosophila suzukii.

[This corrects the article DOI: 10.3389/finsc.2022.1063789.].

Expression of the platencin biosynthetic gene cluster in heterologous hosts yielding new platencin congeners.

Platensimycin (PTM) and platencin (PTN) are potent and selective inhibitors of bacterial and mammalian fatty acid synthases and have emerged as promising drug leads for both antibacterial and antidiabetic therapies. We have previously cloned and sequenced the PTM-PTN dual biosynthetic gene cluster from Streptomyces platensis MA7327 and the PTN biosynthetic gene cluster from S. platensis MA7339, the latter of which is composed of 31 genes encoding PTN biosynthesis, regulation, and resistance. We have also demonstrated that PTM or PTN production can be significantly improved upon inactivation of the pathway-specific regulator ptmR1 or ptnR1 in S. platensis MA7327 or MA7339, respectively. We now report engineered production of PTN and congeners in a heterologous Streptomyces host. Expression constructs containing the ptn biosynthetic gene cluster were engineered from SuperCos 1 library clones and introduced into five model Streptomyces hosts, and PTN production was achieved in Streptomyces lividans K4-114. Inactivation of ptnR1 was crucial for expression of the ptn biosynthetic gene cluster, thereby PTN production, in S. lividans K4-114. Six PTN congeners, five of which were new, were also isolated from the recombinant strain S. lividans SB12606, revealing new insights into PTN biosynthesis. Production of PTN in a model Streptomyces host provides new opportunities to apply combinatorial biosynthetic strategies to the PTN biosynthetic machinery for structural diversity.

Modeling-informed Engineered Genetic Incompatibility strategies to overcome resistance in the invasive Drosophila suzukii.

Engineered Genetic Incompatibility (EGI) is an engineered extreme underdominance genetic system wherein hybrid animals are not viable, functioning as a synthetic speciation event. There are several strategies in which EGI could be leveraged for genetic biocontrol of pest populations. We used an agent-based model of Drosophila suzukii (Spotted Wing Drosophila) to determine how EGI would fare with high rates of endemic genetic resistance alleles. We discovered a surprising failure mode wherein field-generated females convert an incompatible male release program into a population replacement gene drive. Local suppression could still be attained in two seasons by tailoring the release strategy to take advantage of this effect, or alternatively in one season by altering the genetic design of release agents. We show in this work that data from modeling can be utilized to recognize unexpected emergent phenomena and a priori inform genetic biocontrol treatment design to increase efficacy.

HUGE pipeline to measure temporal genetic variation in Drosophila suzukii populations for genetic biocontrol applications.

Understanding the fine-scale genome sequence diversity that exists within natural populations is important for developing models of species migration, temporal stability, and range expansion. For invasive species, agricultural pests, and disease vectors, sequence diversity at specific loci in the genome can impact the efficacy of next-generation genetic biocontrol strategies. Here we describe a pipeline for haplotype-resolution genetic variant discovery and quantification from thousands of Spotted Wing Drosophila (Drosophila suzukii, SWD) isolated at two field sites in the North-Central United States (Minnesota) across two seasons. We observed highly similar single nucleotide polymorphism (SNP) frequencies at each genomic location at each field site and year. This supports the hypotheses that SWD overwinters in Minnesota, is annually populated by the same source populations or a combination of both theories. Also, the stable genetic structure of SWD populations allows for the rational design of genetic biocontrol technologies for population suppression.