Development Of Genetic Tools Research Articles

Development of genetic tools for the redbait species Pyura herdmani and P. stolonifera, important bioengineers along African coastlines

The development of new genetic resources is key for biological conservation in an era when the preservation of biodiversity is crucial. Here, we report the development of microsatellites and mitochondrial markers for the redbait species Pyura herdmani and P. stolonifera, both important bioengineers along African coastlines. Specifically, we identified microsatellites by means of pyrosequencing and variable regions in the mitochondrial genome using RAD-seq libraries that were generated with Illumina sequencing. A total of 4 339 putative microsatellites were found, of which 32 were tested on individuals collected along the southeast coast of South Africa. Of those, five microsatellites cross-amplified consistently in both species, and these were tested on a total of 260 samples. Additionally, we mapped the partial mitochondrial genome of a small number of individuals from both species and identified the most-variable regions by comparing a small number of individuals from all regions where these species have been recorded (including western South Africa and northwest Africa). The genetic markers developed here can be used to conduct population genetic studies, to monitor range shifts under contemporary climate change, and to help in preserving ecosystem functioning along African coastlines.

Bioconversion of methane by metabolically engineered methanotrophs

Due to abundant availability of shale gas and biogas, methane has been considered as one of the most potential carbon sources for industrial biotechnology. Methanotrophs carrying the native methane monooxygenase are capable of using methane as a sole energy and carbon source, which provides a novel strategy for reducing greenhouse gas emission and substituting edible substrates used in bioconversion processes. With the rapid development of genetic engineering tools and biosynthesis techniques, various strategies for improving the efficiency of methane bioconversion have been achieved to produce a variety of commodity bio-based products. Herein, we summarize several important aspects related with methane utilization and metabolic engineering of methanotrophs, including the modification of methane oxidation pathways, the construction of efficient cell factories, and biosynthesis of chemicals and fuels. Finally, the prospects and challenges of the future development of methane bioconversion are also discussed.

Volatiles Influencing Sensory Attributes and Bayesian Modeling of the Soluble Solids-Sweetness Relationship in Strawberry.

Descriptive analysis via trained sensory panels has great power to facilitate flavor improvement in fresh fruits and vegetables. When paired with an understanding of fruit volatile organic compounds, descriptive analysis can help uncover the chemical drivers of sensory attributes. In the present study, 213 strawberry samples representing 56 cultivars and advanced selections were sampled over seven seasons and subjected to both sensory descriptive and chemical analyses. Principal component analysis and K-cluster analyses of sensory data highlighted three groups of strawberry samples, with one classified as superior with high sweetness and strawberry flavor and low sourness and green flavor. Partial least square models revealed 20 sweetness-enhancing volatile organic compounds and two sweetness-reducing volatiles, many of which overlap with previous consumer sensory studies. Volatiles modulating green, sour, astringent, overripe, woody, and strawberry flavors were also identified. The relationship between soluble solids content (SSC) and sweetness was modeled with Bayesian regression, generating probabilities for sweetness levels from varying levels of soluble solids. A hierarchical Bayesian model with month effects indicated that SSC is most correlated to sweetness toward the end of the fruiting season, making this the best period to make phenotypic selections for soluble solids. Comparing effects from genotypes, harvest months, and their interactions on sensory attributes revealed that sweetness, sourness, and firmness were largely controlled by genetics. These findings help formulate a paradigm for improvement of eating quality in which sensory analyses drive the targeting of chemicals important to consumer-desired attributes, which further drive the development of genetic tools for improvement of flavor.

The genetic architecture of target-site resistance to pyrethroid insecticides in the African malaria vectors Anopheles gambiae and Anopheles coluzzii.

Resistance to pyrethroid insecticides is a major concern for malaria vector control. Pyrethroids target the voltage‐gated sodium channel (VGSC), an essential component of the mosquito nervous system. Substitutions in the amino acid sequence can induce a resistance phenotype. We use whole‐genome sequence data from phase 2 of the Anopheles gambiae 1000 Genomes Project (Ag1000G) to provide a comprehensive account of genetic variation in the Vgsc gene across 13 African countries. In addition to known resistance alleles, we describe 20 other non‐synonymous nucleotide substitutions at appreciable population frequency and map these variants onto a protein model to investigate the likelihood of pyrethroid resistance phenotypes. Thirteen of these novel alleles were found to occur almost exclusively on haplotypes carrying the known L995F kdr (knock‐down resistance) allele and may enhance or compensate for the L995F resistance genotype. A novel mutation I1527T, adjacent to a predicted pyrethroid‐binding site, was found in tight linkage with V402L substitutions, similar to allele combinations associated with resistance in other insect species. We also analysed genetic backgrounds carrying resistance alleles, to determine which alleles have experienced recent positive selection, and describe ten distinct haplotype groups carrying known kdr alleles. Five of these groups are observed in more than one country, in one case separated by over 3000 km, providing new information about the potential for the geographical spread of resistance. Our results demonstrate that the molecular basis of target‐site pyrethroid resistance in malaria vectors is more complex than previously appreciated, and provide a foundation for the development of new genetic tools for insecticide resistance management.

Genetic Manipulation of Non-tuberculosis Mycobacteria.

Non-tuberculosis mycobacteria (NTMs) comprise a large group of organisms that are phenotypically diverse. Analysis of the growing number of completed NTM genomes has revealed both significant intra-genus genetic diversity, and a high percentage of predicted genes that appear to be unique to this group. Most NTMs have not been studied, however, the rise in NTM infections in several countries has prompted increasing interest in these organisms. Mycobacterial research has recently benefitted from the development of new genetic tools and a growing number of studies describing the genetic manipulation of NTMs have now been reported. In this review, we discuss the use of both site-specific and random mutagenesis tools in NTMs, highlighting the challenges that exist in applying these techniques to this diverse group of organisms.

Toward genetic modification of plant-parasitic nematodes: delivery of macromolecules to adults and expression of exogenous mRNA in second stage juveniles.

Plant-parasitic nematodes are a continuing threat to food security, causing an estimated 100 billion USD in crop losses each year. The most problematic are the obligate sedentary endoparasites (primarily root knot nematodes and cyst nematodes). Progress in understanding their biology is held back by a lack of tools for functional genetics: forward genetics is largely restricted to studies of natural variation in populations and reverse genetics is entirely reliant on RNA interference. There is an expectation that the development of functional genetic tools would accelerate the progress of research on plant-parasitic nematodes, and hence the development of novel control solutions. Here, we develop some of the foundational biology required to deliver a functional genetic tool kit in plant-parasitic nematodes. We characterize the gonads of male Heterodera schachtii and Meloidogyne hapla in the context of spermatogenesis. We test and optimize various methods for the delivery, expression, and/or detection of exogenous nucleic acids in plant-parasitic nematodes. We demonstrate that delivery of macromolecules to cyst and root knot nematode male germlines is difficult, but possible. Similarly, we demonstrate the delivery of oligonucleotides to root knot nematode gametes. Finally, we develop a transient expression system in plant-parasitic nematodes by demonstrating the delivery and expression of exogenous mRNA encoding various reporter genes throughout the body of H. schachtii juveniles using lipofectamine-based transfection. We anticipate these developments to be independently useful, will expedite the development of genetic modification tools for plant-parasitic nematodes, and ultimately catalyze research on a group of nematodes that threaten global food security.

Brachionus rotifers as a model for investigating dietary and metabolic regulators of aging.

Because every species has unique attributes relevant to understanding specific aspects of aging, using a diversity of study systems and a comparative biology approach for aging research has the potential to lead to novel discoveries applicable to human health. Monogonont rotifers, a standard model for studies of aquatic ecology, evolutionary biology, and ecotoxicology, have also been used to study lifespan and healthspan for nearly a century. However, because much of this work has been published in the ecology and evolutionary biology literature, it may not be known to the biomedical research community. In this review, we provide an overview of Brachionus rotifers as a model to investigate nutritional and metabolic regulators of aging, with a focus on recent studies of dietary and metabolic pathway manipulation. Rotifers are microscopic, aquatic invertebrates with many advantages as a system for studying aging, including a two-week lifespan, easy laboratory culture, direct development without a larval stage, sexual and asexual reproduction, easy delivery of pharmaceuticals in liquid culture, and transparency allowing imaging of cellular morphology and processes. Rotifers have greater gene homology with humans than do established invertebrate models for aging, and thus rotifers may be used to investigate novel genetic mechanisms relevant to human lifespan and healthspan. The research on caloric restriction; dietary, pharmaceutical, and genetic interventions; and transcriptomics of aging using rotifers provide insights into the metabolic regulators of lifespan and health and suggest future directions for aging research. Capitalizing on the unique biology of Brachionus rotifers, referencing the vast existing literature about the influence of diet and drugs on rotifer lifespan and health, continuing the development of genetic tools for rotifers, and growing the rotifer research community will lead to new discoveries a better understanding of the biology of aging.

Multi-Level Gene Expression in Response to Environmental Stress in Aquatic Invertebrate Chironomids: Potential Applications in Water Quality Monitoring.

In freshwater ecosystems, aquatic invertebrates are influenced continuously by both physical stress and xenobiotics. Chironomids (Diptera; Chironomidae), or non-biting midges, are the most diverse and abundant invertebrates in freshwater habitats. They are a fundamental link in food chains of aquatic ecosystems. Chironomid larvae tolerate stress factors in their environments via various physiological processes. At the molecular level, environmental pollutants induce multi-level gene responses in Chironomus that regulate cellular protection through the activation of defense processes. This paper reviews literature on the transcriptional responses of biomarker genes to environmental stress in chironomids at the molecular level, in studies conducted from 1991 to 2020 (120 selected literatures of 374 results with the keywords "Chironomus and gene expression" by PubMed search tool). According to these studies, transcriptional responses in chironomids vary depending on the type of stress factor and defensive responses associated with antioxidant activity, the endocrine system, detoxification, homeostasis and stress response, energy metabolism, ribosomal machinery, apoptosis, DNA repair, and epigenetics. These data could provide a comprehensive overview of how Chironomus species respond to pollutants in aquatic environments. Furthermore, the transcriptomic data could facilitate the development of genetic tools for water quality and environmental monitoring based on resident chironomid species.

Victoria L. Sork—Recipient of the 2020 Molecular Ecology Prize

Victoria L. Sork—Recipient of the 2020 Molecular Ecology Prize

Brown Algal Model Organisms.

Model organisms are extensively used in research as accessible and convenient systems for studying a particular area or question in biology. Traditionally, only a limited number of organisms have been studied in detail, but modern genomic tools are enabling researchers to extend beyond the set of classical model organisms to include novel species from less-studied phylogenetic groups. This review focuses on model species for an important group of multicellular organisms, the brown algae. The development of genetic and genomic tools for the filamentous brown alga Ectocarpus has led to it emerging as a general model system for this group, but additional models, such as Fucus or Dictyota dichotoma, remain of interest for specific biological questions. In addition, Saccharina japonica has emerged as a model system to directly address applied questions related to algal aquaculture. We discuss the past, present, and future of brown algal model organisms in relation to the opportunities and challenges in brown algal research.

Single nucleotide polymorphism associated with disease resistance in Penaeus vannamei

Despite the considerable number of genetic markers published for Penaeus vannamei, the classification of these markers and their standardization in specific databases is still insufficient. As a consequence, access to these markers is difficult, hampering their application in genetic association studies. In this study, all previously described single nucleotide polymorphisms (SNPs) related to resistance for P. vannamei were revised, and 512 SNPs were identified and classified in detail. We observed that most of the SNPs occurred in the proteins including Toll like receptors 1 and 3, hemocyanin large and small subunits, and anti-lipopolysaccharide factors 1 and 2, allowing to propose to use them as targets in association studies involving resistance in P. vannamei. Additionally, the potential effects of the most frequent non-synonymous coding SNPs in the secondary structure of the main target proteins were evaluated using an in silico approach. These data can serve as the starting point for the development of new genetic and computational tools as well as for the design of new association studies that involve resistance in P. vannamei.

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica.

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to their successful evolutionary radiation on the earth. The application of genetic tools such as genome editing is the key to understanding genetic changes that are responsible for evolutionary transitions in an Evo-Devo approach. In this article, we describe our current protocol for generating and maintaining mutant strains of T. domestica. We report a dry injection method, as an alternative to the reported wet injection method, that allows us to obtain stably high survival rates in injected embryos. We also report an optimized environment setting to mate adults and obtain subsequent generations with high efficiency. Our method underlines the importance of taking each species' unique biology into account for the successful application of genome editing methods to non-traditional model organisms. We predict that these genome editing protocols will help in implementing T. domestica as a laboratory model and to further accelerate the development and application of useful genetic tools in this species.

Identification of antibiotics for use in selection of the chytrid fungi Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans.

Global amphibian populations are being decimated by chytridiomycosis, a deadly skin infection caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). Although ongoing efforts are attempting to limit the spread of these infections, targeted treatments are necessary to manage the disease. Currently, no tools for genetic manipulation are available to identify and test specific drug targets in these fungi. To facilitate the development of genetic tools in Bd and Bsal, we have tested five commonly used antibiotics with available resistance genes: Hygromycin, Blasticidin, Puromycin, Zeocin, and Neomycin. We have identified effective concentrations of each for selection in both liquid culture and on solid media. These concentrations are within the range of concentrations used for selecting genetically modified cells from a variety of other eukaryotic species.

73 - Characterization and Flaws of a New Potential β-Cell-Specific Mouse Line

Development and thorough characterization of genetic tools is necessary for efficient and productive understanding of the beta cell. Over the past few years, multiple mouse lines expressing Cre recombinase in beta cells have been developed, but all have significant caveats towards their use. Our laboratory has invested significant effort into characterizing new and existing lines to provide a model for the beta cell community. Our goal is to provide a mouse that 1) has efficient, inducible Cre expression; 2) only in beta cells and 3) limits the need for drug administration (i.e. tamoxifen and doxycycline) during critical periods of experimentation. In this study, we characterized an inducible Tet-Off system using a combination of a bacterial artificial chromosome (BAC) transgenic Ins1-itTA with the TetO-CreJaw/J line. Unfortunately, in our characterization (including assessment of: gene recombination in multiple tissues; beta cell architecture/mass; in vitro and in vivo glucose-stimulated insulin secretion (GSIS) and whole body glucose homeostasis), we discovered a significant defect in pancreatic and islet insulin content and insulin secretion in mice expressing the Ins1-itTA transgene. Moreover, we found that the rtTA or the tTA proteins themselves decreased expression of the Insulin 1, Insulin 2 and Glut2 genes in INS-1 cells, corresponding to a significant decrease in insulin content. Overall, these results considerably decreased our enthusiasm toward using the Tet-On or Tet-Off systems for future projects in beta cell, but it also emphasizes the importance of complete characterization of new mouse lines and the use of all controls for in vivo studies.

Synthetic biology, systems biology, and metabolic engineering of Yarrowia lipolytica toward a sustainable biorefinery platform.

Yarrowia lipolytica is an oleaginous yeast that has been substantially engineered for production of oleochemicals and drop-in transportation fuels. The unique acetyl-CoA/malonyl-CoA supply mode along with the versatile carbon-utilization pathways makes this yeast a superior host to upgrade low-value carbons into high-value secondary metabolites and fatty acid-based chemicals. The expanded synthetic biology toolkits enabled us to explore a large portfolio of specialized metabolism beyond fatty acids and lipid-based chemicals. In this review, we will summarize the recent advances in genetic, omics, and computational tool development that enables us to streamline the genetic or genomic modification for Y. lipolytica. We will also summarize various metabolic engineering strategies to harness the endogenous acetyl-CoA/malonyl-CoA/HMG-CoA pathway for production of complex oleochemicals, polyols, terpenes, polyketides, and commodity chemicals. We envision that Y. lipolytica will be an excellent microbial chassis to expand nature's biosynthetic capacity to produce plant secondary metabolites, industrially relevant oleochemicals, agrochemicals, commodity, and specialty chemicals and empower us to build a sustainable biorefinery platform that contributes to the prosperity of a bio-based economy in the future.

Distribution and biogeography of Sanguina snow algae: Fine-scale sequence analyses reveal previously unknown population structure.

It has been previously suggested that snow algal species within the genus Sanguina (S. nivaloides and S. aurantia) show no population structure despite being found globally (S. nivaloides) or throughout the Northern Hemisphere (S. aurantia). However, systematic biogeographic research into global distributions is lacking due to few genetic and no genomic resources for these snow algae. Here, using all publicly available and previously unpublished Sanguina sequences of the Internal Transcribed Spacer 2 region, we investigated whether this purported lack of population structure within Sanguina species is supported by additional evidence. Using a minimum entropy decomposition (MED) approach to examine fine‐scale genetic population structure, we find that these snow algae populations are largely distinct regionally and have some interesting biogeographic structuring. This is in opposition to the currently accepted idea that Sanguina species lack any observable population structure across their vast ranges and highlights the utility of fine‐scale (sub‐OTU) analytical tools to delineate geographic and genetic population structure. This work extends the known range of S. aurantia and emphasizes the need for development of genetic and genomic tools for additional studies on snow algae biogeography.

Modulating microbiome metabolites in vivo

A new genetic tool enables mechanistic dissection of microbiome–host interactions

High-efficiency transformation of archaea by direct PCR products with its application to directed evolution of a thermostable enzyme.

SummaryHyperthermophilic archaea with unique biochemical and physiological characteristics are important organisms for fundamental research of life science and have great potential for biotechnological applications. However, low transformation efficiency of foreign DNA molecules impedes developments in genetic modification tools and industrial applications. In this study, we applied prolonged overlap extension PCR (POE‐PCR) to generate multimeric DNA molecules and then transformed them into two hyperthermophilic archaea, Thermococcus kodakarensis KOD1 and Pyrococcus yayanosii A1. This study was the first example to demonstrate the enhanced transformation efficiencies of POE‐PCR products by a factor of approximately 100 for T. kodakarensis KOD1 and 8 for P. yayanosii A1, respectively, relative to circular shuttle plasmids. Furthermore, directed evolution of a modestly thermophilic enzyme, Methanothermococcus okinawensis 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGR), was conducted to obtain more stable ones due to high transformation efficiency of T. kodakarensis (i.e. ~3 × 104 CFU per μg DNA). T. kodakarensis harbouring the most thermostable MoHMGR mutant can grow in the presence of a thermostable antibiotic simvastatin at 85°C and even higher temperatures. This high transformation efficiency technique could not only help develop more hyperthermophilic enzyme mutants via directed evolution but also simplify genetical modification of archaea, which could be novel hosts for industrial biotechnology.

Lipid metabolism of the oleaginous yeast Lipomyces starkeyi.

The oleaginous yeast Lipomyces starkeyi is an excellent sustainable lipid producer, which can convert industrial wastes into lipids and accumulate triacylglycerols (TAG) by > 70% of its dry cell weight. Recent studies using omics technologies applied in L. starkeyi have aided in obtaining greater understanding of the important mechanisms of lipid metabolism in L. starkeyi. Therefore, the development of genetic engineering tools for L. starkeyi has led to accelerated efforts for a highly efficient production of lipids.This review focuses on the aspects of TAG and fatty acid synthesis pathways in L. starkeyi. We also present a quite effective strategy to obtain L. starkeyi mutants accumulating a larger amount of lipids and having a higher lipid production rate than the wild-type strain. The analysis of these mutants exhibiting high lipid production has led to the identification of important genes for achieving highly effective lipid production and thus advanced improvement in lipid production. Herein, our aim was to provide useful information to advance the development of L. starkeyi as a cost-effective TAG feedstock.Key Points•Oleaginous yeast Lipomyces starkeyi is an excellent sustainable lipid producer.•Efficient isolation of lipid-enriched L. starkeyi mutants depends on the low density of lipids.•Increased acyl-CoA synthesis pathway is important for improving lipid productivity.

Recent advances in domesticating non-model microorganisms.

Non-model microorganisms have been increasingly explored as microbial cell factories for production of chemicals, fuels, and materials owing to their unique physiology and metabolic capabilities. However, these microorganisms often lack facile genetic tools for strain development, which hinders their adoption as production hosts. In this review, we describe recent advances in domestication of non-model microorganisms, including bacteria, actinobacteria, cyanobacteria, yeast, and fungi, with a focus on the development of genetic tools. In addition, we highlight some successful applications of non-model microorganisms as microbial cell factories.