Long oligodeoxynucleotides: chemical synthesis, isolation via catching-by-polymerization, verification via sequencing, and gene expression demonstration.

Long oligodeoxynucleotides (ODNs) are segments of DNAs having over one hundred nucleotides (nt). They are typically assembled using enzymatic methods such as PCR and ligation from shorter 20 to 60 nt ODNs produced by automated de novo chemical synthesis. While these methods have made many projects in areas such as synthetic biology and protein engineering possible, they have various drawbacks. For example, they cannot produce genes and genomes with long repeats and have difficulty to produce sequences containing stable secondary structures. Here, we report a direct de novo chemical synthesis of 400 nt ODNs, and their isolation from the complex reaction mixture using the catching-by-polymerization (CBP) method. To determine the authenticity of the ODNs, 399 and 401 nt ODNs were synthesized and purified with CBP. The two were joined together using Gibson assembly to give the 800 nt green fluorescent protein (GFP) gene construct. The sequence of the construct was verified via Sanger sequencing. To demonstrate the potential use of the long ODN synthesis method, the GFP gene was expressed in E. coli. The long ODN synthesis and isolation method presented here provides a pathway to the production of genes and genomes containing long repeats or stable secondary structures that cannot be produced or are highly challenging to produce using existing technologies.

Proteotranscriptomics assisted gene annotation and spatial proteomics of Bombyx mori BmN4 cell line.

BACKGROUND: The process of identifying all coding regions in a genome is crucial for any study at the level of molecular biology, ranging from single-gene cloning to genome-wide measurements using RNA-seq or mass spectrometry. While satisfactory annotation has been made feasible for well-studied model organisms through great efforts of big consortia, for most systems this kind of data is either absent or not adequately precise. RESULTS: Combining in-depth transcriptome sequencing and high resolution mass spectrometry, we here use proteotranscriptomics to improve gene annotation of protein-coding genes in the Bombyx mori cell line BmN4 which is an increasingly used tool for the analysis of piRNA biogenesis and function. Using this approach we provide the exact coding sequence and evidence for more than 6200 genes on the protein level. Furthermore using spatial proteomics, we establish the subcellular localization of thousands of these proteins. We show that our approach outperforms current Bombyx mori annotation attempts in terms of accuracy and coverage. CONCLUSIONS: We show that proteotranscriptomics is an efficient, cost-effective and accurate approach to improve previous annotations or generate new gene models. As this technique is based on de-novo transcriptome assembly, it provides the possibility to study any species also in the absence of genome sequence information for which proteogenomics would be impossible.

A global survey of arsenic-related genes in soil microbiomes.

BACKGROUND: Environmental resistomes include transferable microbial genes. One important resistome component is resistance to arsenic, a ubiquitous and toxic metalloid that can have negative and chronic consequences for human and animal health. The distribution of arsenic resistance and metabolism genes in the environment is not well understood. However, microbial communities and their resistomes mediate key transformations of arsenic that are expected to impact both biogeochemistry and local toxicity. RESULTS: We examined the phylogenetic diversity, genomic location (chromosome or plasmid), and biogeography of arsenic resistance and metabolism genes in 922 soil genomes and 38 metagenomes. To do so, we developed a bioinformatic toolkit that includes BLAST databases, hidden Markov models and resources for gene-targeted assembly of nine arsenic resistance and metabolism genes: acr3, aioA, arsB, arsC (grx), arsC (trx), arsD, arsM, arrA, and arxA. Though arsenic-related genes were common, they were not universally detected, contradicting the common conjecture that all organisms have them. From major clades of arsenic-related genes, we inferred their potential for horizontal and vertical transfer. Different types and proportions of genes were detected across soils, suggesting microbial community composition will, in part, determine local arsenic toxicity and biogeochemistry. While arsenic-related genes were globally distributed, particular sequence variants were highly endemic (e.g., acr3), suggesting dispersal limitation. The gene encoding arsenic methylase arsM was unexpectedly abundant in soil metagenomes (median 48%), suggesting that it plays a prominent role in global arsenic biogeochemistry. CONCLUSIONS: Our analysis advances understanding of arsenic resistance, metabolism, and biogeochemistry, and our approach provides a roadmap for the ecological investigation of environmental resistomes.

SfiNX: a method for assembly of protein coding sequences with high success rates.

OBJECTIVE: Concatenation of two NdeI-XhoI gene fragments via an oligonucleotide linker on a plasmid vector with an SfiI site was performed to evaluate success rates in construction of polycistronic genes expressible in Escherichia coli. RESULTS: A series of plasmids with an SfiI site between the selection marker and the replication origin were constructed. The three wheat eEF1B subunit genes inserted between the NdeI and XhoI sites of pET-22b were transferred to the SfiI-containing plasmid with a spectinomycin-resistance gene. Then, the marker gene in the resultant plasmids was substituted with the ampicillin-resistance gene. These plasmids were used for concatenation of two different genes via a linker oligonucleotide containing a ribosome-binding site. During these operations, 42 clones were picked up out of which 41 had the intended product plasmid. CONCLUSION: This method, named as the SfiNX method, is useful for trial-and-error based testing of different combinations of fusion and co-expression partners for optimization of recombinant protein production.

Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering.

In vitro reconstitution of an artificial metabolic pathway is an emerging approach for the biocatalytic production of industrial chemicals. However, several enzymes have to be separately prepared (and purified) for the construction of an in vitro metabolic pathway, thereby limiting the practical applicability of this approach. In this study, genes encoding the nine thermophilic enzymes involved in a non-ATP-forming chimeric glycolytic pathway were assembled in an artificial operon and co-expressed in a single recombinant Escherichia coli strain. Gene expression levels of the thermophilic enzymes were controlled by their sequential order in the artificial operon. The specific activities of the recombinant enzymes in the cell-free extract of the multiple-gene-expression E. coli were 5.0-1,370 times higher than those in an enzyme cocktail prepared from a mixture of single-gene-expression strains, in each of which a single one of the nine thermophilic enzymes was overproduced. Heat treatment of a crude extract of the multiple-gene-expression cells led to the denaturation of indigenous proteins and one-step preparation of an in vitro synthetic pathway comprising only a limited number of thermotolerant enzymes. Coupling this in vitro pathway with other thermophilic enzymes including the H2 O-forming NADH oxidase or the malate/lactate dehydrogenase facilitated one-pot conversion of glucose to pyruvate or lactate, respectively.

Transcriptome annotation and marker discovery in white bass (Morone chrysops) and striped bass (Morone saxatilis).

Striped bass (Morone saxatilis) and white bass (Morone chrysops) are the parental species of the hybrid striped bass, a major U.S. aquaculture species. Currently, genomic resources for striped bass, white bass, and their hybrid lag behind those of other aquaculture species. Current resources consist of a medium-density genetic linkage map and a well-annotated ovarian transcriptome. A well-annotated transcriptome from across striped bass and white bass tissues is needed to advance both broad-based RNA-seq studies of gene expression as well as aid in more targeted studies of important genes and pathways critical for reproductive physiology and immunity. Here, we carried out Illumina-based transcriptome sequencing and annotation in both species utilizing the trinity and trinotate packages. The assembled Moronid reference transcriptomes and identified SSRs and SNPs should advance ongoing studies of reproduction, physiology, and immunology in these species and provide markers for broodstock management and selection.

When Livestock Genomes Meet Third-Generation Sequencing Technology: From Opportunities to Applications.

Third-generation sequencing technology has found widespread application in the genomic, transcriptomic, and epigenetic research of both human and livestock genetics. This technology offers significant advantages in the sequencing of complex genomic regions, the identification of intricate structural variations, and the production of high-quality genomes. Its attributes, including long sequencing reads, obviation of PCR amplification, and direct determination of DNA/RNA, contribute to its efficacy. This review presents a comprehensive overview of third-generation sequencing technologies, exemplified by single-molecule real-time sequencing (SMRT) and Oxford Nanopore Technology (ONT). Emphasizing the research advancements in livestock genomics, the review delves into genome assembly, structural variation detection, transcriptome sequencing, and epigenetic investigations enabled by third-generation sequencing. A comprehensive analysis is conducted on the application and potential challenges of third-generation sequencing technology for genome detection in livestock. Beyond providing valuable insights into genome structure analysis and the identification of rare genes in livestock, the review ventures into an exploration of the genetic mechanisms underpinning exemplary traits. This review not only contributes to our understanding of the genomic landscape in livestock but also provides fresh perspectives for the advancement of research in this domain.

Modern Approaches to de novo Synthesis of Extended DNA Fragments: Assembly of a Wide Repertoire of Sequences.

The standardization of DNA fragment assembly methods for many tasks of synthetic biology is crucial. This is necessary for synthesizing a wider repertoire of sequences, as well as for further automation and miniaturization of such reactions. In this work, we proposed conditions for the assembly of DNA fragments from chemically synthesized oligonucleotides and we identified the errors occurring in the sequence under these conditions. Additionally, we proposed conditions for further combining synthetic fragments into larger DNA fragments. We showed that the optimized conditions are suitable for the assembly of a wide range of sequences.

Simple and Rapid Non-ribosomal Peptide Synthetase Gene Assembly Using the SEAM-OGAB Method.

Recombination of biosynthetic gene clusters including those of non-ribosomal peptide synthetases (NRPSs) is essential for understanding the mechanisms of biosynthesis. Due to relatively huge gene cluster sizes ranging from 10 to 150 kb, the prevalence of sequence repeats, and inability to clearly define optimal points for manipulation, functional characterization of recombinant NRPSs with maintained activity has been hindered. In this study, we introduce a simple yet rapid approach named "Seamed Express Assembly Method (SEAM)" coupled with Ordered Gene Assembly in Bacillus subtilis (OGAB) to reconstruct fully functional plipastatin NRPS. This approach is enabled by the introduction of restriction enzyme sites as seams at module borders. SEAM-OGAB is then first demonstrated by constructing the ppsABCDE NRPS (38.4 kb) to produce plipastatin, a cyclic decapeptide in B. subtilis. The introduced amino acid level seams do not hinder the NRPS function and enable successful production of plipastatin at a commensurable titer. It is challenging to modify the plipastatin NRPS gene cluster due to the presence of three long direct-repeat sequences; therefore, this study demonstrates that SEAM-OGAB can be readily applied towards the recombination of various NRPSs. Compared to previous NRPS gene assembly methods, the advantage of SEAM-OGAB is that it readily enables the shuffling of NRPS gene modules, and therefore, chimeric NRPSs can be rapidly constructed for the production of novel peptides. This chimeric assembly application of SEAM-OGAB is demonstrated by swapping plipastatin NRPS and surfactin NRPS modules to produce two novel lipopeptides in B. subtilis.

Assessment of enzymatically synthesized DNA for gene assembly.

Phosphoramidite chemical DNA synthesis technology is utilized for creating de novo ssDNA building blocks and is widely used by commercial vendors. Recent advances in enzymatic DNA synthesis (EDS), including engineered enzymes and reversibly terminated nucleotides, bring EDS technology into competition with traditional chemical methods. In this short study, we evaluate oligos produced using a benchtop EDS instrument alongside chemically produced commercial oligonucleotides to assemble a synthetic gene encoding green fluorescent protein (GFP). While enzymatic synthesis produced lower concentrations of individual oligonucleotides, these were available in half the time of commercially produced oligonucleotides and were sufficient to assemble functional GFP sequences without producing hazardous organic chemical waste.

A depth-first search algorithm for oligonucleotide design in gene assembly.

When synthesizing a gene with a long DNA sequence, it is usually necessary to divide it into several fragments. Based on these fragments, a set of oligonucleotides for gene assembly is produced. Each oligonucleotide is synthesized separately by the chemical reaction, and then the obtained oligonucleotides are assembled into the full gene sequence, in a specific environment, by polymerase chain reaction (PCR) or ligase chain reaction (LCR). In this paper, an effective and efficient algorithm to divide long genes into oligonucleotide sets is presented. First, according to the length of the overlapping oligonucleotide region, the long DNA sequence to be synthesized is divided into fragments of approximately equal length. Second, the length of these fragments is iterated to dynamically optimize the length of the overlapping regions to reduce melting temperature fluctuations. Then, the improved depth-first search algorithm is used according to the design principle of pruning optimization to obtain a uniform set of oligonucleotides with very close melting temperatures. This will decrease the errors in gene assembly with PCR or LCR. Lastly, the oligonucleotides that have homologous melting temperatures needed for PCR-based synthesis and two-step assembly of the target gene are deduced and outputted.

Golden Gate Multigene Assembly Method for Yarrowia lipolytica.

The oleaginous yeast Yarrowia lipolytica has emerged as a powerful alternative for biolipid production due to its high capacity for lipid accumulation. Genetic engineering and synthetic biology are promoted forward to improve production and reroute metabolism for high-value compound synthesis. In this context, efficient, modular, and high-throughput compatible cloning and expression system are required to speed up and rationalize research in this field. Here, we present the fast and modular Golden Gate cloning strategy for the construction of multigene expression vectors and their transformation into Y. lipolytica. As an example, we used the heterologous expression of the carotenoid pathway by cloning three genes involved in this pathway in only one vector allowing reaching production of ß-carotene after a single transformation.

Construction of DNA Tools for Hyperexpression in Marchantia Chloroplasts.

Chloroplasts are attractive platforms for synthetic biology applications since they are capable of driving very high levels of transgene expression, if mRNA production and stability are properly regulated. However, plastid transformation is a slow process and currently limited to a few plant species. The liverwort Marchantia polymorpha is a simple model plant that allows rapid transformation studies; however, its potential for protein hyperexpression has not been fully exploited. This is partially due to the fact that chloroplast post-transcriptional regulation is poorly characterized in this plant. We have mapped patterns of transcription in Marchantia chloroplasts. Furthermore, we have obtained and compared sequences from 51 bryophyte species and identified putative sites for pentatricopeptide repeat protein binding that are thought to play important roles in mRNA stabilization. Candidate binding sites were tested for their ability to confer high levels of reporter gene expression in Marchantia chloroplasts, and levels of protein production and effects on growth were measured in homoplastic transformed plants. We have produced novel DNA tools for protein hyperexpression in this facile plant system that is a test-bed for chloroplast engineering.

Engineering Yarrowia lipolytica for Production of Fatty Alcohols with YaliBrick Vectors.

Biosynthesis of fatty alcohol holds great promise as substitute to replace petroleum-derived fatty alcohols to mitigate environmental concerns and reduce earth's carbon footprint. In this protocol, we detail the procedures of how to use the YaliBrick gene assembly platform to achieve modular assembly of fatty alcohol pathway in Y. lipolytica. To limit fatty alcohol oxidation, we will also describe the hydroxyurea-based protocols for the efficient disruption of POX1 gene, encoding the fatty acyl coenzyme A in Y. lipolytica, with the homologous arm about 500 bp. We envision that this chapter would improve our ability to engineer microbial cell factories for oleochemical and fatty alcohol production in oleaginous yeast species.

TransGene Stacking II Vector System for Plant Metabolic Engineering and Synthetic Biology.

Efficient stacking of multiple genes is a critical element in metabolic engineering of complex pathways, synthetic biology, and genetic improvement of complex agronomic traits in plants. Here we present a high-efficiency multigene assembly and transformation vector system, TransGene Stacking II (TGS II), for these purposes. The operation process is described in detail, and the successful operation mainly depends on effective reagents, special Escherichia coli strains, and basic molecular biological means without other specific equipments.

Complete Chloroplast Genome Sequence of Erigeron breviscapus and Characterization of Chloroplast Regulatory Elements.

Erigeron breviscapus is a famous medicinal plant. However, the limited chloroplast genome information of E. breviscapus, especially for the chloroplast DNA sequence resources, has hindered the study of E. breviscapus chloroplast genome transformation. Here, the complete chloroplast (cp) genome of E. breviscapus was reported. This genome was 152,164bp in length, included 37.2% GC content and was structurally arranged into two 24,699bp inverted repeats (IRs) and two single-copy areas. The sizes of the large single-copy region and the small single-copy region were 84,657 and 18,109bp, respectively. The E. breviscapus cp genome consisted of 127 coding genes, including 83 protein coding genes, 36 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. For those genes, 95 genes were single copy genes and 16 genes were duplicated in two inverted regions with seven tRNAs, four rRNAs, and five protein coding genes. Then, genomic DNA of E. breviscapus was used as a template, and the endogenous 5' and 3' flanking sequences of the trnI gene and trnA gene were selected as homologous recombinant fragments in vector construction and cloned through PCR. The endogenous 5' flanking sequences of the psbA gene and rrn16S gene, the endogenous 3' flanking sequences of the psbA gene, rbcL gene, and rps16 gene and one sequence element from the psbN-psbH chloroplast operon were cloned, and certain chloroplast regulatory elements were identified. Two homologous recombination fragments and all of these elements were constructed into the cloning vector pBluescript SK (+) to yield a series of chloroplast expression vectors, which harbored the reporter gene EGFP and the selectable marker aadA gene. After identification, the chloroplast expression vectors were transformed into Escherichia coli and the function of predicted regulatory elements was confirmed by a spectinomycin resistance test and fluorescence intensity measurement. The results indicated that aadA gene and EGFP gene were efficiently expressed under the regulation of predicted regulatory elements and the chloroplast expression vector had been successfully constructed, thereby providing a solid foundation for establishing subsequent E. breviscapus chloroplast transformation system and genetic improvement of E. breviscapus.

phyloFlash: Rapid Small-Subunit rRNA Profiling and Targeted Assembly from Metagenomes.

The small-subunit rRNA (SSU rRNA) gene is the key marker in molecular ecology for all domains of life, but it is largely absent from metagenome-assembled genomes that often are the only resource available for environmental microbes. Here, we present phyloFlash, a pipeline to overcome this gap with rapid, SSU rRNA-centered taxonomic classification, targeted assembly, and graph-based binning of full metagenomic assemblies. We show that a cleanup of artifacts is pivotal even with a curated reference database. With such a filtered database, the general-purpose mapper BBmap extracts SSU rRNA reads five times faster than the rRNA-specialized tool SortMeRNA with similar sensitivity and higher selectivity on simulated metagenomes. Reference-based targeted assemblers yielded either highly fragmented assemblies or high levels of chimerism, so we employ the general-purpose genomic assembler SPAdes. Our optimized implementation is independent of reference database composition and has satisfactory levels of chimera formation. phyloFlash quickly processes Illumina (meta)genomic data, is straightforward to use, even as part of high-throughput quality control, and has user-friendly output reports. The software is available at https://github.com/HRGV/phyloFlash (GPL3 license) and is documented with an online manual.IMPORTANCE To track organisms across all domains of life, the SSU rRNA gene is the gold standard. Many environmental microbes are known only from high-throughput sequence data, but the SSU rRNA gene, the key to visualization by molecular probes and link to existing literature, is often missing from metagenome-assembled genomes (MAGs). The easy-to-use phyloFlash software suite tackles this gap with rapid, SSU rRNA-centered taxonomic classification, targeted assembly, and graph-based linking to MAGs. Starting from a cleaned reference database, phyloFlash profiles the taxonomic diversity and assembles the sorted SSU rRNA reads. The phyloFlash design is domain agnostic and covers eukaryotes, archaea, and bacteria alike. phyloFlash also provides utilities to visualize multisample comparisons and to integrate the recovered SSU rRNAs in a metagenomics workflow by linking them to MAGs using assembly graph parsing.

Systematic Tools for Reprogramming Plant Gene Expression in a Simple Model, Marchantia polymorpha.

We present the OpenPlant toolkit, a set of interlinked resources and techniques to develop Marchantia as testbed for bioengineering in plants. Marchantia is a liverwort, a simple plant with an open form of development that allows direct visualization of gene expression and dynamics of cellular growth in living tissues. We describe new techniques for simple and efficient axenic propagation and maintenance of Marchantia lines with no requirement for glasshouse facilities. Marchantia plants spontaneously produce clonal propagules within a few weeks of regeneration, and lines can be amplified million-fold in a single generation by induction of the sexual phase of growth, crossing, and harvesting of progeny spores. The plant has a simple morphology and genome with reduced gene redundancy, and the dominant phase of its life cycle is haploid, making genetic analysis easier. We have built robust Loop assembly vector systems for nuclear and chloroplast transformation and genome editing. These have provided the basis for building and testing a modular library of standardized DNA elements with highly desirable properties. We have screened transcriptomic data to identify a range of candidate genes, extracted putative promoter sequences, and tested them in vivo to identify new constitutive promoter elements. The resources have been combined into a toolkit for plant bioengineering that is accessible for laboratories without access to traditional facilities for plant biology research. The toolkit is being made available under the terms of the OpenMTA and will facilitate the establishment of common standards and the use of this simple plant as testbed for synthetic biology.

Manipulating ATP supply improves in situ CO2 recycling by reductive TCA cycle in engineered Escherichia coli.

The reductive tricarboxylic acid (rTCA) cycle was reconstructed in Escherichia coli by introducing pGETS118KAFS, where kor (encodes α-ketoglutarate:ferredoxin oxidoreductase), acl (encodes ATP-dependent citrate lyase), frd (encodes fumarate reductase), and sdh (encodes succinate dehydrogenase) were tandemly conjugated by the ordered gene assembly in Bacillus subtilis (OGAB). E. coli MZLF (E. coli BL21(DE3) Δzwf, Δldh, Δfrd) was employed so that the C-2/C-1 [(ethanol + acetate)/(formate + CO2)] ratio can be used to investigate the effectiveness of the recombinant rTCA for in situ CO2 recycling. It has been shown that supplying ATP through the energy pump (the EP), where formate donates electron to nitrate to form ATP, elevates the C-2/C-1 ratio from 1.03 ± 0.00 to 1.49 ± 0.02. Similarly, when ATP production is increased by the introduction of the heterologous ethanol production pathway (pLOI295), the C-2/C-1 ratio further increased to 1.79 ± 0.02. In summary, the ATP supply is a rate-limiting step for in situ CO2 recycling by the recombinant rTCA cycle. The decrease in C-1 is significant, but the destination of those recycled C-1 is yet to be determined.

Identification and reconstruction of novel antibiotic resistance genes from metagenomes.

BACKGROUND: Environmental and commensal bacteria maintain a diverse and largely unknown collection of antibiotic resistance genes (ARGs) that, over time, may be mobilized and transferred to pathogens. Metagenomics enables cultivation-independent characterization of bacterial communities but the resulting data is noisy and highly fragmented, severely hampering the identification of previously undescribed ARGs. We have therefore developed fARGene, a method for identification and reconstruction of ARGs directly from shotgun metagenomic data. RESULTS: fARGene uses optimized gene models and can therefore with high accuracy identify previously uncharacterized resistance genes, even if their sequence similarity to known ARGs is low. By performing the analysis directly on the metagenomic fragments, fARGene also circumvents the need for a high-quality assembly. To demonstrate the applicability of fARGene, we reconstructed ß-lactamases from five billion metagenomic reads, resulting in 221 ARGs, of which 58 were previously not reported. Based on 38 ARGs reconstructed by fARGene, experimental verification showed that 81% provided a resistance phenotype in Escherichia coli. Compared to other methods for detecting ARGs in metagenomic data, fARGene has superior sensitivity and the ability to reconstruct previously unknown genes directly from the sequence reads. CONCLUSIONS: We conclude that fARGene provides an efficient and reliable way to explore the unknown resistome in bacterial communities. The method is applicable to any type of ARGs and is freely available via GitHub under the MIT license.