Human DDX6 regulates translation and decay of inefficiently translated mRNAs.

Recent findings indicate that the translation elongation rate influences mRNA stability. One of the factors that has been implicated in this link between mRNA decay and translation speed is the yeast DEAD-box helicase Dhh1p. Here, we demonstrated that the human ortholog of Dhh1p, DDX6, triggers the deadenylation-dependent decay of inefficiently translated mRNAs in human cells. DDX6 interacts with the ribosome through the Phe-Asp-Phe (FDF) motif in its RecA2 domain. Furthermore, RecA2-mediated interactions and ATPase activity are both required for DDX6 to destabilize inefficiently translated mRNAs. Using ribosome profiling and RNA sequencing, we identified two classes of endogenous mRNAs that are regulated in a DDX6-dependent manner. The identified targets are either translationally regulated or regulated at the steady-state-level and either exhibit signatures of poor overall translation or of locally reduced ribosome translocation rates. Transferring the identified sequence stretches into a reporter mRNA caused translation- and DDX6-dependent degradation of the reporter mRNA. In summary, these results identify DDX6 as a crucial regulator of mRNA translation and decay triggered by slow ribosome movement and provide insights into the mechanism by which DDX6 destabilizes inefficiently translated mRNAs.

Subtractive proteomics-based vaccine targets annotation and reverse vaccinology approaches to identify multiepitope vaccine against Plesiomonas shigelloides.

Plesiomonas shigelloides, an aquatic bacterium belonging to the Enterobacteriaceae family, is a frequent cause of gastroenteritis with diarrhea and gastrointestinal severe disease. Despite decades of research, discovering a licensed and globally accessible vaccine is still years away. Developing a putative vaccine that can combat the Plesiomonas shigelloides infection by boosting population immunity against P. shigelloides is direly needed. In the framework of the current study, the entire proteome of P. shigelloides was explored using subtractive genomics integrated with the immunoinformatics approach for designing an effective vaccine construct against P. shigelloides. The overall stability of the vaccine construct was evaluated using molecular docking, which demonstrated that MEV showed higher binding affinities with toll-like receptors (TLR4: 51.5 ± 10.3, TLR2: 60.5 ± 9.2) and MHC receptors(MHCI: 79.7 ± 11.2 kcal/mol, MHCII: 70.4 ± 23.7). Further, the therapeutic efficacy of the vaccine construct for generating an efficient immune response was evaluated by computational immunological simulation. Finally, computer-based cloning and improvement in codon composition without altering amino acid sequence led to the development of a proposed vaccine. In a nutshell, the findings of this study add to the existing knowledge about the pathogenesis of this infection. The schemed MEV can be a possible prophylactic agent for individuals infected with P. shigelloides. Nevertheless, further authentication is required to guarantee its safeness and immunogenic potential.

An important consideration when expressing mAbs in Escherichiacoli.

Monoclonal antibodies (mAbs) are a driving force in the biopharmaceutical industry. Therapeutic mAbs are usually produced in mammalian cells, but there has been a push towards the use of alternative production hosts, such as Escherichia coli. When the genes encoding for a mAb heavy and light chains are codon-optimized for E. coli expression, a truncated form of the heavy chain can form along with the full-length product. In this work, the role of codon optimization in the formation of a truncated product was investigated. This study used the amino acid sequences of several therapeutic mAbs and multiple optimization algorithms. It was found that several algorithms incorporate sequences that lead to a truncated product. Approaches to avoid this truncated form are discussed.

A strategy for successful dual-species protein expression of genes with non-optimal codon usage destined for bacterial and yeast cell factories.

Recombinant protein expression on an industrial scale traditionally utilizes one of two microbial workhorses: Escherichia coli or Saccharomyces cerevisiae. Additionally, random protein engineering of enzymes and proteins aimed for expression in S. cerevisiae are often mutagenized and pre-screened in E. coli before expression in yeast. This introduces artificial bottlenecks as the bacterial expression vector needs to be substituted for a yeast expression vector via sub-cloning, and the new library re-evaluated before a final screening in yeast. Here, we put forward a protein expression and engineering strategy that involves the use of a dual-host shuttle vector (pYB-Dual) designed with both a strong inducible yeast promoter (pGAL1), and a strong inducible bacterial promoter (pT7-RNAP), which allows for inducible protein expression in both species. Additionally, we demonstrate that by transforming the pYB-Dual vector into the E. coli strain Rosetta 2, which has elevated levels of 7 rare tRNAs, we can achieve high-level protein expression in both yeast and bacteria, even when using a mNeonGreen gene codon optimized for yeast. This dual expression vector is expected to remove bottlenecks during protein engineering of commercially important enzymes destined for high-titer expression in yeast.

Codon Optimization-based Whole-gene Scanning Identifies Hidden Nucleotides Essential for Bombyx mori Nucleopolyhedrovirus polyhedrin Hyperexpression.

During the late stage of infection, alphabaculoviruses produce many occlusion bodies (OBs) in the nuclei of the insect host's cells through the hyperexpression of polyhedrin (POLH), a major OB component encoded by polh. The strong polh promoter has been used to develop a baculovirus expression vector system for recombinant protein expression in cultured insect cells and larvae. However, the relationship between POLH accumulation and the polh coding sequence remains largely unelucidated. This study aimed to assess the importance of polh codon usage and/or nucleotide sequences in POLH accumulation by generating a baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) expressing mutant polh (co-polh) optimized according to the codon preference of its host insect. Although the deduced amino acid sequence of CO-POLH was the same as that of wild-type POLH, POLH accumulation was significantly lower in cells infected with the co-polh mutant. This reduction was due to decreased polh mRNA levels rather than translational repression. Analysis of mutant viruses with chimeric polh revealed that a 30 base-pair (bp) 5' proximal polh coding region was necessary for maintaining high polh mRNA levels. Sequence comparison of wild-type polh and co-polh identified five nucleotide differences in this region, indicating that these nucleotides were critical for polh hyperexpression. Furthermore, luciferase reporter assays showed that the 30 bp 5' coding region was sufficient for maintaining the polh promoter-driven high level of polh mRNA. Thus, our whole-gene scanning by codon optimization identified important hidden nucleotides for polh hyperexpression in alphabaculoviruses.

Optimizing interleukin-6 and 8 expression, clarification and purification in plant cell packs and plants for application in advanced therapy medicinal products and cellular agriculture.

Healthcare and nutrition are facing a paradigm shift in light of advanced therapy medicinal products (ATMPs) and cellular agriculture options respectively. Both options heavily rely on some sort of animal cell culture, e.g. autologous stem cells. These cultures require various growth factors, such as interleukin-6 and 8 (IL-6/8), in a pure, safe and sustainable form that can be provided in a scalable manner. Plants seem well suited for this task because purification of small proteins can be readily achieved by membrane separation, human/animal pathogens do not replicate in plants and production can be scaled up using in-door farming or agricultural practices. Here, we illustrate this capacity by first optimizing the codon usage of IL-6/8 for translation in Nicotiana spp., as well as testing the effect of untranslated regions and product targeting to different sub-cellular compartments on expression in a high-throughput plant cell pack (PCP) assay. In the chloroplast, IL-6 accumulated up to 6.9±3.8 (SD, n=2) and 14.4±7.4 mg kg-1 (SD, n=5) were observed in case of IL-8. When transferring IL-8 expression into whole plants, accumulation was 12.3±1.5 mg kg-1 (SD, n=3). After extraction and clarification, IL-8 was purified using a two-stage process consisting of an ultrafiltration/diafiltration step with 100 kDa and 10 kDa cut off membranes followed by an IMAC polishing step. The purity, yield and recovery were 97.8%, 6.6 mg kg-1 and 38%, respectively. We evaluated the ability of the proposed purification process to remove endotoxins to ensure the compatibility of plant-made growth factors with cell culture.

High-level expression of codon-optimized Taq DNA polymerase under the control of rhaBAD promoter.

A DNA polymerase from Thermus aquaticus remains the most popular among DNA polymerases. It was widely applied in various fields involving the application of polymerase chain reaction (PCR), implying the high commercial value of this enzyme. For this reason, an attempt to obtain a high yield of Taq DNA polymerase is continuously conducted. In this study, the l-rhamnose-inducible promoter rhaBAD was utilized due to its ability to produce recombinant protein under tight control in E. coli expression system. Instead of full-length Taq polymerase, an N-terminal deletion of Taq polymerase was selected. To obtain a high-level expression, we attempted to optimize the codon by reducing the rare codon and GC content, and in a second attempt, we optimized the culture conditions for protein expression. The production of Taq polymerase using the optimum culture condition improved the level of expression by up to 3-fold. This approach further proved that a high level of recombinant protein expression could be achieved by yielding a purified Taq polymerase of about 8.5 mg/L of culture. This is the first research publication on the production of Taq polymerase with N-terminal deletion in E. coli with the control of the rhaBAD promoter system.

An Integer Linear Programming Model to Optimize Coding DNA Sequences By Joint Control of Transcript Indicators.

A Coding DNA Sequence (CDS) is a fraction of DNA whose nucleotides are grouped into consecutive triplets called codons, each one encoding an amino acid. Because most amino acids can be encoded by more than one codon, the same amino acid chain can be obtained by a very large number of different CDSs. These synonymous CDSs show different features that, also depending on the organism the transcript is expressed in, could affect translational efficiency and yield. The identification of optimal CDSs with respect to given transcript indicators is in general a challenging task, but it has been observed in recent literature that integer linear programming (ILP) can be a very flexible and efficient way to achieve it. In this article, we add evidence to this observation by proposing a new ILP model that simultaneously optimizes different well-grounded indicators. With this model, we efficiently find solutions that dominate those returned by six existing codon optimization heuristics.

Codon-optimization in gene therapy: promises, prospects and challenges.

Codon optimization has evolved to enhance protein expression efficiency by exploiting the genetic code's redundancy, allowing for multiple codon options for a single amino acid. Initially observed in E. coli, optimal codon usage correlates with high gene expression, which has propelled applications expanding from basic research to biopharmaceuticals and vaccine development. The method is especially valuable for adjusting immune responses in gene therapies and has the potenial to create tissue-specific therapies. However, challenges persist, such as the risk of unintended effects on protein function and the complexity of evaluating optimization effectiveness. Despite these issues, codon optimization is crucial in advancing gene therapeutics. This study provides a comprehensive review of the current metrics for codon-optimization, and its practical usage in research and clinical applications, in the context of gene therapy.

Synthetic biology approach revealed enhancement in haeme oxygenase-1 gene expression by codon pair optimization while reduction by codon deoptimization.

Haem oxygenase-1 (HO-1) is a ubiquitously expressed gene involved in cellular homoeostasis, and its imbalance in expression results in various disorders. To alleviate such disorders, HO-1 gene expression needs to be modulated. Codon usage bias results from evolutionary forces acting on any nucleotide sequence and determines the gene expression. Like codon usage bias, codon pair bias also exists, playing a role in gene expression. In the present study, HO-1 gene was recoded by manipulating codon and codon pair bias, and four such constructs were made through codon/codon pair deoptimization and codon/codon pair optimization to reduce and enhance the HO-1 gene expression. Codon usage analysis was done for these constructs for four tissues brain, heart, pancreas and liver. Based on codon usage in different tissues, gene expression of these tissues was determined in terms of the codon adaptation index. Based on the codon adaptation index, minimum free energy, and translation efficiency, constructs were evaluated for enhanced or decreased HO-1 expression. The analysis revealed that for enhancing gene expression, codon pair optimization, while for reducing gene expression, codon deoptimization is efficacious. The recoded constructs developed in the study could be used in gene therapy regimens to cure HO-1 over or underexpression-associated disorders.

Identification of a novel MT-ND3 variant and restoring mitochondrial function by allotopic expression of MT-ND3 gene.

Mitochondrial diseases are caused by nuclear, or mitochondrial DNA (mtDNA) variants and related co-factors. Here, we report a novel m.10197G > C variant in MT-ND3 in a patient, and two other patients with m.10191 T > C. MT-ND3 variants are known to cause Leigh syndrome or mitochondrial complex I deficiency. We performed the functional analyses of the novel m.10197G > C variant that significantly lowered MT-ND3 protein levels, causing complex I assembly and activity deficiency, and reduction of ATP synthesis. We adapted a previously described re-engineering technique of delivering mitochondrial genes into mitochondria through codon optimization for nuclear expression and translation by cytoplasmic ribosomes to rescue defects arising from the MT-ND3 variants. We constructed mitochondrial targeting sequences along with the codon-optimized MT-ND3 and imported them into the mitochondria. To achieve the goal, we imported codon-optimized MT-ND3 into mitochondria in three patients with m.10197G > C and m.10191 T > C missense variants in the MT-ND3. Nuclear expression of the MT-ND3 gene partially restored protein levels, complex I deficiency, and significant improvement of ATP production indicating a functional rescue of the mutant phenotype. The codon-optimized nuclear expression of mitochondrial protein and import inside the mitochondria can supplement the requirements for ATP in energy-deficient mitochondrial disease patients.

Expanding the synthetic biology toolbox of Cupriavidus necator for establishing fatty acid production.

The Gram-negative betaproteobacterium Cupriavidus necator is a chemolithotroph that can convert carbon dioxide into biomass. Cupriavidus necator has been engineered to produce a variety of high-value chemicals in the past. However, there is still a lack of a well-characterized toolbox for gene expression and genome engineering. Development and optimization of biosynthetic pathways in metabolically engineered microorganisms necessitates control of gene expression via functional genetic elements such as promoters, ribosome binding sites (RBSs), and codon optimization. In this work, a set of inducible and constitutive promoters were validated and characterized in C. necator, and a library of RBSs was designed and tested to show a 50-fold range of expression for green fluorescent protein (gfp). The effect of codon optimization on gene expression in C. necator was studied by expressing gfp and mCherry genes with varied codon-adaptation indices and was validated by expressing codon-optimized variants of a C12-specific fatty acid thioesterase to produce dodecanoic acid. We discuss further hurdles that will need to be overcome for C. necator to be widely used for biosynthetic processes.

Alter codon bias of the P. pastoris genome to overcome a bottleneck in codon optimization strategy development and improve protein expression.

Apart from its role in translation, codon bias is also an important mechanism to regulate mRNA levels. The traditional frequency-based codon optimization strategy is rather efficient in organisms such as N. crassa, but much less in yeast P. pastoris which is a popular host for heterologous protein expression. This is because that unlike N. crassa, the preferred codons of P. pastoris are actually AU-rich and hence codon optimization for extremely low GC content comes with issues of pre-mature transcriptional termination or low RNA stability in spite of translational advantages. To overcome this bottleneck, we focused on three reporter genes in P. pastoris first and confirmed the great advantage of GC-prone codon optimization on mRNA levels. Then we altered the codon bias profile of P. pastoris by introducing additional rare tRNA gene copies. Prior to that we constructed IPTG-regulated tRNA species to enable chassis cells to switch between different codon bias status. As demonstrated again with reporter genes, protein yield of luc and 0788 was successfully increased by 4-5 folds in chassis cells. In summary, here we provide an alternative codon optimization strategy for genes with unsatisfactory performance under traditional codon frequency-based optimization.

Rescue of Recombinant Newcastle Disease Virus Expressing Heterologous Genes.

Reverse genetics allows for the generation of recombinant infectious viruses from viral sequences or complete viral genomes cloned into plasmids. Using reverse genetics, it is then possible to introduce changes in the genome of infectious viruses for multiple applications.Newcastle disease virus (NDV) is a non-segmented, negative-sense RNA virus that has been amenable to manipulation by reverse genetics for more than two decades. Since then, recombinant NDVs have been extensively used as viral vectors to express heterologous proteins. We describe the key steps required to design and introduce an additional transcription unit in the genome of the Newcastle disease virus for the efficient expression of a heterologous gene.

Codon optimization of a gene encoding DNA polymerase from Pyrococcus furiosus and its expression in Escherichia coli.

BACKGROUND: DNA polymerase is an essential component in PCR assay for DNA synthesis. Improving DNA polymerase with characteristics indispensable for a powerful assay is crucial because it can be used in wide-range applications. Derived from Pyrococcus furiosus, Pfu DNA polymerase (Pfu pol) is one of the excellent polymerases due to its high fidelity. Therefore, we aimed to develop Pfu pol from a synthetic gene with codon optimization to increase its protein yield in Escherichia coli. RESULTS: Recombinant Pfu pol was successfully expressed and purified with a two-step purification process using nickel affinity chromatography, followed by anion exchange chromatography. Subsequently, the purified Pfu pol was confirmed by Western blot analysis, resulting in a molecular weight of approximately 90 kDa. In the final purification process, we successfully obtained a large amount of purified enzyme (26.8 mg/L). Furthermore, the purified Pfu pol showed its functionality and efficiency when tested for DNA amplification using the standard PCR. CONCLUSIONS: Overall, a high-level expression of recombinant Pfu pol was achieved by employing our approach in the present study. In the future, our findings will be useful for studies on synthesizing recombinant DNA polymerase in E. coli expression system.

Maximizing Heterologous Expression of Engineered Type I Polyketide Synthases: Investigating Codon Optimization Strategies.

Type I polyketide synthases (T1PKSs) hold enormous potential as a rational production platform for the biosynthesis of specialty chemicals. However, despite great progress in this field, the heterologous expression of PKSs remains a major challenge. One of the first measures to improve heterologous gene expression can be codon optimization. Although controversial, choosing the wrong codon optimization strategy can have detrimental effects on the protein and product levels. In this study, we analyzed 11 different codon variants of an engineered T1PKS and investigated in a systematic approach their influence on heterologous expression in Corynebacterium glutamicum, Escherichia coli, and Pseudomonas putida. Our best performing codon variants exhibited a minimum 50-fold increase in PKS protein levels, which also enabled the production of an unnatural polyketide in each of these hosts. Furthermore, we developed a free online tool (https://basebuddy.lbl.gov) that offers transparent and highly customizable codon optimization with up-to-date codon usage tables. In this work, we not only highlight the significance of codon optimization but also establish the groundwork for the high-throughput assembly and characterization of PKS pathways in alternative hosts.

An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system.

Saccharomyces cerevisiae is one of the most extensively used biosynthetic systems for the production of diverse bioproducts, especially biotherapeutics and recombinant proteins. Because the expression and insertion of foreign genes are always impaired by the endogenous factors of Saccharomyces cerevisiae and nonproductive procedures, various technologies have been developed to enhance the strength and efficiency of transcription and facilitate gene editing procedures. Thus, the limitations that block heterologous protein secretion have been overcome. Highly efficient promoters responsible for the initiation of transcription and the accurate regulation of expression have been developed that can be precisely regulated with synthetic promoters and double promoter expression systems. Appropriate codon optimization and harmonization for adaption to the genomic codon abundance of S. cerevisiae are expected to further improve the transcription and translation efficiency. Efficient and accurate translocation can be achieved by fusing a specifically designed signal peptide to an upstream foreign gene to facilitate the secretion of newly synthesized proteins. In addition to the widely applied promoter engineering technology and the clear mechanism of the endoplasmic reticulum secretory pathway, the innovative genome editing technique CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated system) and its derivative tools allow for more precise and efficient gene disruption, site-directed mutation, and foreign gene insertion. This review focuses on sophisticated engineering techniques and emerging genetic technologies developed for the accurate metabolic regulation of the S. cerevisiae expression system.

A Machine Learning Method for Genome Engineering Design Tool Attribution.

As the ability to engineer biological systems improves with increasingly advanced technology, the risk of accidental or intentional release of a dangerous genetically modified organism becomes greater. It is important that authorities can carry out attribution for the source of a genetically modified biological agent release. In the absence of evidence that ties a release directly to the individuals responsible, attribution can be carried out in part by discovering the in silico tools used to design the engineered genetic components, which can leave a signature in the DNA of the organism. Previous attribution methods have focused on identifying the laboratory of origin of an engineered organism using machine learning on plasmid signatures. The next logical step is to address attribution using signatures from the tools that are used to create the engineered modifications. A random forest classifier was developed that discriminates between design tools used to optimize coding regions for incorporation into the genome of another organism. To this end, tens of thousands of genes were optimized with 4 different codon optimization methods and relevant features from these sequences were generated for a machine learning classifier. This method achieves more than 97% accuracy in predicting which tools were used to design codon optimized genes for expression in other organisms. The methods presented here lay the groundwork for the creation of effective organism engineering attribution techniques. Such methods can act both as deterrents for future attempts at creating dangerous organisms as well as tools for forensic science.

A Systematic Review of the Potential of Pichia pastoris (Komagataella phaffii) as an Alternative Host for Biologics Production.

The methylotrophic yeast Pichia pastoris is garnering interest as a chassis cell factory for the manufacture of recombinant proteins because it effectively satisfies the requirements of both laboratory and industrial set up. The optimisation of P. pastoris cultivation is still necessary due to strain- and product-specific problems such as promoter strength, methanol utilisation type, and culturing conditions to realize the high yields of heterologous protein(s) of interest. Techniques integrating genetic and process engineering have been used to overcome these problems. Insight into the Pichia as an expression system utilizing MUT pathway and the development of methanol free systems are highlighted in this systematic review. Recent developments in the improved production of proteins in P. pastoris by (i) diverse genetic engineering such as codon optimization and gene dosage; (ii) cultivating tactics including co-expression of chaperones; (iii) advances in the use of the 2A peptide system, and (iv) CRISPR/Cas technologies are widely discussed. We believe that by combining these strategies, P. pastoris will become a formidable platform for the production of high value therapeutic proteins.

Heterologous Expression of Codon-Optimized Azurin Transferred by Magnetofection Method in MCF-10A Cells.

Transfection efficiency of the immortalized human breast epithelial cell line MCF-10A remains an issue that needs to be resolved. In this study, it was aimed to deliver a recombinant DNA (pCMV-Azu-GFP) to the MCF-10A cells by the magnetofection method using magnetic nanoparticles (MNPs) and a simple magnet to accelerate the DNA delivery. Surface positively modified silica-coated iron oxide MNPs (MSNP-NH2) were produced and characterized via TEM, FTIR, and DLS analyses. The recombinant DNA (rDNA) was obtained by the integration of codon-optimized azurin to produce a fusion protein. Then, rDNA cloned in Escherichia coli cells was validated by sequence analysis. The electrostatically conjugated rDNA on MSNP-NH2 with an enhancer polyethyleneimine (PEI) was studied by agarose gel electrophoresis and the optimum conditions were determined to apply to the cell. A dose-dependent statistical difference was observed on treated cells based on the MTS test. The expression of the fusion protein after magnetofection was determined using laser scanning confocal microscope imaging and western blot analysis. It was observed that the azurin gene could be transferred to MCF-10A cells by magnetofection. Thus, when the azurin gene is used as a breast cancer treatment agent, it can be expressed in healthy cells without toxic effects.