Effect Of Codon Optimization Research Articles

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.

Effects of codon optimization on expression in Escherichia coli of protein-coding DNA sequences from the protozoan Eimeria

The objective of this study was to compare the levels of recombinant protein from three Eimeria genes before and after optimization of codons for expression in Escherichia coli. Protein coding sequences from Eimeria maxima (EmaxSO7, EmaxIMP1) and Eimeria acervulina (EAH00033530) were cloned with or without prior codon optimization and expressed as polyHis fusion proteins. All three outcomes: higher, lower, or no change in the yield of amount of recombinant protein were observed suggesting that codon optimization alone for expression in E. coli does not inevitably lead to higher expression levels. Analysis of codon usage for each gene sequence revealed that, similar to other organisms, Eimeria intersperses rare and frequently used codons in protein-coding sequences. However, no relationship was observed between the predicted protein structure and the location of major and minor codons, suggesting that codon selection in this apicomplexan parasite is influenced by factors other than regional secondary protein structure.

Effects of codon optimization, N-terminal truncation and gene dose on the heterologous expression of berberine bridge enzyme

Morphine, sanguinarine and chelerythrine are benzylisoquinoline alkaloids (BIAs), and these compounds possess strong biological activities. (S)-scoulerine is a commonly shared precursor of these compounds, and berberine bridge enzyme (BBE) is a key rate-limiting enzyme in the synthesis of (S)-scoulerine. We isolated the BBE gene from Macleaya cordata (McBBE) and used CEN.PK2-1C as a chassis strain. We compared the catalytic efficiency of five codon-optimized McBBE genes in Saccharomyces cerevisiae and finally obtained a yeast strain (YH03) that exhibited a 58-fold increase in yield (1.12mg/L). Then, we truncated the N-terminus of McBBE by 8 and 22 amino acids and found that with the increase in the number of N-terminal truncated amino acids, the production of (S)-scoulerine gradually decreased. Additionally, we used CRISPR-Cas9 to integrate the McBBE gene at the delta site of the S. cerevisiae genome to achieve stable genetic inheritance and found that the yield of (S)-scoulerine was not significantly increased in the integrated strain. In conclusion, our work achieved high-efficiency expression of McBBE in S. cerevisiae, explored the influence of N-terminal truncation on the yield of (S)-scoulerine, and obtained a genetically stable S. cerevisiae strain with high McBBE expression. This study provides a reference for further complex metabolic engineering optimization and lays a foundation for the efficient biosynthesis of BIAs.

Effects of codon optimization on coagulation factor IX translation and structure: Implications for protein and gene therapies

Synonymous codons occur with different frequencies in different organisms, a phenomenon termed codon usage bias. Codon optimization, a common term for a variety of approaches used widely by the biopharmaceutical industry, involves synonymous substitutions to increase protein expression. It had long been presumed that synonymous variants, which, by definition, do not alter the primary amino acid sequence, have no effect on protein structure and function. However, a critical mass of reports suggests that synonymous codon variations may impact protein conformation. To investigate the impact of synonymous codons usage on protein expression and function, we designed an optimized coagulation factor IX (FIX) variant and used multiple methods to compare its properties to the wild-type FIX upon expression in HEK293T cells. We found that the two variants differ in their conformation, even when controlling for the difference in expression levels. Using ribosome profiling, we identified robust changes in the translational kinetics of the two variants and were able to identify a region in the gene that may have a role in altering the conformation of the protein. Our data have direct implications for codon optimization strategies, for production of recombinant proteins and gene therapies.

Effects of codon optimization and glycosylation on the high-level production of hydroxynitrile lyase from Chamberlinius hualienensis in Pichia pastoris.

A hydroxynitrile lyase (HNL) from the millipede Chamberlinius hualienensis has high potential for industrial use in the synthesis of cyanohydrins. However, obtaining sufficient amounts of millipedes is difficult, and the production of the Chamberlinius hualienensis HNL (ChuaHNL) in E. coli has not been very successful. Therefore, we investigated the conditions required for high-yield heterologous production of this enzyme using Pichia pastoris. When we employed P. pastoris to express His-ChuaHNL, the yield was very low (22.6 ± 3.8U/L culture). Hence, we investigated the effects of ChuaHNL codon optimization and the co-production of two protein disulfide isomerases (PDIs) [from P. pastoris (PpPDI) and C. hualienensis (ChuaPDI1, ChuaPDI2)] on His-ChuaHNL production. The productivity of His-ChuaHNL was increased approximately 140 times per unit culture to 3170 ± 144.7U/L by the co-expression of codon-optimized ChuaHNL and PpPDI. Moreover, we revealed that the N-glycosylation on ChuaHNL had a large effect on the stability, enzyme secretion, and catalytic properties of ChuaHNL in P. pastoris. This study demonstrates an economical and efficient approach for the production of HNL, and the data show that glycosylation has a large effect on the enzyme properties and the P. pastoris expression system.

Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots.

SummaryPlant synthetic biology and cereal engineering depend on the controlled expression of transgenes of interest. Most engineering in plant species to date has relied heavily on the use of a few, well‐established constitutive promoters to achieve high levels of expression; however, the levels of transgene expression can also be influenced by the use of codon optimization, intron‐mediated enhancement and varying terminator sequences. Most of these alternative approaches for regulating transgene expression have only been tested in small‐scale experiments, typically testing a single gene of interest. It is therefore difficult to interpret the relative importance of these approaches and to design engineering strategies that are likely to succeed in different plant species, particularly if engineering multigenic traits where the expression of each transgene needs to be precisely regulated. Here, we present data on the characterization of 46 promoters and 10 terminators in Medicago truncatula, Lotus japonicus, Nicotiana benthamiana and Hordeum vulgare, as well as the effects of codon optimization and intron‐mediated enhancement on the expression of two transgenes in H. vulgare. We have identified a core set of promoters and terminators of relevance to researchers engineering novel traits in plant roots. In addition, we have shown that combining codon optimization and intron‐mediated enhancement increases transgene expression and protein levels in barley. Based on our study, we recommend a core set of promoters and terminators for broad use and also propose a general set of principles and guidelines for those engineering cereal species.

Optimizing a Human Papillomavirus Type 16 L1-Based Chimaeric Gene for Expression in Plants.

Human papillomaviruses (HPVs) are the causative agents of cervical cancer, the fourth most prevalent cancer in women worldwide. The major capsid protein L1 self-assembles into virus-like particles (VLPs), even in the absence of the minor L2 protein: such VLPs have successfully been used as prophylactic vaccines. There remains a need, however, to develop cheaper vaccines that protect against a wider range of HPV types. The use of all or parts of the L2 minor capsid protein can potentially address this issue, as it has sequence regions conserved across several HPV types, which can elicit a wider spectrum of cross-neutralizing antibodies. Production of HPV VLPs in plants is a viable option to reduce costs; the use of a L1/L2 chimera which has previously elicited a cross-protective immune response is an option to broaden cross-protection. The objective of this study was to investigate the effect of codon optimization and of increasing the G+C content of synthetic L1/L2 genes on protein expression in plants. Additionally, we replaced varying portions of the 5′ region of the L1 gene with the wild type (wt) viral sequence to determine the effect of several negative regulatory elements on expression. We showed that GC-rich genes resulted in a 10-fold increase of mRNA levels and 3-fold higher accumulation of proteins. However, the highest increase of expression was achieved with a high GC-content human codon-optimized gene, which resulted in a 100-fold increase in mRNA levels and 8- to 9-fold increase in protein levels. Changing the 5′ end of the L1 gene back to its wt sequence decreased mRNA and protein expression. Our results suggest that the negative elements in the 5′ end of L1 are inadvertently destroyed by changing the codon usage, which enhances protein expression.

Effect of codon optimization on the enhancement of the β-carotene contents in rice endosperm

A β-carotene is the most well-known dietary source as provitamin A carotenoids. Among β-carotene-producing Golden Rice varieties, PAC (Psy:2A:CrtI) rice has been previously developed using a bicistronic recombinant gene that linked the Capsicum Psy and Pantoea CrtI genes by a viral 2A sequence. To enhance β-carotene content by improving this PAC gene, its codon was optimized for rice plants (Oryza sativa L.) by minimizing the codon bias between the transgene donor and the host rice and was then artificially synthesized as stPAC (stPsy:2A:stCrtI) gene. The GC content (58.7 from 50.9%) and codon adaptation index (0.85 from 0.77) of the stPAC gene were increased relative to the original PAC gene with 76% DNA identity. Among 67 T1 seeds of stPAC transformants showing positive correlations between transgene copy numbers (up to three) and carotenoid contents, three stPAC lines with a single intact copy were chosen to minimize unintended insertional effects and compared to the representative line of the PAC transgene with respect to their codon optimization effects. Translation levels were stably increased in all three stPAC lines (3.0-, 2.5-, 2.9-fold). Moreover, a greater intensity of the yellow color of stPAC seeds was correlated with enhanced levels of β-carotene (4-fold, 2.37 μg/g) as well as total carotenoid (2.9-fold, 3.50 μg/g) relative to PAC seeds, suggesting a β-branch preference for the stPAC gene. As a result, the codon optimization of the transgene might be an effective tool in genetic engineering for crop improvement as proven at the enhanced levels of translation and carotenoid production.

Expression of Codon-Optimized Plant GlycosyltransferaseUGT72B14inEscherichia coliEnhances Salidroside Production

Salidroside, a plant secondary metabolite in Rhodiola, has been demonstrated to have several adaptogenic properties as a medicinal herb. Due to the limitation of plant source, microbial production of salidroside by expression of plant uridine diphosphate glycosyltransferase (UGT) is promising. However, glycoside production usually remains hampered by poor expression of plant UGTs in microorganisms. Herein, we achieved salidroside production by expression of Rhodiola UGT72B14 in Escherichia coli (E. coli) and codon optimization was accordingly applied. UGT72B14 expression was optimized by changing 278 nucleotides and decreasing the G+C content to 51.05% without altering the amino acid sequence. The effect of codon optimization on UGT72B14 catalysis for salidroside production was assessed both in vitro and in vivo. In vitro, salidroside production by codon-optimized UGT72B14 is enhanced because of a significantly improved protein yield (increased by 4.8-fold) and an equivalently high activity as demonstrated by similar kinetic parameters (K M and V max), compared to that by wild-type protein. In vivo, both batch and fed-batch cultivation using the codon-optimized gene resulted in a significant increase in salidroside production, which was up to 6.7 mg/L increasing 3.2-fold over the wild-type UGT72B14.

Rational Design for Enhanced Gene Therapy with DNA Transposons

Several genetic diseases caused by deficiencies in enzymes required throughout the body can be treated by enzyme replacement therapy at costs of upward of hundreds of thousands of dollars per year per patient. In the long run, such costs are prohibitive. Gene therapy is therefore an appealing option in that its aim is to treat patients after only a single or very few interventions. Clinical-grade viral vectors can efficiently deliver therapeutic expression cassettes, but such viruses can still be expensive to prepare. By contrast, clinical-grade nonviral nucleic acids are relatively inexpensive to produce but difficult to deliver into cells. In this issue of Molecular Therapy, two reports from the same group describe improvements to two limiting factors of nonviral gene therapy using DNA transposons delivered to the liver.1,2 The authors substantially increased efficiency of delivery of transgenes to the target organ and the amount of therapeutic protein exported per cell following transgene delivery.

Effects of codon optimization on the mRNA levels of heterologous genes in filamentous fungi

Filamentous fungi, particularly Aspergillus species, have recently attracted attention as host organisms for recombinant protein production. Because the secretory yields of heterologous proteins are generally low compared with those of homologous proteins or proteins from closely related fungal species, several strategies to produce substantial amounts of recombinant proteins have been conducted. Codon optimization is a powerful tool for improving the production levels of heterologous proteins. Although codon optimization is generally believed to improve the translation efficiency of heterologous genes without affecting their mRNA levels, several studies have indicated that codon optimization causes an increase in the steady-state mRNA levels of heterologous genes in filamentous fungi. However, the mechanism that determines the low mRNA levels when native heterologous genes are expressed was poorly understood. We recently showed that the transcripts of heterologous genes are polyadenylated prematurely within the coding region and that the heterologous gene transcripts can be stabilized significantly by codon optimization, which is probably attributable to the prevention of premature polyadenylation in Aspergillus oryzae. In this review, we describe the detailed mechanism of premature polyadenylation and the rapid degradation of mRNA transcripts derived from heterologous genes in filamentous fungi.

Effect of codon optimization on expression levels of human cystatin C in Pichia pastoris.

Human cystatin C (CysC) is a cysteine proteinase inhibitor with many potential applications. To facilitate further studies of the functions and applications of CysC, we improved the heterologous expression of CysC using a basic codon optimization method. In this study, we cloned the high-GC content wild-type sequence of the CysC gene and also designed a slightly AT-biased sequence, with codons optimized for expression in the Pichia pastoris GS115 strain. Our results showed that the optimized coding sequence of human CysC increased the expression and secretion of the CysC protein by approximately 3- to 5-fold (90-96 mg CysC/L) in yeast, compared with the expression levels of the native CysC gene (17.9-18.4 mg CysC/L). We designed, constructed, and applied an optimized version of the CysC gene for the Pichia expression system. Our results demonstrate that the optimized coding sequence provides a higher yield of secreted CysC than that produced using the wild-type gene. Our data also serve as a practical example demonstrating a rational design strategy for the heterologous expression of secreted proteins.

Effect of codon optimization on the expression of Trichoderma reesei endoglucanase 1 in Pichia pastoris

Trichoderma reesei cellulases are important biocatalysts for a wide range of industrial applications that include the paper, feed, and textile industries. T. reesei endoglucanase 1 (egl1) was successfully expressed as an active and stable catalyst in Pichia pastoris for the first time. Codon optimization was applied to egl1 of T. reesei to enhance its expression levels in P. pastoris. When compared with the originally cloned egl1 gene of T. reesei, the synthetic codon optimized egl1 gene (egl1s) was expressed at a higher level in P. pastoris. Batch fermentations of both clones with the same copy number under controlled conditions indicated that codon optimized EGI enzyme activity increased to 1.24 fold after 72 h of methanol induction. Our research indicated that P. pastoris is a suitable host for cellulase production.

Effect of codon optimization and subcellular targeting on Toxoplasma gondii antigen SAG1 expression in tobacco leaves to use in subcutaneous and oral immunization in mice

BackgroundCodon optimization and subcellular targeting were studied with the aim to increase the expression levels of the SAG178-322 antigen of Toxoplasma gondii in tobacco leaves. The expression of the tobacco-optimized and native versions of the SAG1 gene was explored by transient expression from the Agrobacterium tumefaciens binary expression vector, which allows targeting the recombinant protein to the endoplasmic reticulum (ER) and the apoplast. Finally, mice were subcutaneously and orally immunized with leaf extracts-SAG1 and the strategy of prime boost with rSAG1 expressed in Escherichia coli was used to optimize the oral immunization with leaf extracts-SAG1.ResultsLeaves agroinfiltrated with an unmodified SAG1 gene accumulated 5- to 10-fold more than leaves agroinfiltrated with a codon-optimized SAG1 gene. ER localization allowed the accumulation of higher levels of native SAG1. However, no significant differences were observed between the mRNA accumulations of the different versions of SAG1. Subcutaneous immunization with leaf extracts-SAG1 (SAG1) protected mice against an oral challenge with a non-lethal cyst dose, and this effect could be associated with the secretion of significant levels of IFN-γ. The protection was increased when mice were ID boosted with rSAG1 (SAG1+boost). This group elicited a significant Th1 humoral and cellular immune response characterized by high levels of IFN-γ. In an oral immunization assay, the SAG1+boost group showed a significantly lower brain cyst burden compared to the rest of the groups.ConclusionTransient agroinfiltration was useful for the expression of all of the recombinant proteins tested. Our results support the usefulness of endoplasmic reticulum signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The results showed that this plant-produced protein has potential for use as vaccine and provides a potential means for protecting humans and animals against toxoplasmosis.

Improved Production of (R)-1-phenyl-1,2-ethanediol by a Codon-optimized R-specific Carbonyl Reductase from Candida parapsilosis in Escherichia coli

An R-specific carbonyl reductase from Candida parapsilosis (CprCR) catalyzes the transformation of (R)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone. The gene rcr coding CprCR contains a few codons rarely used by Escherichia coli. In order to improve chiral alcohol production, three codon variants Delta24, aRCR, and mRCR of CprCR were designed through truncation of 4-27 bp disorder sequence at the 5'-terminus or/and adaption of nine rare codons. The effects of codon optimization on enzyme activity, protein production, and biotransformation were studied. Among these three types, the disorder sequence-truncated and rare codon-adapted variant mRCR presents the highest enzyme activity. When compared with CprCR, mRCR showed an increase of 35.6% in the total activity of cell-free extracts. The specific activity of mRCR presented similar increase in the cell-free extract with purified protein, which suggested that the codon optimization caused positive effect on protein productivity of variant enzyme. When microbial cells concentration was 30% (w/v), the molar conversion yield and enantiomeric excess of the mRCR variant reached 86.4% and 93.6%, which were increased 36.5% and 15.8% than those of wild-type at a high substrate concentration of 5 g/L. The work will supply a new method for improving chiral alcohol preparation with codon engineered microorganisms.

DNA/MVA Vaccine for HIV Type 1: Effects of Codon-Optimization and the Expression of Aggregates or Virus-Like Particles on the Immunogenicity of the DNA Prime

Recently, a vaccine consisting of DNA priming followed by boosting with modified vaccinia Ankara (MVA) has provided long-term protection of rhesus macaques against a virulent challenge with a chimera of simian and human immunodeficiency viruses. Here, we report studies on the development of the DNA component for a DNA/MVA HIV vaccine for humans. Specifically, we assess the ability of a codon-optimized Gag-expressing DNA and two noncodon-optimized Gag-Pol-Env-expressing DNAs to prime the MVA booster dose. The codon-optimized DNA expressed virus-like particles (VLPs), whereas one of the noncodon-optimized DNAs expressed VLPs and the other expressed aggregates of HIV proteins. The MVA boost expressed Gag-Pol and Env and produced VLPs. Immunogenicity studies in macaques used one intramuscular prime with 600 microg of DNA and two intramuscular boosts with 1 x 10(8) pfu of MVA at weeks 8 and 30. The codon-optimized and noncodon-optimized DNAs proved similar in their ability to prime anti-Gag T cell responses. The aggregate and VLP-expressing Gag-Pol-Env DNAs also showed no significant differences in their ability to prime anti-Env Ab responses. The second MVA booster dose did not increase the peak CD4 and CD8 T cell responses, but increased anti-Env Ab titers by 40- to 90-fold. MVA-only immunizations elicited 10-100 times lower frequencies of T cells and 2-4 lower titers of anti-Env Ab than the Gag-Pol-Env DNA/MVA immunizations. Based on the breadth of the T cell response and a trend toward higher titers of anti-Env Ab, we are moving forward with human trials of the noncodon-optimized VLP-expressing DNA.

Effects of codon-optimization on protein expression by the human herpesvirus 6 and 7 U51 open reading frame

Codon-optimization refers to the alteration of gene sequences, to make codon usage match the available tRNA pool within the cell/species of interest. Codon-optimization has emerged as a powerful tool to increase protein expression by genes from small RNA and DNA viruses, which commonly contain overlapping reading frames as well as structural elements that are embedded within coding regions; these features are not widespread among large DNA viruses. We therefore examined whether codon-optimization might influence protein expression from a herpesvirus gene. We focused on the U51 gene from human herpesviruses-6 and -7, which was cloned in both native and codon-optimized form, with an N-terminal HA epitope tag to allow protein detection. Codon-optimization was associated with a profound (10–100 fold) increase in U51 expression in human (293A, HSG, K562) or hamster (CHO) cell lines, suggesting this may represent a valuable tool to facilitate functional studies on recalcitrant herpesvirus genes. Finally, it is postulated that the suboptimal expression of native U51 may reflect a regulatory mechanism that controls viral gene expression.