Synonymous codon usage pattern in model legume Medicago truncatula

Synonymous codon usage pattern presumably reflects gene expression optimization as a result of molecular evolution. Though much attention has been paid to various model organisms ranging from prokaryotes to eukaryotes, codon usage has yet been extensively investigated for model legume Medicago truncatula. In present study, 39 531 available coding sequences （CDSs） from M. truncatula were examined for codon usage bias （CUB）. Based on analyses including neutrality plots, effective number of codons plots, and correlations between optimal codons frequency and codon adaptation index, we conclude that natural selection is a major driving force in M. truncatula CUB. We have identified 30 optimal codons encoding 18 amino acids based on relative synonymous codon usage. These optimal codons characteristically end with A or T, except for AGG and TTG encoding arginine and leucine respectively. Optimal codon usage is positively correlated with the GC content at three nucleotide positions of codons and the GC content of CDSs. The abundance of expressed sequence tag is a proxy for gene expression intensity in the legume, but has no relatedness with either CDS length or GC content. Collectively, we unravel the synonymous codon usage pattern in M. truncatula, which may serve as the valuable information on genetic engineering of the model legume and forage crop.

The codon-optimized capsid gene of duck circovirus can be highly expressed in yeast and self-assemble into virus-like particles

The capsid （Cap） protein, which is the only structural protein of duck circovirus （DuCV）, is the most important antigen for the development of vaccines against DuCV and the virus＇s serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCVCap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid （Opt-Cap） gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pas- toris GSl15. The strains that displayed the phenotype of Mut~ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% （v/v） methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What＇s more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles （VLPs）. These VLPs, with a diameter of 15-20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the resultsdemonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.

Characterization and comparison of chloroplast genomes from two sympatric Hippophae species(Elaeagnaceae)

The genus Hippophae includes deciduous shrubs or small trees,which provide many ecological,economic,and social benefi ts.We assembled and annotated the chloroplast genomes of sympatric Hippophae gyantsensis(Rousi)Lian and Hippophae rhamnoides Linn subsp.yunnanensis Rousi and comparatively analyzed their sequences.The fulllength chloroplast genomes of H.gyantsensis and H.rhamnoides subsp.yunnanensis were 155,260 and 156,415 bp,respectively;both featured a quadripartite structure with two copies of a large inverted repeat(IR)separated by small(SSC)and large(LSC)single-copy regions.Each Hippophae chloroplast genome contained 131 genes,comprising 85 protein-coding,8 ribosomal RNA,and 38 transfer RNA genes.Of 1302 nucleotide substitutions found between these twogenomes,824(63.29%)occurred in the intergenic region or intron sequences,and 478(36.71%)were located in the coding sequences.The SSC region had the highest mutation rate,followed by the LSC region and IR regions.Among the protein-coding genes,three had a ratio of nonsynonymous to synonymous substitutions(Ka/Ks)>1 yet none were signifi-cant,and 66 had Ka/Ks<1,of which 46 were signifi cant.We found 20 and 16 optimal codons,most of which ended with A or U,for chloroplast protein-coding genes of H.gyantsensis and H.rhamnoides subsp.yunnanensis,respectively.Phylogenetic analysis of fi ve available whole chloroplast genome sequences in the family Elaeagnaceae—using one Ziziphus jujube sequence as the outgroup—revealed that all fi ve plant species formed a monophyletic clade with two subclades:one subclade consisted of three Hippophae species,while the other was formed by two Elaeagnus species,supported by 100%bootstrap values.Together,these results suggest the chloroplast genomes among Hippophae species are conserved,both in structure and gene composition,due to general purifying selection;like many other plants,a signifi cant AT preference was discerned for most proteincoding genes in the Hippophae chloroplast genome.This study provides a valuable reference tool for future research on the general characteristics and evolution of chloroplast genomes in the genus Hippophae.

Prokaryotic expression of goldfish Tgf2 transposase with optimal codons and its enzyme activity

Tgf2 transposase(Tgf2-TPase),a hAT transposase from goldfish,plays an important role in fish transgenic applications.Previously,the production of the recombinant Tgf2-TPase protein required rigorous fermentation at low temperatures(22℃)and early log phase induction(OD600=0.3–0.4)in Rosetta 1(DE3)Escherichia coli lines.In order to better express the Tgf2-TPase and detect its enzyme activity,83 rare codons in Tgf2-TPase were optimized and designated Tgf2-TPase^(83).The expression results showed that the soluble recombinant Tgf2-TPase83 was highly expressed at 30℃ and was inducible at an OD600 of 0.5–0.6 in the same prokaryotic expression system.After purification by affinity chromatography,Tgf2-TPase83 with codon optimization had higher enzyme activity than the Tgf2-TPase control.Comparison of different preservation methods(freezedrying at−80℃,storage in 20%-glycerol,8%-sucrose,4%-mannitol),revealed storage of Tgf2-TPase^(83) in glycerol helped to preserve its DNase digestion activity.Furthermore,size exclusion chromatography suggested that the purified Tgf2-TPase^(83) could recognize and bind to DNA probes containing a terminal inverted repeat(TIR)and a subterminal repeat(STR)sequence of the Tgf2 transposon.Overall,the results showed that optimizing the 83 codons of Tgf2 transposase can simplify the fermentation process and improve the enzyme activity.We propose that the production of the Tgf2-Tpase83 protein in a soluble and active form could provide an alternative tool for genetic modification of fish.

Efficient hepatic delivery and protein expression enabled by optimized mRNA and ionizable lipid nanoparticle

mRNA is a novel class of therapeutic modality that holds great promise in vaccination,protein replacement therapy,cancer immunotherapy,immune cell engineering etc.However,optimization of mRNA molecules and efficient in vivo delivery are quite important but challenging for its broad application.Here we present an ionizable lipid nanoparticle(iLNP)based on iBL0713 lipid for in vitro and in vivo expression of desired proteins using codon-optimized mRNAs.mRNAs encoding luciferase or erythropoietin(EPO)were prepared by in vitro transcription and formulated with proposed iLNP,to form iLP171/mRNA formulations.It was revealed that both luciferase and EPO proteins were successfully expressed by human hepatocellular carcinoma cells and hepatocytes.The maximum amount of protein expression was found at 6 h post-administration.The expression efficiency of EPO with codon-optimized mRNA was significantly higher than that of unoptimized mRNA.Moreover,no toxicity or immunogenicity was observed for these mRNA formulations.Therefore,our study provides a useful and promising platform for mRNA therapeutic development.

Heterologous expression of bovine lactoferrin C‑lobe in Bacillus subtilis and comparison of its antibacterial activity with N‑lobe

The natural concentration of bovine lactoferrin C-lobe is low and its separation by proteolytic enzyme digestion is difcult.Here,we expressed the codon-optimized fragment of C-lobe on plasmid pMA0911 with the P_(veg) promoter in Bacillus subtilis 168 at 20℃.The yield was 7.5 mg/L,and 90.6%purity was achieved using ammonium sulfate precipitation,Ni–NTA and molecular exclusion.The C-lobe at 10 mg/mL completely inhibited cell growth of Escherichia coli JM109(DE3)and Pseudomonas aeruginosa CGMCC 1.6740,and 48.4%of growth of Staphylococcus aureus CGMCC 1.282,the result is similar to that of 200 ng/mL N-lobe.The minimum inhibitory concentrations of C-lobe were 4,8 and 16 mg/mL,while those of N-lobe were 128,256 and 512μg/mL for E.coli,P.aeruginosa and S.aureus,respectively.This is the frst report on bovine lactoferrin C-lobe expression and the comparative resistance of the recombinant N-and C-lobes in a food-safe strain of B.subtilis.Our fndings ofer the potential to study the structure–function relationship of the N-and C-lobes recombinantly produced in the same host.

Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity

Surface display is effectively utilized to construct a whole-cell biocatalyst.Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast.Here,the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae,and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor,recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed.Compared with the wild-type ROL-displaying yeast,the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate.To our knowledge,this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction.Consequently,the yeast whole-cell ROL biocatalyst was constructed with high activity.The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C.Furthermore,this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.