Analysis Of Factors Associated With Translation Efficiency And Application Research In Enhancing Protein Expression Levels

Protein synthesis,a fundamental molecular process in biological cells,requires considerable resources including energy and raw materials.In bacteria,proteins constitute60% to 80% of their dry weight,with protein synthesis consuming over 50% of total energy.Natural selection optimizes both translation efficiency and accuracy given this substantial energy investment.Studies reveal that codon usage bias(CUB)and amino acid composition(AACB)impact protein translation efficiency,and optimizing these factors can enhance protein yield.However,our understanding of the role of translation efficiency in protein synthesis remains limited,hindering the development of effective regulatory elements for efficient protein production.Drawing upon current research on translation efficiency and utilizing the genome-wide codon usage bias and the coadaptability of tRNA genes with amino acid composition,this thesis systematically investigates translation efficiency across organisms spanning diverse evolutionary lineages.This thesis encompasses the following four research objectives.Firstly,utilizing the measurements of Relative Synonymous Codon Usage(RSCU),we observed both similarities and differences in CUB between highly expressed genes and the overall genome in fast-growing bacteria.By integrating genome-wide CUB with AACB,we constructed a multiple linear regression model to predicting bacterial doubling time.This model fits the available minimal doubling time data well,achieving an adjusted R-squared value of 0.69.Compared to the most recent model for predicting bacterial generation time,ours enhances the adjusted coefficient of determination by 5 percentage points and exhibits a lower mean squared error,indicating a more precise estimation of bacterial growth rate through our integrated approach.Furthermore,a noteworthy negative correlation is observed between the tRNA gene and amino acid composition coadaptation index(TAAI)and bacterial doubling time.Secondly,plasmids have long served as models for genetic engineering vector construction,and a deeper understanding of plasmid translation efficiency could pave the way for novel approaches in expressing foreign proteins in genetic engineering.This thesis analyzed genomic data from over 30,000 symbiotic plasmids and more than 2,000bacteria-plasmid pairs.Our findings reveal that plasmid-encoded tRNA genes are not only correlated with the number of protein-coding genes carried by plasmids but also significantly associated with plasmid mobility types.While plasmids are conventionally believed not to carry essential genes,this thesis revealed that certain symbiotic plasmids do harbor essential translation-related genes,notably with 5.1% of plasmid genomes carring at least one tRNA gene.This suggests that hosts utilize plasmid-encoded translation elements to meet the demands of protein synthesis under varying environmental pressures.Furthermore,symbiotic plasmids exhibit significant similarities and distinct differences in CUB compared to host bacteria.Futhermore,a significant correlation exists between translation efficiency and the life history traits(LHTs)of mammals.We compiled a dataset for mammalian translation efficiency analysis by collected 150 high-quality mammalian genome sequences along with LHTs.After adjusting for phylogenetic relationships among mammals,TAAI shows significant correlations with generation time(PICs: r =-0.36,p < 0.001)and lifespan(PICs: r = 0.32,p < 0.001).Moreover,TAAI displays significant associations with protein translation efficiency(r = 0.16,p < 0.001)and the selection pressure indicator Ka/Ks(r=-0.10,p < 0.001).Constrained by natural selection,the co-adaptation between tRNA genes and amino acid composition can maximize translation efficiency and accuracy,thus meeting the demands for efficient protein synthesis to adapt to various environmental conditions.This analysis of translation selection and LHTs provides new insights into the relationship between mammalian growth-to-aging.Following the analysis of translation efficiency,this thesis constructed two genetically engineered strains,Seven Box and UNION,based on the theoretical framework of TAAI,using Escherichia coli BL21(DE3)as the host organism.The Seven Box strain integrates seven tRNA genes to enhance TAAI,while the UNION strain incorporates an additional six tRNA genes for rare codons.These engineered strains were evaluated using two exogenous protein genes,showing a significant increase in expression compared to the wild-type strain.This offers a straightforward approach to enhance exogenous protein expression in genetic engineering.In summary,this thesis employs regression analysis and other methods to investigate the factors associated with protein translation efficiency in bacteria,plasmids,and mammals,based on various features such as CUB and AACB.For bacteria,a multiple linear regression model combining genome-wide CUB and AACB accurately predicts bacterial growth rate.Compared to the current state-of-the-art bacterial doubling time models,this combined model demonstrates superior predictive performance.Regarding plasmids,the thesis reveals that some symbiotic plasmids carry essential components related to the "translation machinery",with 5.1% of plasmid genomes carrying at least one tRNA gene.Results from codon usage analysis also suggest that hosts utilize plasmidencoded translation elements to meet protein synthesis demands in different environments.As for mammals,the proposed translation efficiency metric TAAI shows significant correlations with individual average generation time and lifespan.The comprehensive analysis further indicates that protein co-adaptation balances the different requirements for translation efficiency and accuracy in organisms.Finally,guided by TAAI,we used genome editing techniques to obtain Escherichia coli BL21(DE3)genetically modified strains with some specific tRNA gene amplification.Compared to wild-type strains,the engineered strains significantly enhance the expression of exogenous genes.This research deepens the comprehensive understanding of the determinants and influencing factors of protein translation efficiency in living organisms and contributes to the industrial application of increased expression of exogenous genes.