Computational codon optimization of synthetic gene for protein expression

Abstract

BackgroundThe construction of customized nucleic acid sequences allows us to have greater flexibility in gene design for recombinant protein expression. Among the various parameters considered for such DNA sequence design, individual codon usage (ICU) has been implicated as one of the most crucial factors affecting mRNA translational efficiency. However, previous works have also reported the significant influence of codon pair usage, also known as codon context (CC), on the level of protein expression.ResultsIn this study, we have developed novel computational procedures for evaluating the relative importance of optimizing ICU and CC for enhancing protein expression. By formulating appropriate mathematical expressions to quantify the ICU and CC fitness of a coding sequence, optimization procedures based on genetic algorithm were employed to maximize its ICU and/or CC fitness. Surprisingly, the in silico validation of the resultant optimized DNA sequences for Escherichia coli, Lactococcus lactis, Pichia pastoris and Saccharomyces cerevisiae suggests that CC is a more relevant design criterion than the commonly considered ICU.ConclusionsThe proposed CC optimization framework can complement and enhance the capabilities of current gene design tools, with potential applications to heterologous protein production and even vaccine development in synthetic biotechnology.

Optimizing assembly and production of native bispecific antibodies by codon de-optimization

“…[18][19][20] A variety of software are available for synthetic gene design, as well as computational procedures to evaluate the effect of codon optimization. [21][22][23][24][25][26] Using such strategies, increases in protein yields have been reported. [27][28][29][30][31] However, although the final protein encoded by an optimized DNA sequence has the same amino acid composition, the modifications that are introduced might alter proper protein folding, solubility and activity by modifying the dynamics of protein translation.…”

Gene and Protein Sequence Optimization for High-Level Production of Fully Active and Aglycosylated Lysostaphin in Pichia pastoris

“…Gene sequence optimization has been a widely effective measure to increase heterologous protein expression levels in P. pastoris (32)(33)(34)(35)(36), but the mechanism behind this strategy has seldom been studied. The results presented herein are consistent with prior insights that premature transcription termination may result from short segments of severe nucleotide bias within some native genes (37)(38)(39).…”

Exploring codon context bias for synthetic gene design of a thermostable invertase in Escherichia coli

“…CV design was performed by a commercial vendor, considering several parameters including codon usage, codon context, RNA secondary structure, and codon-anticodon pair interactions according to their claim. The codon context distribution used for CC design was computed from the top 5% of highly expressed genes identified using microarray data [33]. To design the SS sequence, we first obtain the secondary structure annotation of E. coli proteins from PDB [36].…”

“…A principal component analysis plot of codon context shows the differences between the secondary structure regions. Subsequently, the target protein, invertase, with the protein shown and its secondary structure regions identified as obtained from the Protein Data Bank (PDB ID: 1UYP; http://www.rcsb.org/pdb/) [33,34], is optimized based on the codon context profile derived for each secondary structure from the hosts' genes. established as reference inputs to the algorithm [36].…”

“…For an average sized protein of 300 amino acids, the number of possible codon coding sequences is in the order of 10 100 [33]. Therefore, the genetic algorithm was employed to obtain a good feasible solution within an acceptable amount of time [34].…”