Translation Engineering and Synthetic Biology
David A. Roth, Liza S.Z. Larsen and G. Wesley Hatfield
We have developed a toolbox approach for protein engineering to enhance protein expression yield and functionality by manipulating ribosomal “pause” signals correlated with over-represented codon pairs contained in the open reading frame of genes. CODA’s Translation Engineering algorithms simultaneously optimize each gene design for both organism-specific codon usage and for organism-specific codon pair usage. To further implement Translation Engineering, CODA has developed propriety gene software, SpeedPlotTM, a design tool that predicts and graphically displays the positions of translational pause sites in genes expressed in native and heterologous biological hosts. For high protein expression levels, SpeedPlot information is used to design Hot-rodTM genes with all predicted pause signals removed for maximal translation elongation rates. For improved protein function, Speedplot information is used to rationally design gene sets using Hot-rod genes as parental templates, from which directed variant genes are constructed to contain specified combinations of pause signals (Planned Pause Gene SetsTM). Each defined gene set can be tested in parallel for optimal expressors that encode proteins with premium activities. These Translation Engineering methods are enabled by CODA’s proprietary Computationally Optimized DNA Assembly (CODA) technology to assemble full-length genes by thermodynamic necessity. In combination, these technologies make it possible to deliberately alter the predicted translation kinetics patterns of genes without altering their amino acid sequences. In this chapter, we discuss the application of these tools for the design and assembly of synthetic genes optimized for expression and function in any biological host or cell-free protein synthesis system.