Optimal encoding rules for synthetic genes: the need for a community effort

A paradigm shift is underway within the methodology of heterologous protein expression. Specifically, researchers are moving away from conventional techniques of cloning genes from cDNA libraries and moving toward the rational design and de novo synthesis of entire protein-coding sequences from pre-annealed oligonucleotides (Libertini and Di Donato, 1992; Gustafsson et al, 2004). It was the invention of polymerase chain reaction (PCR) that allowed efficient construction of synthetic genes. Since then, the steadily increasing accuracy and decreasing cost of oligonucleotide synthesis (now as low as $0.10 per base; Carlson, 2003; Carr et al, 2004; Kong et al, 2007, see Figure 1) has created a research environment in which gene synthesis offers three main advantages over molecular cloning: cost efficiency, scope and flexibility of redesign (Libertini and Di Donato, 1992). As a result, the emerging field of synthetic biology is highly motivated to improve this approach, as it seeks to expand the sophistication of human-engineered genetic architectures, leading ultimately to the synthesis of entire genomes (Yount et al, 2000; Smith et al, 2003). Current research into synthetic gene construction has focused largely on improving PCR-based methods. Areas under active investigation include the following: increasing the accuracy of gene products by reducing errors in oligonucleotide construction and PCR synthesis/amplification (Ciccarelli et al, 1991; Young and Dong, 2004), reducing the relatively high cost of post-synthesis sequencing (Young and Dong, 2004), increasing the length of genes that can be synthesized (Kodumal et al, 2004), developing microchip-based technology and/or microfluidic devices that allow for the simultaneous assembly of multiple genes (Tian et al, 2004; Zhou et al, 2004; Kong et al, 2007), and automating the whole pipeline from gene design to synthetic gene screening (Cox et al, 2007). All frontiers show signs of rapid improvement (e.g., Xiong et al, 2004; Engels, 2005; Wu et al, 2006a), therefore the current challenges for gene synthesis are essentially optimizations of existing concepts. In stark contrast, it appears that we have much left to learn when it comes to the conceptual design of gene sequences. A significant fraction of the biologically and commercially important genes that have been redesigned report little or no success in increasing protein expression (e.g., see Alexeyev and Winkler, 1999; Flick et al, 2004; Wu et al, 2004; Hillier et al, 2005). More surprising, some of these ‘improvements’ have led to a direct and observable reduction in protein production (Griswold et al, 2003). Even those that do report increased protein yield require careful scrutiny, because many have not controlled for altered mRNA levels in their system (e.g., Deng, 1997; Alexeyev and Winkler, 1999; Feng et al, 2000; Humphreys et al, 2000; Nalezkova et al, 2005). Thus, although excellent progress in the practice of gene synthesis enables experimental implementation of the technique, the scientific community remains far from a complete understanding of what constitutes a rational design strategy for a protein-coding gene. Instead, the very concept of a ‘translationally optimal codon’ has grown to incorporate dimensions of translational speed, translational accuracy and sustainability of yield that could vary from one experiment to another. Meanwhile, we have learned that a codon’s position within a coding sequence, its ‘neighborhood’ of other codons, its structural role within the mRNA sequence and the nature of the genomic system in which it is to be expressed can all influence the effects of ‘synonymous’ codon choices. Given that we can physically construct any gene, what rules define the appropriate sequence to manufacture? Here, we examine current progress and emerging challenges in both theory and practice, showing how this topic exemplifies the interdisciplinary challenges of 21st century biology.