Instead of the NNK codon for saturation mutagenesis, one can use the NNS codon. Is one a better choice? Maybe. K represents G or T, while S represents G or C. The sixteen codons that contain G in the last position are identical in both choices of degenerate codon. The remaining sixteen codons are NNT for NNK degenerate codon, but are NNC for NNS degenerate codon. These sixteen codons are synonymous, so they encode the same amino acids. They do differ at the level of nucleotides - the NNS-containing primer has a higher GC content. While the NNK degenerate primer contains five rare codons, it includes synonymous non-rare codons. In contrast, the NNS degenerate primer lacks a synonymous non-rare codon for arginine, so variants with this amino acid substitution may express poorly. (This potential problem only occurs in yeast; no problems predicted for expression in E. coli.) The melting temperature of the NNS degenerate primer will be slightly higher than the NNK degenerate primer, which, depending on the partner primer, may be an advantage. Choosing between NNK and NNS may also minimize formation of primer hairpins or primer dimers.
The current cycle of carbon atoms in fuels is too slow to be sustainable. Fuels and chemicals come from petroleum. Fuels are burned releasing the carbon as carbon dioxide. Plastics are used, then landfilled. To reuse these carbon atoms, the carbon dioxide and landfill contents must be converted back to plants, but currently this is too slow. Too much carbon dioxide is released and the conversion of landfill to plant nutrients is too slow. The cycle is not turning. Even if these problems could be solved, an even bigger one remains. Converting plants to petroleum takes eons.
The broken carbon cycle.
One solution is new biocatalysis reactions. One set is biofuels and biorefinery processes to convert plants directly to fuels and chemicals. The second set is biodegradation of plastics and other chemicals to plant nutrients. Inventing these reactions using synthetic biology and protein engineering is an important goal of biocatalysis today.
￼A sustainable carbon cycle needs new biocatalysis processes.
The extent of changes made in the course of a protein improvement has increased dramatically in the past decade. In the early 2000's one or two mutations were typical, while by 2010, 30-40 amino acid substitutions are not unusual. For example, directed evolution of halohydrin dehalogenase for manufacture of the atorvastatin (Lipitor) side chain changed at least 35 of the 254 amino acids (>14%; Fox et al. 2007) and directed evolution of the transaminase for sitagliptin manufacture changed 27 of the 330 amino acids (8.2%; Savile et al. 2010). Similarly, computational design of a retro aldolase required 8 or 12 amino acid substitutions (4-6%) in the starting enzyme, a 197-aa xylanase (Jiang et al., 2008).
Amino acid sequences of proteins in mice and human typically differ by 13% (Waterston et al., 2002), so this laboratory evolution of enzymes is equivalent to compressing the 75 million-year-evolution from an early mammal to today's mice and humans into several months of laboratory work.
Fox, R. J. et al. (2007), Improving catalytic function by ProSAR-driven enzyme evolution, Nature Biotechnol., 25, 338-344.
Jiang, L. et al. (2008), De novo computational design of retro-aldol enzymes, Science, 319, 1387-1391.
Savile, C. K. et al. (2010), Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture, Science, 329, 305-309.
Waterston, R. H. et al. (2002) Initial sequencing and comparative analysis of the mouse genome. Nature, 420, 520-62.