As biomedical technology advances, the probability increases that evolution guided, constrained, or facilitated by scientists will be relevant to medicine. Of particular interest in this context is the increasing ability of investigators to engineer microbes to produce gene products of benefit to individuals in need of specific treatments or for the general maintenance of health. Applications of a more industrial nature are also readily conceivable.
There are different possible paths to the eventual goal of tailored microbial genomes. One approach is the de novo synthesis of whole bacterial genomes followed by transplantation into selected cells previously rendered genome-free (Gibson et al. Science, 2010). I have previously expressed doubt that this scheme is necessarily the most likely means to achieve the goal of engineering bacteria to express gene products and functions of our choosing (Greenspan, 2010). The alternative approaches I had in mind were based on the reasonable supposition that it would ultimately be easier and more efficient for most researchers to employ enhanced versions of already well-developed technologies based on mutation and selection of existing microbial strains.
Church and colleagues (Isaacs et al. Science, 2011) have now obliged by demonstrating the potential for advances in microbial genome engineering based on enhancements in current methods applied to existing bacterial strains and their genomes. More specifically, Isaacs et al. developed new strategies for introducing multiple mutations into the genomes of E. coli cells. First they employed what they call “multiplex automated genome engineering” (MAGE; Wang et al., 2009) to generate many mutations in parallel at precise locations across the E. coli genome. In this instance they replaced all 314 TAG stop codons with TAA stop codons in genomes for reasons to be explained below. These mutations were introduced, using oligonucleotides of 90 base pairs, in groups of ten (except in one case where only four were introduced) to create 32 separate E. coli strains permitting the investigators to determine if any particular allelic substitutions were problematic. Due care was taken to confirm all targeted codon replacements and to assess the extent of unintended secondary mutations.
The authors then used a newly developed method, hierarchical “conjugative assembly genome engineering” (CAGE) to combine up to 80 of these 314 mutations into single genomes. This exercise was ultimately successful, but it required a substantial number of steps and extensive quality control efforts. These developments offer the prospect of creating new strains of E. coli that permit complete reassignment of the TAG codon to a new use, such as the encoding of an unnatural amino acid, thereby substantially expanding the universe of possible biosynthetically-produced proteins. As the authors note, their “methods treat the chromosome as both an editable and an evolvable template, permitting the exploration of vast genetic landscapes.”
Also of interest are the comments of Church and colleagues on the methods preferred by Venter and his colleagues at the J. Craig Venter Institute (JCVI). They point out that so far the only templates used for de novo synthesis of bacterial genomes have been based directly on existing genomes with the introduction of at most minor variations in nucleotide sequence. I suggest that it remains to be demonstrated that it is more efficient to obtain microbial cells with desired biosynthetic or other functional properties through de novo genome design (as Venter and colleagues propose) than through generation of substantial genomic diversity followed by selection (as advocated by Church and colleagues). My intuition is that methods based on or related to those of Church and his associates will be adopted by the majority of investigators interested in generating microbes useful for medical and other applications.
Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY, Algire MA, Benders GA, Montague MG, Ma L, Moodie MM, Merryman C, Vashee S, Krishnakumar R, Assad-Garcia N, Andrews-Pfannkoch C, Denisova EA, Young L, Qi ZQ, Segall-Shapiro TH, Calvey CH, Parmar PP, Hutchison CA 3rd, Smith HO, Venter JC. Creation of a bacterial cell controlled by a chemically synthesized genome. Science. 2010 Jul 2;329(5987):52-6. Epub 2010 May 20. PubMed PMID: 20488990.
Greenspan, N.S. Genes that promote self-promotion. The Huffington Post, May 27, 2010. http://www.huffingtonpost.com/neil-s-greenspan/genes-that-promote-self-p_b_589710.html
Isaacs FJ, Carr PA, Wang HH, Lajoie MJ, Sterling B, Kraal L, Tolonen AC, Gianoulis TA, Goodman DB, Reppas NB, Emig CJ, Bang D, Hwang SJ, Jewett MC, Jacobson JM, Church GM. Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science. 2011 Jul 15;333(6040):348-53. PubMed PMID: 21764749.
Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, Church GM. Programming cells by multiplex genome engineering and accelerated evolution. Nature. 2009 Aug 13;460(7257):894-8. Epub 2009 Jul 26. PubMed PMID: 19633652.