Genome shuffling

Zhang Y-X, Perry K, Vinci VA, Powell K, Stemmer WPC, del Cardayre SB. (2(X)2) Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature 415 644-646. 

Two major advances describe the construction of mutants with an apparently significantly higher probability of improved phenotype. The method involves whole-genome shuffling - with protoplast fusion - of a small number of parental strains... 

A recently introduced technique that also targets the phenotype is whole genome shuffling. This latter approach involves the amplification of the genetic diversity within a population through genetic recombination [19, 20]. Next, similar to the traditional procedure, the newly created library is screened for mutants with the desired properties. Its main advantage is that improvement is much faster than is possible with classical mutation and selection as described above. 

Acid-tolerant lactic acid bacteria would solve this latter problem and have indeed been obtained from screening [44] and via whole genome shuffling [45]. Alternatively, a lactate dehydrogenase gene could be inserted in a yeast, such as S. cerevisiae. These grow well at low pH and efficiently channel carbon into the glycolysis pathway under anaerobic conditions [46, 47]. Such constructs were indeed found to produce up to 55 g L-1 of lactic acid at pH 3.6 [46]. 

One of the drawbacks in the current commercial fermentation process is that the predominant form of the product is the deprotonated lactate rather than lactic acid, requiring more expensive and wasteful product purification steps. This is because the Lactobacillus fermentation operates at a minimum pH of 5.0-5.5 which is above the pA a of lactic acid (3.87). To overcome this limitation, a powerful strain improvement method, genome shuffling, was used to improve the acid tolerance of a poorly characterized industrial strain of LactobacillusA population of strains with subtle improvement in pH tolerance was isolated using classical strain improvement methods such as chemostats, and were then shuffled by recursive pool-wise protoplast fusion to create mutant strains that grow at substantially lower pH than does the wild-type strain. 

Genome shuffling of Lactobacillus for improved acid tolerance. Nat. Biotechnol. 2002, 20 (7), 707-712. 

Otte B, Grunwaldt E, Mahmoud O, Jennewein S. (2009). Genome shuffling in Clostridium diolis DSM 15410 for improved 1,3-propanediol production. Appl Environ Microbiol, 75, 7610-7616. 

Wang M, Liu S, li Y, Xu R, Lu C, Shen Y (2010) Protoplast mutation and genome shuffling induce the endophytic fungus Tubercularia sp. TF5 to produce new compounds. Curr Microbiol 61 254-260. doi 10.1007/s00284-010-9604-7... 

John, R., Gangadharan, D., and Nampoothiri, K. (2008) Genome shuffling of Lactobacillus delbrueddi mutant and Bacillus amyloliquefaciens through protoplasmic fusion for L-lactic acid production from starchy wastes. Bioresource... 

Petri R, Schmidt-Dannert C (2004) Dealing with complexity evolutionary engineering and genome shuffling. CurrOpin Biotechnol 15 298-304... 

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