For millennia, selective breeding, on the basis of biparental mating, has led to the successful improvement of plants and animals to meet societal needs. At a molecular level, DNA shuffling mimics, yet accelerates, evolutionary processes, and allows the breeding and improvement of individual genes and subgenomic DNA fragments. We describe here whole-genome shuffling; a process that combines the advantage of multi-parental crossing allowed by DNA shuffling with the recombination of entire genomes normally associated with conventional breeding. We show that recursive genomic recombination within a population of bacteria can efficiently generate combinatorial libraries of new strains. When applied to a population of phenotypically selected bacteria, many of these new strains show marked improvements in the selected phenotype. We demonstrate the use of this approach through the rapid improvement of tylosin production from Streptomyces fradiae. This approach has the potential to facilitate cell and metabolic engineering and provide a non-recombinant alternative to the rapid production of improved organisms.

Fermentation-based bioprocesses rely extensively on strain improvement for commercialization. Whole-cell biocatalysts are commonly limited by low tolerance of extreme process conditions such as temperature, pH, and solute concentration. Rational approaches to improving such complex phenotypes lack good models and are especially difficult to implement without genetic tools. Here we describe the use of genome shuffling to improve the acid tolerance of a poorly characterized industrial strain of Lactobacillus. We used classical strain-improvement methods to generate populations with subtle improvements in pH tolerance, and then shuffled these populations by recursive pool-wise protoplast fusion. We identified new shuffled lactobacilli that grow at substantially lower pH than does the wild-type strain on both liquid and solid media. In addition, we identified shuffled strains that produced threefold more lactic acid than the wild type at pH 4.0. Genome shuffling seems broadly useful for the rapid evolution of tolerance and other complex phenotypes in industrial microorganisms.

times (4, 12, 24, 48, 72, and 144 h). pH1N1 was distributed onto N95 coupons (3.8 to 4.2 cm 2 ) and extracted by a vortex-centrifugation-filtration process, and the ability of the remaining virus to replicate was quantified using an enzyme-linked immunosorbent assay (ELISA) to determine the log 10 concentration of the infectious virus per coupon. Overall, pH1N1 remained infectious for 6 days, with an approximately 1-log 10 loss of virus concentrations over this time period. Time and AH both affected virus survival. We found significantly higher (P < 0.01) reductions in virus concentrations at time points beyond 24 to 72 h (؊0.52-log 10 reduction) and 144 h (؊0.74) at AHs of 6.5 ؋ 10 5 mPa (؊0.53) and 14.6 ؋ 10 5 mPa (؊0.47). This research supports discarding respirators after close contact with a person with suspected or confirmed influenza infection due to the virus's demonstrated ability to persist and remain infectious.