Abstract:Abstract: [Objective] To improve the tolerance of main metabolites, we used genome shuffling to achieve high 1,3-propanediol producing mutants. [Methods] Based on 96 deep-well palates containing prepared ended fed-batch broth as an efficient selection method, genome shuffling has been applied in strain improvement. [Results] Five high producers were obtained after genome shuffling (LSG1, LSG2, LSG4, LSG5 and LSG6). During batch fermentation (3 L), the 1,3-propanediol production of the five mutants were improved 17.0%,19.0%,12.9%,23.9% and18.0%, compared with the parent strain; the conservations from glycerol were improved 17.7%,20.0%,13.3%,24.4% and 17.7%. [Conclusion] Genome shuffling was an efficient approach for strain improvement, and 96 deep-well palates containing fed-batch broth has been demonstrated as an efficient selection approach.