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Breeding of 1, 3-propanediol-overproducing Strain by Genome Shuffling

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Tutor: MengQingXiong
School: Kunming University of Science and Technology
Course: Biochemical Engineering
Keywords: 1,3-propanediol,Klebsiella pneumoniae,genome shuffling,protoplast mutagesis
CLC: TQ923
Type: Master's thesis
Year:  2011
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1,3-propanediol is acknowledged as one of six new petrochemical products currently in the world. Its main function is as an important monomer to synthesize a new type of polyester, polyether, polyurethane. Compared with chemical synthesis, microbial production of 1,3-propanediol (1,3-PD) have many obvious advantages, and thus become the foucuses of research. Klebsiella pneumoniae used as a srain for producing 1,3-PD has attracted increasing attention due to that high glycerol conversity, high 1,3-PD concentration and productivity during the fermentation process. Aims for improved the tolerance of main metabolites and substrats, protoplast UV mutagenesis and genome shuffling were applied for achieved high 1,3-propanediol producing mutants. The main contents of this study as follows:1) This study used Klebsiella pneumoniae as original strain and examined the process of protoplast prepration and protoplast mutagenesis. We applied the millipore creatively in filtering the original strain in order to simplify the enzymolysis process and enhance the regeneration rate and formation rate during the process of protoplast preparation. After protoplast mutagenesis, aimed at the tolerance of high concentrations of glycerol and 1,3-propanediol and high productivity of acid, we have gained three high yield mutants Kp-1, Kp-4 and Kp-5. In fed-batch fermentation, the volumetric productivity of above strains were 70.24 g/L,65.21 g/L and 75.51 g/L, respectively, increased by 25.92%,16.91% and 35.37% compared with wild type strain.2) To improve the tolerance of main metabolites, this study attempt to use genome shuffling for achieved high 1,3-propanediol producing mutants. 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.5 high producers were achieved after genome shuffling (LSG1, LSG2, LSG4, LSG5, LSG6). During batch fermentation (3 L), the 1,3-propanediol productions of five mutants were improved 17.0%,19.0%,12.9%, 23.9%and18.0%, respectively, compared with parent strain; the conservations from glycerol were improved 17.7%,20.0%,13.3%,24.4%,17.7%, respectively. Genome shuffling was a novel and efficient approach of strain improvement, whereas 96 deep-well palates containing fed-batch broth has been demonstrated as an efficient selection approach.
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