Enhancing 1,3-propanediol Biosynthesis By Multi-strategy Modifications On Klebsiella Pneumoniae

1,3-Propanediol(1,3-PDO),an important three-carbon compound and industrial chemical widely used for fibres,medicines and cosmetics,especially as monomer of a new polyester fiber polytrimethylene terephthalate(PTT),possesses huge market potentiality.Many researches showed that K.pneumoniae was an excellent cell factory for the production of 1,3-PDO.However,byptoducts accumulation and reducing power supply were limitations of 1,3-PDO production.A new strategy by abolishing byproduct synthesis pathways,redepressing the TCA cycle activity,and realizing co-fermentation with glycerol and glucose was applied to modify the producer and enhance 1,3-PDO biosynthesis.To decrease byproducts during the 1,3-PDO production,the poxB,pta-ack,ldhA,budR and budB genes,which were responsible for acetate,lactate,2,3-butanediol synthesis,were disrupted,respectively.The corresponding mutant strains K.pneumoniae W001,K.pneumoniae W002,K.pneumoniae W003,K.pneumoniae W004,K.pneumoniae W005 were then constructed.Fermentation results showed that the recombinant strains deficient of bypathways achieved decreased acetate,lactate,2,3-butanediol accumulations.Compared with the control strain,the 1,3-PDO titers of K.pneumoniae W001 and K.pneumoniae W003 increased to 18.90 g·L-1 and 20.17 g·L-1,the molar conversion ratio was 0.59 mol·mol-1,0.63 mol·mol-1,respectively,increased by 10% and 17%,respectively.To further decrease the byproduct accumulations,the pox B,pta-ack,ldhA genes were deleted simultaneously.The corresponding mutant K.pneumoniae W006 showed better performance and the 1,3-PDO production was up to 20.91 g·L-1.To derepress the NADH shortage for 1,3-PDO production,which resulted from the limited TCA cycle under micro-aerobic condition,the arcA gene regulating the TCA cycle activity was removed from K.pneumoniae W006.qRT-PCR was performed,and the results showed that the key genes gltA,icd,sucC,sudC involved in the TCA cycle from mutant strain K.pneumoniae W007 were all up-regulated 3-folds,the key genes cydA and cydD of Electron Transfer Chains were up-regulated 2.39 and 3.03-folds.These results demonstrated that the TCA cycle activity was enhanced,leading to an increased cellular NADH/NAD+ ratio by 54%.Flask fermentation results also showed that 1,3-PDO production increased by 8%,up to 22.49 g·L-1 under microaerobic condition.To relieve the negative effects of glucose metabolism on glycerol usage,and improve the 1,3-PDO production,the ptsG,crr genes of the phosphate transferase system from the K.pneumoniae were deleted,respectively.The corresponding PTS-mutant strains were constructed,and named K.pneumoniae W008,K.pneumoniae W009,respectively.Flask fermentation results showed that only K.pneumoniae W009 could enhance 1,3-PDO production,increased by 36%.Next,the crr gene was disrupted from K.pneumoniae W007 and K.pneumoniae W010 was constructed.K.pneumoniae W010 relieved the negative effects of glucose metabolism on glycerol usage,and the 1,3-PDO production increased by 6%,up to 23.82 g·L-1,which was a high level as reported.The glycerol conversion ratio also reached 0.73 mol·mol-1,increased by 5%.These results demonstrated that the co-fermentation of glucose and glycerol by the PTS mutant strain constructed here was realized and more glycerol was shifted for 1,3-PDO production,which provided a new method for the 1,3-PDO production.