Metabolic Pathway Modification Of Strains Producing Trans-4-Hydroxyproline

Trans-4-Hydroxyproline?P4H?is widely used in pharmaceutical,chemical synthesis,food and cosmetic fields,so the P4 H production has attracted extensive attention at home and abroad.Though microbial method has been used for industrial production of P4 H abroad,the research is very rare in China.In recent years,our research group has successfully constructed strains to produce P4 H with no exogenous L-proline addition,but the yield is low.This thesis attempted to adopt some strategies,i.e.,multigene co-expression,gene knock-out,fermentation optimization and fed-batch fermentation,to improve the yield of P4 H.The recombinant plasmid pUC19-proB2A-ptrp2-hyp?pUC19-BH?containing a mutant gene proB2 A that can alleviate the feedback inhibition of L-proline was constructed,so it helped lead the strain to produce P4 H without extra L-proline addition.By using the Red homologous recombination system,the acetyl glutamic acid kinase gene?argB?of Escherichia coli JM109 was knocked out and the arginine metabolic pathway was cut off.The plasmid pUC19-BH was transformed into E.coli JM109?argB to construct a recombinant strain E.coli JM109?argB/pUC19-BH.The yield of E.coli JM109?argB/pUC19-BH was about 918.7 mg·L^-1,which was higher than that of the original strain.The fermentation was optimized at a shake-flask level.The optimized fermentation medium was as follows: 10 g·L^-1 maltose,5 g·L^-1 glycerol,22 g·L^-1 trypsin,4 g·L^-1 K2HPO4,5 mmol·L^-1 FeSO4,1.7 g·L^-1 MgSO4,3 g·L^-1 NaCl and 0.005 g·L^-1 CaCl2.The fermentation conditions were optimized as below: inoculum size 4%?v/v?,initial pH8.5,culture temperature 30°C.After 24 h of the fermentationg,the yield of P4 H achieved at 1602.8 mg·L^-1,which was about 1.7 times of that before optimization.During the fermentation,a positive correlation between P4 H production and bacterial cell biomass was found.In order to facilitate the dissolved oxygen in the fermentation process and improve the cell growth,the recombinant vector pUC19-proB2A-ptrp2-hyp-VHB?pUC19-BHV?containing the vitrescilla hemoglobin gene VHB was constructed.At shake flask fermentation level,the P4 H production by E.coli JM109?argB/pUC19-BHV increased slightly as compared to that with no VHB insertion.To further compare the difference of these two strains with or without gene VHB at growth and metabolism,the incubation was carried out in a 7 L fermenter.The maximum yield of P4 H by the recombinant strains E.coli JM109?argB/pUC19-BH and E.coli JM109?argB/pUC19-BHV was 15.9 g·L^-1 at 60 h and 18.4 g·L^-1 at 56 h,respectively,indicating of an active role of the gene VHB introduction in the P4 H production recombinant.