Construction Of Recombinanted Escherichia Coli And Optimization Of Shake-Flask Fermentation Process Which Biosynthesize Trans-4-hydroxyproline

The trans-4-hyrixyproline is a chiral amino acid which used as an important raw material in a wide range of applications.This paper constructed a recombinant E.coli which can efficient expressed the trans-4-proline hydroxylase gene. The recombinanted strain E.coli DH5a/pUC-19-pH was further optimized with shake flask fermentation process. The main result are as follows:1, Opimize the coding region sequence:Using JCAT software to optimized the coding area sequence of trans-4-proline hydroxylase gene. We changed 141 bases (Replaced 138 synonymous codons) and the CAI value was optimized from 0.3965 to 0.9277; the ratio of the C/G was optimized from 73.6263% to 59.7069% which improved the extending efficiency of the mRNA transcription.2, Opimize the non-coding region sequence:We added the g10-L translational enhance sequence in the front of the RBS sequence to increase the expression quantity of exogenous gene. By reconstructured the RBS sequence,we made it contained a powerful SD sequence which included an AAGGA core. This step made the mRNA combined with procaryotic organism ribosome with high-efficiency. Meanwhile,we placed the T7 g10-L sequence in SD sequence upstream and improved the expression level of the down stream coding gene.To enhanced the transcritption initiating efficiency of the mRNA,we used the RNA structure software to optimized the secondary structure of the translation initiation region and the free energy ?G was raised form-12.8 to-7.6.3, Using tryptophan promoter and simplify it:To protect the stability of gene sequence, we added TrpLABCDE transcription termination sequence which date from PET carrier in tail end of trans-4-proline hydroxylase gene. In order to facilitate subsequent experiment, we added the Sal I restriction enzyme cutting site in the 5'end of express box and Nde I in the 3'end;Using t-vector plasmid pUC-19 to constructed recombinant bacteria DH5?/pUC-19-pH,optimized the host bacteria by compared with E.coli JM109,C43,BL21 and XL-1, compared pCDFDuet-1,pACYCDuet-1,pETDuet-l,pRSFDuet-1,pCOLAduet-1 to optimized the vector plasmid,the final recombinanted E.coli strain DH5?/pUC-19-pH with the highest specific enzyme activity of 0.0108 U/mg.4, Measured growth and fermentation curve of DH5a/pUC-19-pH,optimized single factor of shaking flask fermentation,the optimal values were as follows:Temperature is 37?,Initial L-proline concentration 200 mmol/L,Ferrous sulfate 2 mmol/L,Magnesium sulfate is 0.02 g/L,Ammonium sulfate 10 g/L,Tryptone 8 g/L,C/N ratiois 1. Medium componentas follows:Glucose 20 g/L,Tryptone 8 g/L,L-proline 200 mmol/L,Ferrous sulfate 2 mmol/L,Magnesium sulfate 0.2 g/L, Ammonium sulfate 10 g/L, Dipotassium phosphate 1 g/L,Sodium chloride 2 g/L,Citric acid 2 g/L,Calcium chloride 0.015 g/L.Specific enzyme activityafter 16 h fermentation achieved to 0.301 U/mg,which increased 45.4% than recent report.