Construction And Fermentation Of Recombinant Escherichia Coli Producing Trans-4-hydroxyproline

The proline4-hydroxylase from Dactylosporangium sp.strain catalyzes the hydroxylation of L-proline to4-hydroxyproline. To improve the expression level of proline4-hydroxylase and its conversion efficiency of L-proline in E. coli, the DNA sequence encoding proline4-hydroxylase was redesigned and synthesized based on the codon bias of E.coli. Comparing the native and adjusted proline4-hydroxylase genes, the Codon Adaptation Index (CAI) of the latter was increased from0.3992to0.9941. Furthermore, the GC content was reduced from73.62%to59.34%, close to the GC content of other high-expression genes in E. coli. In addition, the mRNA secondary structure and the free energy (AG) of mRNA folding was predicted by RNA-folding analysis software. The mRNA secondary structure near the AUG start codon was adjusted by changing synonymous codons. The modified secondary structure around AUG was relatively opened to ensure efficient translation of the mRNA. The codons encoding the272amino acids of the enzyme were futher optimized. Finally, compared with the wild-type,139nucleotides of the proline4-hydroxylase gene were changed, the GC content was decreased from73.62%to59.27%.The sequence of the optimized optimized proline-4-hydroxylase gene and a tryptophan tandem promoter was synthesized and inserted into pAMP to form pAMP-ptrp2-Hyp. In order to overexpress the proline4-hydroxylase gene and obtain a high yield of trans-4-hydroxyproline, the selection of host strain and prokaryotic expressing vector was conducted. Shaking-flask tests showed that, among all transformants, E. coli BL21(DE3)/pUC19-ptrp2-Hyp reached the highest whole-cell proline4-hydroxylase activity which was0.075±0.004U/mg.In shake flask, the proline4-hydroxylase was expressed in BL21(DE3)/pUC19-ptrp2-Hyp in tryptophan-deficient medium and convert L-proline to trans-hydroxyproline. In order to improve the production of trans-hydroxyproline, the fermentation culture medium and condition for recombinant BL21(DE3)/pUC19-ptrp2-Hyp were optimized by single factor analysis and orthogonal design. The optimal medium was composed as follows:glucose10g/L, glycerol5g/L, tryptone8g/L, ammonium sulfate5g/L, dipotassium phosphate1g/L, magnesium sulfate0.2g/L, ferrous sulfate3mM, calcium chloride0.0015%. The yield of trans-hydroxyproline reached to1396±21mg/L, which was2.77times of that under the initial fermentation conditions.During the4-hydroxyproline fermentation process, an appropriate feeding strategy is essential. For an enhanced4-hydroxyproline production by E.coli BL21(DE3)/pUC19-ptrp2-Hyp, the effect of fed-batch strategy on4-hydroxyproline production at fermentor level was investigated. Through constant glucose feeding approach with a flow speed at18g/h, a maximum4-hydroxyproline titer of42.5g/L with a high4-hydroxyproline productivity of0.966g/(L·h) was achieved.The consumption of the intracellular oxygen by the proline4-hydroxylase biocatalysis, which interferes with metabolic activity of the cell, is responsible for the low level of dissolved oxygen during fermentation. With an aim to increase the oxygen supply, the VHb was expressed in recombinant E. coli. The recombinant E. coli with VHb had a peak at419nm and a valley in436nm in the CO difference spectra. The trans-4-hydroxyproline production of the recombinant E. coli with VHb reached6.19±0.28g/L in shake flask, which was2.57times of the control group. Meanwhile, the OD600of recombinant E. coli reached9.26±0.42, which was1.56times of control group in64h.