Construction of Recombinant Escherichia coli Strains Producing cis-3-Hydroxyproline and Preliminary Optimization of the Fermentation Conditions

Abstract:

The cis-3-hydroxy-L-proline can be used to synthesize many anticancer drugs with important commercial value. At present，cis-3-hydroxyproline is produced with IPTG-inducible recombinants in two steps. The subject of this study is to construct a recombinant with high proline-3-hydroxylase activity and generate cis-3-hydroxyproline without adding IPTG which is expensive and toxic .We constructed the recombinant plasmid pES-Ptrp2-P3H and transformed E. coli BL21（DE3） successfully. Compared with the original gene sequence of proline 3-hydroxylase，168 nucleotides were changed and the GC percentage was reduced from 64.83 % to 49.31%. The preliminarily optimized medium was 1 g/dL glucose，0.125 g/dL glycerinum，1.6 g/dL tryptone，0.5 g/dL（NH4）2SO4，0.1 g/dL K2HPO4，0.2 g/dL NaCl，1 mmol/L FeSO4，0.5 g/dL MgSO4，0.015 g/dL CaCl2，10 g/L L-proline，and pH valve at 7.5. At shaking flask level，cis-3-hydroxyproline was accumulated to about 0.8 g/L in 24 h，which was one times higher than before. It provides the basis for the industrialization of cis-3-hydroxyproline bioproduction .