Construction Of Recombinant Escherichia Coli Strains Producing Cis-3-Hydroxyproline And Optimization Of The Fermentation Conditions

The cis-3-hydroxy-L-proline(cis-3-hydroxyproline) can be used to synthesize many anticancer drugs with important commercial value. Since the concentration of cis-3-hydroxyproline is extremely rare in nature, researchers from all over the world are looking for a kind of economic and effective method to obtain a large amount of it. The proline-3-hydroxylase from Streptomycete sp. strain TH1 catalyzes the hydroxylation of L-proline to 3-hydroxyproline. The DNA sequence encoding proline-3-hydroxylase was redesigned and a recombinant Escherichia coli producing cis-3-hydroxyproline was constructed. The conversion efficiency of L-proline by the recombinant E. coli strain was improved through the optimization of fermentation condition.The codon bias of P3 H and E. coli gene varies widely. We redesigned P3 H with the strategy of synonymous substitution, secondary structure optimization and enzyme digestion site design. Compared with the original gene sequence of the proline 3-hydroxylase, 187 nucleotides were changed and the GC percentage was reduced from 64.83% to 49.31%. Besides, the Condon Adaptation Index(CAI) of the latter was increased from 0.331 to 0.998 which was close to the optimal value. The synthesized P3 H gene with adjusted gene sequese was integrated to the vector p ES.To construct a recombinant with a high proline-3-hydroxylase activity and the capability of generating cis-3-hydroxyproline without inducers, the tryptophan tandem promoter(Ptrp2) was synthesized and inserted into p ES-P3 H. We constructed the recombinant plasmid p ES-Ptrp2-P3 H and transformed E. coli BL21(DE3) successfully. In order to overexpress the proline-3-hydroxylase gene and obtain a high yield of cis-3-hydroxyproline, the selection of host strain and prokaryotic expressing vector was conducted. The whole cell enzyme activity of the recombinant E. coli BL21(DE3)/p ET21a-Ptrp2-P3 H was 722.807 U/g, the highest value among different recombinant E. coli strains constructed in this study.At shake flask level, cis-3-hydroxyproline was accumulated to about 0.4 g/L in 24 h by the BL21(DE3)/p ET21a-Ptrp2-P3 H. To further improve the production of cis-3-hydroxyproline, the fermentation culture medium and conditions for recombinant BL21(DE3)/p ET21a-Ptrp2-P3 H were optimized by single factor analysis and orthogonal design. The optimal medium was composed as follows: 1.5% glucose, 0.125% glycerol, 1.6% tryptone, 0.05% ammonium sulfate, 0.05% dipotassium phosphate, 0.2% sodium chloride, 0.5 m M ferrous sulfate, 0.5% magnesium sulfate, 0.015% calcium chloride, 0.1 m M L-ascorbic acid. The yield of cis-3-hydroxyproline reached to 0.8 g/L, which was 2 times of that under the initial fermentation conditions. It provided basis for the industrialization of cis-3-hydroxyproline bioproduction.In order to further reduce the costs of production cis-3-hydroxyproline, we introduced pro BA’ which is the key enzyme in L-proline metabolism at the molecular level. We constructed the recombinant E. coli JM109/p ET21a-Ptrp2-pro BA’-Ptrp2-P3 H as well as the recombinant E. coli with p ET21a-Ptrp2-P3 H and p ET28a-Ptrp2-pro BA’ successfully. The recombinant E. coli can convert glucose to cis-3-hydroxyproline. At shake flask level, the cis-3-hydroxyproline productivity of the JM109/p ET21a-Ptrp2-pro BA’-Ptrp2-P3 H and the double-plasmid recombinant E. coli were 100 and 60 mg/L, respectively.