The Improvement Of Trans-4-Hydroxy-L-proline Producing Escherichia Coli And Priliminary Research

Trans-4-Hydroxy-L-proline is widely applied in the pharmaceutical, chemical, food and cosmetic products. In this paper, the constructed trans-4-hydroxy-L-proline-producing E.coli BL21(DE3)/p UHT4 strain was employed as the original strain. In order to improve production efficiency and reduce production cost, strain improvement and medium optimization were carried out. This article mainly includes the following aspects:The plasmid p UHVT4 coexpressing proline-4-hydroxylase gene and Vitreoscilla hemoglobin gene(vgb gene) was constructed, on the basis of the vector p UHT4. The shake flask results showed that, after 24 h fermentation, the OD600 of E.coli BL21(DE3)/p UHVT4 culture and hydroxyproline yield are 10.25±0.37 and 2.18±0.11 g/L, respectively. Compared with E.coli BL21(DE3)/p UHT4, biomass and hydroxyproline yield were increased by 47% and 29%, respectively.The corn steep liquor was used to replaced tryptone in GT medium. Single factor and orthogonal experiments were carried out to obtain the most optimal medium compositions: glucose 8 g/L, glycerol 10 g/L, corn steep liquor 8 g/L, ammonium sulfate 13 g/L, potassium hydrogen phosphate 1.5 g/L, sodium chloride 2 g/L, magnesium sulfate 0.3 g/L, calcium chloride 0.015 g/L, ferrous sulfate 4 m M. The optimized medium was named GC medium. After cultured 24 h in GC medium, the hydroxyproline yield of E.coli BL21(DE3)/p UHVT4 reached 1.27±0.05 g/L, increased by 41%, compared with the unoptimized medium.In order to interrupt the degradation pathway of proline in E.coli during the biosynthesis process of hydroxyproline, the Red/ET homologous recombination system was employed to knockout the put A genes of E.coli BL21(DE3) and W3110. The shake flask culture showed that, the put A gene mutants were not able to degrade proline.The influence of put A gene deletion on proline transformation was studied in GT and GC media. The shake flask fermentation results showed that, in terms of put A gene mutants, all of the consumed proline was transformed to hydroxyproline, the effective transformation rate of proline was 100%. While the value was only 52%-65% in wild types. The effect of vgb gene on hydroxyproline in put A gene mutants was also tested. The results indicated that, the presence of vgb gene significantly improved the hydroxyproline yield. And all the experiments proved that, E.coli W3110 is not a suitable strain for hydroxyproline production. Finally, the culture of E.coli BL21(DE3)Δ put A/p UHVT4 in a 7 L fermenter was performed, using GT and GC media, respectively. The results indicated that, the feed rate of glucose in 1.6 g/(L?h) was not appropriate for GT medium, but was suitable for GC medium, after cultured 80 h in GC medium, the hydroxyproline yield reached 45.23 g/L.