Recombinant Expression And Functional Study Of Coenzyme Dependent Oxidoreductase (CHIE4) In Marine Streptomyces

Contrast with terrestrial Streptomyces,marine Streptomyces could produce diverse secondary metabolites,which arises from the more complex,higher salt,higher pressure,lower temperature,lower oxygen,lower nutrients and other marine extreme environmental factors and also enables the marine Streptomyces to be an important source of new bioactive substances.Streptomyces griseoaurantiacus M045 isolated from Jiaozhou Bay in China produces manumycin A,chinikomycins A and B and other antibiotics.Chinikomycins A is produced from the precursor manumycin A,which is catalyzed by the coenzyme dependent oxidoreductase CHIE4.Chinikomycins were shown to inhibit the occurrence of melanoma and breast cancer,and also have antimicrobial effects on crop pathogenic fungi.Therefore,chinikomycins has a good value on researching and application.In this study,we employed the multiple recombinant expression systems to study expression and purification the CHIE4.The CHIE4 was expressed as inclusion body in pET28 expression system,and the soluble CHIE4 was obtained by dialysis renaturation experiment,but activity test showed that the the catalytic activity of CHIE4 got lost.Using the pMAL-p5 x and pMAL-c5 x expression vectors,we obtained CHIE4 as soluble protein fused with maltose bindind protein tags.This fusion protein was purified with MBP affinity chromatography and subjected to the TEV digestion and Ni affinity chromatography to generate the recombinant CHIE4.The catalytic activity of CHIE4 was confirmed by the activity test.The result showed that the CHIE4 could not be successfully expressed using the eukaryotic expression vector pPIC9 K and Pichia Pastoris expression system..FAD-dependent oxidoreductase CHIE4 encoding gene has high content of GC,and CHIE4 is apt to form inclusion body in the process of recombinant expression in E.coli.This CHIE4 usually lose its activity after the dialysis and renaturation,which is a limition to the production and application of CHIE4.In this study,we successfully obtain the soluble and functional CHIE4 through the MBP fusion expression,TVE treatment and Ni affinity chromatography,which provides the theory and methodology for the development and the utilization of the chinikomycins.