ObjectivesTo improve the bioproductivity of secondary metabolites of marine derived Nocardiopsis flavescens CGMCC 4.5723 by enhancing its riboflavin supplement.ResultsThe NfRibA, type II guanosine triphosphate (GTP) cyclohydrolase (GCH II) of Nocardiopsis flavescens CGMCC 4.5723, was biochemically identified and showed that NfRibA could efficiently catalyze the first step of riboflavin biosynthesis to hydrolyze GTP into 2, 5-diamino-6-ribosylamino-4(3H)-pyrimidinedione 5-phosphate (DARPP) with K-m value of 160.11 +/- 26.81 mu M in vitro. The overexpression of NfribA could obviously increase riboflavin bioproduction to the titers of 0.41 +/- 0.19mg/l by comparing with the wild type counterpart. Consequently, this rise of riboflavin bioproduction did not disturb the expression of genes involved in marinacarboline A biosynthesis, but could significantly enhance its bioproduction with the titer of 5.5 +/- 0.17mg/l through comparing with wild type control.Conclusions Optimization of riboflavin supplement could be a new promising strategy in actinomycetic marinacarboline A exploitation.