| Indirubin,a bis-indole alkaloid,is the main active ingredient of Chinese traditional medicine "Danggui Longhui Wan".It is also a red isomer that accompanies the synthesis of the dye indigo.It can be used for the treatment of various diseases including chronic myeloid leukemia,however,indirubin has long been used as a by-product of indigo production,and its microbial production has not attracted much attention.In this paper,first,the recombinant E.coli engineering strain expressing flavin monooxygenase(fmo)was constructed by screening,so that it can produce indirubin with tryptophan as the substrate,and the yield was 2.3 mg/L.In order to further the production of indirubin,metabolic engineering was applied to change the pathway of engineering bacteria,and the yield of indirubin was increased to 8.7 mg/L.Afterwards,the enzyme fmo was randomly mutated by directed evolution,error-prone PCR technology to construct mutation libraries,by the effective screening mutant strains L70 I and S321 T were abtained,the maximum production of indirubin reached 29.1mg/L.The molecular docking calculation indicate that the mutation may change the regioselectivity for indole hydroxylation,which shift the preference for indole hydroxylation from C-3 to C-2,resulting in increased production of indirubin.Finally,after optimizing the cultivation conditions,the production of indirubin can reach 40.1mg/L after the 5 L tank scale-up culture.Modern microbial biosynthesis of high value-added compounds must not only focus on the improvement of yield,but also the regional selective transformation of engineered enzymes to meet the application needs has gradually become an important research goal.In this paper,the directed evolution of the fmo enzyme to change its regioselectivity not only provides new information for the enzyme,but also provides a possible method for improving the production of indirubin,which lays the foundation for the production and application of microorganism indirubin. |
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