Biosynthetic Mechanism Of Streptonigron And High Production Of 10'-Desmethoxystreptonigrin

Streptonigrin?STN?is a highly functionalized aminoquinone alkaloid produced by Streptomyces flocculus ATCC 13257.STN,lavendamycin,streptonigron,10?-desmethoxylstreptonigrin,and related congeners constitute the“streptonigrinoids”-family of natural products.Members of this family have shown antiviral activity,potent and broad spectrum activities against bacteria and fungi,as well as potent activity against a wide range of tumors.Therefore,they have attracted considerable attention.As a part of our ongoing study of the biosynthesis of streptonigrin,we have identified the complete biosynthetic gene cluster in Streptomyce flocculus CGMCC4.1223,and both lavendamycin and 10?-desmethoxylstreptonigrin have been confirmed to be the biosynthetic intermediates of STN.However,efforts to find out the relationship between STN and streptonigron have been hampered by the lack of knowledge about their biosynthesis.We assume that streptonigron is biosynthesized via an oxidative decarboxylation of STN.In nature,most enzymes catalyzing the oxidative decarboxylation of aromatic carboxylic acids are flavin containing proteins,such as salicylate-1-monooxygenase and 6-hydroxynicotinate-3-monooxygenase.On the basis of bioinformatic analysis,one of the four flavin-dependent enzymes was proposed to be responsible for the transformation of STN to streptonigron,so their inactivation was firstly performed.However,the biochemical characterization of these four proteins?StnD,StnH₂,StnH₃,and StnK₁?revealed that the oxidative decarboxylation of STN generating streptonigron does not require the involvement of any flavin-dependent enzyme,but FAD is only required.After a variety of reaction parameters were screened,the optimal reaction condition for the flavin-cataltyzed oxidative decarboxylation of STN was determined to include STN?100?M?,FAD?10?M?,flavin reductase Fre?2?M?,and NADH?5mM?in phosphate buffer?pH 7.0-9.0?at room temperature for 10 min.Other two natural flavins such as flavin mononucleotide?FMN?and riboflavin were tested and found to be capable of catalyzing the conversion of STN to streptonigron with similar efficiency as FAD.When dithiothreitol?DTT?was used to replace flavin reductase to generate FADH₂ for activation of oxygen,the reaction efficiency was found about two-fold lower than under the standard conditions.Under the standard reaction conditions,FAD also can catalyze the oxidative decarboxylation of STN analogues including 10?-desmethoxylstreptonigrin,6,10?-didesmethoxylstreptonigrin,9?-desmethyl-10?-desmethoxylstreptonigrin,and10?-desmethylstreptonigrin with the similar efficiency with STN,generating the corresponding streptonigron analogues.Surprisingly,the oxidative decarboxylation also worked for lavedamycin that comprises of a tricyclic pyrido[3,4-b]indole ring,different from that of streptonigrin.These results hint that this non-enzymatic flavin-catalyzed oxidative decarboxylation may be applied to wider range substrates.In order to elucidate the chemical mechanism of flavin-catalyzed oxidative decarboxylation of STN,we designed a series of putative substrates to probe the mechanism.Firstly,amino-substituted and hydroxyl picolinic acids were tested that bear structural similarity to the multi-substituted pyridine-2-carboxylic acid moiety of streptonigrin.Complete conversion was found for 3-aminopicolinic acid,84%conversion for 5-aminopicolinic acid,and 58%conversion for 3-hydroxypicolinic acid,but no conversion was detected for 4-and 6-aminosubstituted picolinic acids.Subsequently,the corresponding benzoic acids bearing hydroxyl or amino groups at the ortho position and both ortho-and para-positions were tested.But,none of the expected products could be detected.When picolinic acid,pyridine-2,3-dicarboxylic acid and 3-nitropyridine-2-carboxylic acid with electron withdrawing groups were used as putative substrates,but no conversion could be detected.Moreover,no conversion was found for pyridin-3-amine.These findings indicated that the electron donating groups at the ortho-or para-positions and 2-carboxylic group are necessary and that flavin-catalyzed oxidative decarboxylation of STN is in line with the reactivity patterns of electrophilic aromatic substitutions.In order to examine whether hydrogen peroxide or superoxide radical produced by oxidized flavin is involved in the flavin-catalyzed oxidative decarboxylation of STN,H₂O₂,m-choroperoxy-benzoic acid and tert-butyl hydroperoxide was used as a putative oxidant with or without FAD,respectively.No conversion confirmed that H₂O₂ is not involved in the flavin-catalyzed oxidative decarboxylation.No effect of5,5-dimethyl-1-pyrroline N-oxide?DMPO?and SOD,commonly used radical scavengers,on the reaction,and no conversion of STN to streptonigron when hypoxanthine/xanthine oxidase and potassium superoxide to generate superoxides,instead of flavin,ruled out the possible involvement of radicals in the flavin-catalyzed oxidative decarboxyaltion.Based on these results,we proposed the mechanism of the oxidation of picolinic acids.The initial driving force of the oxidative decarboxylation process is anticipated to be the electron-donating properties of the-NH₂ or-OH group and the poor aromaticity of the pyridine moiety.In the first step,the attack at the flavin peroxide gives the resonance-stabilized arenium ion??complex?.Subsequently,the resultant intermediate is deprotonated by flavin oxide and further undergoes a?-keto acid type decarboxylation under release of the 2-hydroxy-substituted pyridine which can tautomerize to the corresponding 2-pyridone.10?-desmethoxystreptonigrin is a member of the streptonigriniods family that exhibits the ability to inhibit farnesylation of ras oncogene p²¹ protein.However,the production of 10?-desmethoxystreptonigrin is very low in streptomyces albus.In this study,10?-desmethoxystreptonigrin has been obtained on a large scale by continuous hydrolysis of methyl ester of 10?-desmethoxystreptonigrin which was produced in?35?stnA fermentation combining macroporous resin adsorption.Here two approaches were utilized to improve the yields of methyl ester of10?-desmethoxystreptonigrin:?a?optimization of the culture time and?b?removal of the toxic effects and a reduction in the feedback repression associated with synthesis using resin adsorption.Upon introduction of 10%?w/v?HP2MGL resin to the culture,the production of methyl ester of 10?-desmethoxystreptonigrin reached a maximum titer of 600 mg/l.This value was 24-times greater than the titer that was observed in the absence of the resin.The carboxylesterase gene?stnA?was cloned from streptomyces flocculus CGMCC4.1223 and the recombinant protein was shown to hydrolyze methyl ester of10?-desmethoxystreptonigrin to produce 10?-desmethoxystreptonigrin,efficiently.It is noteworthy that purified recombinant StnA remained stable at room temperature for12 days,demonstrating 90%residual activity after this period.It also demonstrated stability in the presence of water-miscible organic solvents such as 40%DMSO?v/v?.Since StnA exhibited both thermal and chemical stability,the enzyme was immobilized and continuously used for several days to facilitate the large-scale manufacture of 10?-desmethoxystreptonigrin.