An A-factor-like Signaling Cascade Involved In Validamycin Fermentation

About two-thirds of known antibiotics are produced by the Gram-positive bacteria withhigh mol%G+C content. Actinobacteria have gained considerable interest, because of theirgreat potential for antibiotics production and complicated morphological differentiation.Quorum sensing is a widespread phenomenon in the bacterial world, which could control awide range of biological functions, including biofilm formation, secondary metabolism andcell differentiation. γ-Butyrolactones, such as A-factor, are one type of quorum sensingmolecular produced by Streptomyces species and have been reported to regulated secondarymetabolism and sporulation. At least60%of Streptomyces species appear to produce-butyrolactone, however, the regulatory mechanisms of such signaling molecular in differentStreptomyces are not well-understood.Therefore, to understand the regulatory mechanismof-butyrolactone on secondary metabolism in Streptomyces may help to improve theproduction of valuable fermentation products by engineering of the-butyrolactoneregulatory mechanisms as well as metabolic engineering approach.Validamycin, an anti-fungal aminocyclitol containing antibiotic produced byStreptomyces hygroscopicus5008, has been widely used in Asia because of its high efficacyagainst rice sheath blight. Additionally, validamycin can also be used as the source forvalienamine, a pharmaceutically important precursor for the synthesis of the antidiabeticvoglibose. In2005, the whole gene cluster responsible for validamycin biosynthesis wascloned, and eight essential genes located in three operons were identified. Very recently thecomplete genome sequence of S. hygroscopicus5008was clarified by our laboratory, andthe afsA, arpA and adpA homologues involvement of the A-factor cascade in the regulationof cellular metabolism in S. hygroscopicus5008.In the first part of this work, we initially observed the accumulation of putative-butyrolactone autoregulator in fermentation broth of S. hygroscopicus5008. Then,1,4- butyrolactone (1,4-BL) as an accessible and cheap structural analogue of A-factor, wasapplied in validamycin fermentation. The bacterial growth, sugar consumption, validamycinproduction and gene transcription were all monitored during the fermentation. Interestingly,addition of1,4-BL into the fermentation broth enhanced the antibiotic production by morethan30%both in shaking flasks and bioreactors, whereas the cell growth and sugarconsumption were not affected. The transcriptional levels of genes for validamycinbiosynthesis were significantly increased. Moreover, enhanced transcriptions of afsA, andadpA homologues involved in the regulatory cascade of A-factor were observed. Moreover,addition of the1,4-BL also improved the validamycin production in a high-yielding strainTL01. This work demonstrates that the addition of1,4-BL is a new strategy for improvingthe antibiotic yield.The second part of this work was carried out to clarify the role of1,4-BL in theregulation of validamycin production and the regulatory mechanism of A-factor-likecascade on antibiotics biosynthesis in S. hygroscopicus5008. Among the three A-factorreceptor homologous genes identified in the genome of S. hygroscopicus5008, shbR3wasproved to be responsible for the inducing activity of1,4-BL by gene disruption and circulardichroism analysis, and ShbR3could bind to the promoter region of adpA–H as indicatedby EMSA analysis. Furthermore, the mutation of adpA–H abolished the transcription ofvalidamycin biosynthetic genes and validamycin productivity. In EMSA analysis, AdpA–H could directly bind to the promoter regions of validamycin gene cluster. The resultsshowed that1,4-BL could stimulate A-factor-like cascade and subsequently enhancevalidamycin production in S. hygroscopicus5008.Genome-wide analysis revealed that the coexistence of multiple afsA-arpAhomologues is a widespread phenomenon in Streptomyces species. However, no previousstudies have investigated such an interesting phenomenon. The third part of this work wascarried out to study the regulatory mechanism of multiple afsA-arpA homologues onsecondary metabolism. In S. hygroscopicus5008, the transcriptional pattern of three pairsof afsA-arpA homologues were investigated, and it was found that each gene exhibited asingle transcriptional peak at a different stage of log phase in a sequential pattern.Subsequent gene inactivation studies demonstrated that the deletion of afsA homologues decreased the production of validamycin by60~90%, and the deletion of arpA homologuesincreased the production of validamycin by7~30%. The transcriptional and EMSAanalyses showed that ShbR1and ShbR3could repress the transcription of adpA-H bydirectly binding to its promoter region in a sequential pattern, and ShbR1could also up-regulate the transcripts of shbR3by directly binding to its promoter region. Based on theseresults, a double mutant of shbR1and shbR3was constructed, and the production ofvalidamycin was finally increased more than50%compared with the wild type. In theindustrial high-yield strain TL01, by double deletion of shbR1and shbR3, the production ofvalidamycin was enhanced from19g/L to23g/L. In summary, this work demonstrated theregulatory relationship between two arpA homologues involved in A-factor like cascade,and also successfully enhanced the antibiotic production by gene engineering approach.