The Mechanism Of Structural Variation And The Editing Funtion Of Type Ⅱ Thioesterase In FR-008/Candicidin Biosynthesis

FR-008/candicidin, a heptaene macrolide with antifungal activity, is produced by Streptomyces sp. FR-008 or Streptomyces griseus IMRU3570. Three compounds, FR-008-III, -V, and -VI, represent the natural components of FR-008/candicidin mixture. The other three compounds, FR-008-II, -1, and -IV, were identified, based LC-MS and NMR analysis, as the products respectively converted from the component III, V, and VI as the opening of hemiketal ring between C-15 and C-19 in FR-008/candicidin aglycone. FR-008-III, -V, and -VI were characterized by NMR, and the difference of their chemical structure lies in the group variations in polyol region of aglycone, in which III and V have C-9 hydroxyl, while C-9 methylene presents in VI; III and VI have C-3 ketone, while V carries C-3 hydroxyl; compared to the C-5 methylene and C-13 hydroxyl in III and V, component VI bears C-5 hydroxyl and C-13 methylene, which is unexpected according to the domains organization in polyketid assembly 16^th-20^th, and assumed to be resulted in by a untypical program of group modifying by functional domain KR, DH, and ER. According to FR-008/candicidin polyketide assembly line, coexistence of ketone or hydroxyl at C-3, and also methylene or hydroxyl at C-9 in FR-008 aglycone could be generated respectively as the incomplete activities of KR21 and DH18 during polyketide biosynthesis. Additionally, one monooxygenase, encoded by fscO localized at the left end of the FR-008 gene cluster, was assumed as a candidate for restoring C-9 hydroxyl at tailoring modification step. For exploring the hypothesis above, fscO was firstly knocked out and proved to be irrelevant to group variations in FR-008/candicidin. Subsequently, the site-directed mutagenesis analysis of DH18 and KR21 brought out exciting result: inactivation of KR21and DH18 respectively abolished production of component V carrying a C-3 hydroxyl, and VI carrying a C-9 methylene, while combined mutagenesis of KR21 and DH18 created a mutant producing only III, with a C-3 ketone and a C-9 hydroxyl. Incomplete activities of KR21 and DH18 were, therefore, unambiguously identified to be directly involved in co-production of three FR-008 analogues. Furthermore, bioassay analysis indicated component III having the antifungal activity superior to V and comparable to VI. Plus better water-solubility of component III than VI, III appeared to be excellent in pharmacological properties. The mutant ZYJ-6 (combined mutation of KR21 and DH18) singly accumulating component III, therefore, has a distinctive significance of industry application. DH, KR, or ER domains playing as modification domains in PKS contributed basically to the group variations in the carbon frame-work of polyketid product. The modification domains in a PKS can be manipulated effectively to generate series of structural derivatives provided for superior drugs screening.Type I thioesterase (TEI) functions as a catalysis domain fused at the C-terminal of PKS/NRPS assembly protein, while the type II thioesterase (TEII) plays its role as a discrete protein. TEIIs were also found within many type I PKSs and NRPS gene clusters. Disruption of the TEII genes was documented to greatly reduce product yields and thus implying TEII crucial to efficiency of product biosynthesis. Biochemical evidences indicate TEII can remove the nonelongatable substrates bond to ACP or PCP. However, no convince evidence, hitherto, revealed TEII with a capability of selectively removing the nonelongatable residues from the unprocessed PKS proteins, and thus ensuring efficient polyketide biosynthesis.One TEII gene (fscTE) was also found in FR-008/candicidin gene cluster encoding a typical type I PKS biosynthetic pathway. Streptomyces sp. FR-008 is an excellent material for researching on TEII as its convenient genetic manipulation, high yield metabolites production, and short growth-period. Deletion of fscTE reduced approximately 90 % of the FR-008/candicidin production, while the production level was well restored (75%) when fscTE constructively promoted by ermE*p was added back to the mutant in trans. As a negative control of complementation, the mutated fscTE (S129A) was also introduced to the mutant and identified to have no contribution to production restoring, and therefore revealed that the activity of FscTE was crucial to effective FR-008 polyketide biosynthesis. To explore the substrate specificities of TEII in PKS, The FscTE was expressed as a fusion protein tagged with an N-terminal hexa-histidine and purified to homogeneity. As the N-acetylcysteamine thioester (SNAC) well used as the model substrates of the acyl chains bound to the phosphopantetheine arm of the ACP/or PCP in thioesterase hydrolysis assays, acetyl-SNAC and propionyl-SNAC, in this study, were prepared to mimic the aberrant acyl-ACP substrates, and malonyl-SNAC and methylmalonyl-SNAC were used to represent the correct substrates in PKS condensing reaction. Direct biochemical analysis demonstrated FscTE with remarkable preferences of hydrolyzing acyl-thioesters, i.e. propionyl-SNAC over methylmalonyl-SNAC and acetyl-SNAC over malonyl-SNAC, and thus concluded that TEII could maintain effective polyketide biosynthesis by selectively removing the nonelongatable residues bound to ACPs. For further confirming the substrate recognition of TEII, another well described PKS TEII (TylO) from tylosin biosynthetic pathway was also expressed and tested using the same SNAC substrates, and proved to have the same substrates specificities as that of FscTE. Moreover, p-aminobenzoyl-SNAC was also prepared to model the p-aminobenzoyl-ACP substrate in initiation of FR-008/candicidin biosynthesis. Only low hydrolysis activity of FscTE on p-aminobenzoyl-SNAC was detected, and hereby ruled out the possible involvement of FscTE in the initiation of FR-008/candicidin biosynthesis.High level of FscTE expression by introducing extra copy of fscTE under constructive promoter ermE*p did not suppress Candicidin/FR-008 formation, and thus supporting the notion that FscTE only specifically removed the aberrant residues from ACP rather than disturbed the normal process.TEIIs were generally believed not to be involved in releasing of maturely elongated chain mainly based on the biochemical evidences that TEIIs possess very low hydrolysis activities to long chain thioesters of polyketide or polypeptide, while the excised TEI domain can self-efficiently do the work. Considering no genetic evidences support the opinion that TEII can not substitute TEI for chain terminal release, the TEI domain in FscF was inactivated by site-directed mutagenesis (S1887A & S1888A). The TEI mutation resulted in total disruption of Candicidin/FR-008 production, and thus implied that TEII can not exert TEI's role for terminal release. Furthermore, the evolutionary distance between the TEIIs from PKS and NRPS was reflected distinctively by their different complementation efficiencies to the fscTE mutant, in which the PKS-type TEII tylO can partially restore FR-008/candididin production (30%), and the NRPS-type TEII tycF and srfAD were proved to be idle. The result is well correspondent to the documented biochemical evidences, in which NRPS-type TEII can only recognize PCP but not ACP, and PKS-type TEII can recognize the ACPs of different origins. Furthermore, the evidence of TylO improving Candicidin/FR-008 biosynthesis is well consistent to the substrate specificities of TylO in vitro, and further supporting the conclusion: type I1 thioesterase ensures efficient polyketide biosynthesis by selectively removing the aberrant extender units.