Table_1.doc

<p>AdpA, an AraC/XylS family protein, had been proved as a key regulator for secondary metabolism and morphological differentiation in Streptomyces griseus. Here, we identify AdpA_ch, an ortholog of AdpA, as a “higher level” pleiotropic regulator of natamycin biosynthesis with bidirectional regulatory ability in Streptomyces chattanoogensis L10. DNase I footprinting revealed six AdpA_ch-binding sites in the scnRI–scnRII intergenic region. Further analysis using the xylE reporter gene fused to the scnRI–scnRII intergenic region of mutated binding sites demonstrated that the expression of scnRI and scnRII was under the control of AdpA_ch. AdpA_ch showed a bi-stable regulatory ability where it firstly binds to the Site C and Site D to activate the transcription of the two pathway-specific genes, scnRI and scnRII, and then binds to other sites where it acts as an inhibitor. When Site A and Site F were mutated in vivo, the production of natamycin was increased by 21% and 25%, respectively. These findings indicated an autoregulatory mechanism where AdpA_ch serves as a master switch with bidirectional regulation for natamycin biosynthesis.</p

Image_1_Substrate Specificity of Acyltransferase Domains for Efficient Transfer of Acyl Groups.TIF

<p>Acyltransferase domains (ATs) of polyketide synthases (PKSs) are critical for loading of acyl groups on acyl carrier protein domains (A) via self- and trans-acylation reactions, to produce structurally diverse polyketides. However, the interaction specificity between ATs and unusual acyl units is rarely documented. In Streptomycestsukubaensis YN06, we found that AT4_FkbB [an AT in the fourth module of tacrolimus (FK506) PKS] transferred both allylmalonyl (allmal) and emthylmalonyl (ethmal) units to ACPs, which was supposed responsible for the production of both FK506 and its analog FK520, respectively. Mutations of five residues in AT4_FkbB (Q119A, L185I-V186D-V187T, and F203L) caused decreased efficiency of allmal transfer, but a higher ratio of ethmal transfer, supposedly due to less nucleophilic attacks between Ser599 in the active site of AT4_FkbB and the carbonyl carbon in the allmal unit, as observed from molecular dynamics simulations. Furthermore, reverse mutations of these five residues in ethmal-specific ATs to the corresponding residues of AT4_FkbB increased its binding affinity to allmal-CoA. Among these residues, Val187 of AT4_FkbB mainly contributed to allmal recognition, and V187K mutant produced less FK520 than wild type. Our findings thus suggested that five critical residues within AT4_FkbB were important for AT functionality in polyketide extension and potentially for targeting biosynthesis by generating desirable products and eliminating undesirable analogs.</p

Image_3_Substrate Specificity of Acyltransferase Domains for Efficient Transfer of Acyl Groups.TIF

<p>Acyltransferase domains (ATs) of polyketide synthases (PKSs) are critical for loading of acyl groups on acyl carrier protein domains (A) via self- and trans-acylation reactions, to produce structurally diverse polyketides. However, the interaction specificity between ATs and unusual acyl units is rarely documented. In Streptomycestsukubaensis YN06, we found that AT4_FkbB [an AT in the fourth module of tacrolimus (FK506) PKS] transferred both allylmalonyl (allmal) and emthylmalonyl (ethmal) units to ACPs, which was supposed responsible for the production of both FK506 and its analog FK520, respectively. Mutations of five residues in AT4_FkbB (Q119A, L185I-V186D-V187T, and F203L) caused decreased efficiency of allmal transfer, but a higher ratio of ethmal transfer, supposedly due to less nucleophilic attacks between Ser599 in the active site of AT4_FkbB and the carbonyl carbon in the allmal unit, as observed from molecular dynamics simulations. Furthermore, reverse mutations of these five residues in ethmal-specific ATs to the corresponding residues of AT4_FkbB increased its binding affinity to allmal-CoA. Among these residues, Val187 of AT4_FkbB mainly contributed to allmal recognition, and V187K mutant produced less FK520 than wild type. Our findings thus suggested that five critical residues within AT4_FkbB were important for AT functionality in polyketide extension and potentially for targeting biosynthesis by generating desirable products and eliminating undesirable analogs.</p

Table_1_Substrate Specificity of Acyltransferase Domains for Efficient Transfer of Acyl Groups.DOC

<p>Acyltransferase domains (ATs) of polyketide synthases (PKSs) are critical for loading of acyl groups on acyl carrier protein domains (A) via self- and trans-acylation reactions, to produce structurally diverse polyketides. However, the interaction specificity between ATs and unusual acyl units is rarely documented. In Streptomycestsukubaensis YN06, we found that AT4_FkbB [an AT in the fourth module of tacrolimus (FK506) PKS] transferred both allylmalonyl (allmal) and emthylmalonyl (ethmal) units to ACPs, which was supposed responsible for the production of both FK506 and its analog FK520, respectively. Mutations of five residues in AT4_FkbB (Q119A, L185I-V186D-V187T, and F203L) caused decreased efficiency of allmal transfer, but a higher ratio of ethmal transfer, supposedly due to less nucleophilic attacks between Ser599 in the active site of AT4_FkbB and the carbonyl carbon in the allmal unit, as observed from molecular dynamics simulations. Furthermore, reverse mutations of these five residues in ethmal-specific ATs to the corresponding residues of AT4_FkbB increased its binding affinity to allmal-CoA. Among these residues, Val187 of AT4_FkbB mainly contributed to allmal recognition, and V187K mutant produced less FK520 than wild type. Our findings thus suggested that five critical residues within AT4_FkbB were important for AT functionality in polyketide extension and potentially for targeting biosynthesis by generating desirable products and eliminating undesirable analogs.</p

Image_4_Substrate Specificity of Acyltransferase Domains for Efficient Transfer of Acyl Groups.TIF

<p>Acyltransferase domains (ATs) of polyketide synthases (PKSs) are critical for loading of acyl groups on acyl carrier protein domains (A) via self- and trans-acylation reactions, to produce structurally diverse polyketides. However, the interaction specificity between ATs and unusual acyl units is rarely documented. In Streptomycestsukubaensis YN06, we found that AT4_FkbB [an AT in the fourth module of tacrolimus (FK506) PKS] transferred both allylmalonyl (allmal) and emthylmalonyl (ethmal) units to ACPs, which was supposed responsible for the production of both FK506 and its analog FK520, respectively. Mutations of five residues in AT4_FkbB (Q119A, L185I-V186D-V187T, and F203L) caused decreased efficiency of allmal transfer, but a higher ratio of ethmal transfer, supposedly due to less nucleophilic attacks between Ser599 in the active site of AT4_FkbB and the carbonyl carbon in the allmal unit, as observed from molecular dynamics simulations. Furthermore, reverse mutations of these five residues in ethmal-specific ATs to the corresponding residues of AT4_FkbB increased its binding affinity to allmal-CoA. Among these residues, Val187 of AT4_FkbB mainly contributed to allmal recognition, and V187K mutant produced less FK520 than wild type. Our findings thus suggested that five critical residues within AT4_FkbB were important for AT functionality in polyketide extension and potentially for targeting biosynthesis by generating desirable products and eliminating undesirable analogs.</p

Identification and Biosynthetic Characterization of Natural Aromatic Azoxy Products from <i>Streptomyces chattanoogensis</i> L10

Aromatic azoxy compounds recently attracted wide interest for their unique liquid crystalline properties. However, biosynthetic pathways of natural azoxy products have rarely been reported. Three novel aromatic azoxy compounds, azoxymycins A, B, and C, have been isolated and identified from <i>Streptomyces chattanoogensis</i> L10, and their biosynthetic pathways have been reported

Efficient Biosynthesis of Fungal Polyketides Containing the Dioxabicyclo-octane Ring System

Aurovertins are fungal poly­ketides that exhibit potent inhibition of adenosine triphosphate synthase. Aurovertins contain a 2,6-dioxa­bicyclo­[3.2.1]­octane ring that is proposed to be derived from a polyene precursor through regio­selective oxidations and epoxide openings. In this study, we identified only four enzymes required to produce auro­vertin E. The core poly­ketide synthase produces a polyene α-pyrone. Following pyrone <i>O-</i>methylation by a methyl­transferase, a flavin-dependent mono-oxygenase and an epoxide hydrolase can iteratively transform the terminal triene portion of the precursor into the dioxa­bicyclo­[3.2.1]­octane scaffold. We demonstrate that a tetra­hydro­furanyl polyene is the first stable intermediate in the transformation, which can undergo epoxidation and anti-Baldwin 6-<i>endo</i>-tet ring opening to yield the cyclic ether product. Our results further demonstrate the highly concise and efficient ways in which fungal biosynthetic pathways can generate complex natural product scaffolds