scholarly journals Elucidating the mechanism of fluorinated extender unit loading for improved production of fluorine-containing polyketides

2017 ◽  
Vol 114 (5) ◽  
pp. E660-E668 ◽  
Author(s):  
Omer Ad ◽  
Benjamin W. Thuronyi ◽  
Michelle C. Y. Chang
Keyword(s):  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Type System ◽  
Large Family ◽  
Product Yield ◽  
Building Blocks ◽  
Chain Extension ◽  
Chain Elongation ◽  
Single Chain ◽  
Extender Unit

Polyketides are a large family of bioactive natural products synthesized by polyketide synthase (PKS) enzyme complexes predominantly from acetate and propionate. Given the structural diversity of compounds produced using these two simple building blocks, there has been longstanding interest in engineering the incorporation of alternative extender units. We have been investigating the mechanism of fluorinated monomer insertion by three of the six different modules of the PKS involved in erythromycin biosynthesis (6-deoxyerythronolide B synthase, DEBS) to begin understanding the contribution of different steps, such as enzyme acylation, transacylation, C–C bond formation, and chain transfer, to the overall selectivity and efficiency of this process. In these studies, we observe that inactivation of acis-acyltransferase (AT) domain to circumvent its native extender unit preference leads concurrently to a change of mechanism in which chain extension with fluorine-substituted extender units switches largely to an acyl carrier protein (ACP)-independent mode. This result suggests that the covalent linkage between the growing polyketide chain and the enzyme is lost in these cases, which would limit efficient chain elongation after insertion of a fluorinated monomer. However, use of a standalonetrans-acting AT to complement modules with catalytically deficient AT domains leads to enzyme acylation with the fluoromalonyl-CoA extender unit. Formation of the canonical ACP-linked intermediate with fluoromalonyl-CoA allows insertion of fluorinated extender units at 43% of the yield of the wild-type system while also amplifying product yield in single chain-extension experiments and enabling multiple chain extensions to form multiply fluorinated products.

scholarly journals Evidence for an iterative module in chain elongation on the azalomycin polyketide synthase

2016 ◽  
Vol 12 ◽  
pp. 2164-2172 ◽  
Author(s):  
Hui Hong ◽  
Yuhui Sun ◽  
Yongjun Zhou ◽  
Emily Stephens ◽  
Markiyan Samborskyy ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Strong Support ◽  
Artificial Chromosome ◽  
Chain Extension ◽  
Chain Elongation ◽  
Starter Unit ◽  
Polyketide Chain ◽  
Streptomyces Olivaceus ◽  
Streptomyces Malaysiensis

The assembly-line synthases that produce bacterial polyketide natural products follow a modular paradigm in which each round of chain extension is catalysed by a different set or module of enzymes. Examples of deviation from this paradigm, in which a module catalyses either multiple extensions or none are of interest from both a mechanistic and an evolutionary viewpoint. We present evidence that in the biosynthesis of the 36-membered macrocyclic aminopolyol lactones (marginolactones) azalomycin and kanchanamycin, isolated respectively from Streptomyces malaysiensis DSM4137 and Streptomyces olivaceus Tü4018, the first extension module catalyses both the first and second cycles of polyketide chain extension. To confirm the integrity of the azl gene cluster, it was cloned intact on a bacterial artificial chromosome and transplanted into the heterologous host strain Streptomyces lividans, which does not possess the genes for marginolactone production. When furnished with 4-guanidinobutyramide, a specific precursor of the azalomycin starter unit, the recombinant S. lividans produced azalomycin, showing that the polyketide synthase genes in the sequenced cluster are sufficient to accomplish formation of the full-length polyketide chain. This provides strong support for module iteration in the azalomycin and kanchanamycin biosynthetic pathways. In contrast, re-sequencing of the gene cluster for biosynthesis of the polyketide β-lactone ebelactone in Streptomyces aburaviensis has shown that, contrary to a recently-published proposal, the ebelactone polyketide synthase faithfully follows the colinear modular paradigm.

Dibenzochrysene Enables Tightly Controlled Docking and Stabilizes Photoexcited States in Dual-Pore Covalent Organic Frameworks

2019 ◽  
Author(s):  
Torben Sick ◽  
Niklas Keller ◽  
Nicolai Bach ◽  
Andreas Koszalkowski ◽  
Julian Rotter ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Photophysical Properties ◽  
Visible Spectrum ◽  
Large Family ◽  
Building Blocks ◽  
Band Gaps ◽  
Docking Site ◽  
Covalent Organic Frameworks ◽  
Connected Node ◽  
Optoelectronic Applications

Covalent organic frameworks (COFs), consisting of covalently connected organic building units, combine attractive features such as crystallinity, open porosity and widely tunable physical properties. For optoelectronic applications, the incorporation of heteroatoms into a 2D COF has the potential to yield desired photophysical properties such as lower band gaps, but can also cause lateral offsets of adjacent layers. Here, we introduce dibenzo[g,p]chrysene (DBC) as a novel building block for the synthesis of highly crystalline and porous 2D dual-pore COFs showing interesting properties for optoelectronic applications. The newly synthesized terephthalaldehyde (TA), biphenyl (Biph), and thienothiophene (TT) DBC-COFs combine conjugation in the a,b-plane with a tight packing of adjacent layers guided through the molecular DBC node serving a specific docking site for successive layers. The resulting DBC-COFs exhibit a hexagonal dual-pore kagome geometry, which is comparable to COFs containing another molecular docking site, namely 4,4′,4″,4‴-(ethylene-1,1,2,2-tetrayl)-tetraaniline (ETTA). In this context, the respective interlayer distances decrease from about 4.60 Å in ETTA-COFs to about 3.6 Å in DBC-COFs, leading to well-defined hexagonally faceted single crystals sized about 50-100 nm. The TT DBC-COFs feature broad light absorption covering large parts of the visible spectrum, while Biph DBC-COF shows extraordinary excited state lifetimes exceeding 10 ns. In combination with the large number of recently developed linear conjugated building blocks, the new DBC tetra-connected node is expected to enable the synthesis of a large family of strongly p-stacked, highly ordered 2D COFs with promising optoelectronic properties.

scholarly journals Engineering the acyltransferase domain of epothilone polyketide synthase to alter the substrate specificity

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Huimin Wang ◽  
Junheng Liang ◽  
Qianwen Yue ◽  
Long Li ◽  
Yan Shi ◽  
...  
Keyword(s):  
Parental Strain ◽  
Assembly Line ◽  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Critical Factor ◽  
Catalytic Domains ◽  
Malonyl Coa ◽  
Epothilone D ◽  
Careful Design ◽  
Selection Of

Abstract Background Polyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal. Results The substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS. Conclusions These results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.

scholarly journals Sticky swinging arm dynamics: studies of an acyl carrier protein domain from the mycolactone polyketide synthase

2016 ◽  
Vol 473 (8) ◽  
pp. 1097-1110 ◽  
Author(s):  
Steven Vance ◽  
Olga Tkachenko ◽  
Ben Thomas ◽  
Mona Bassuni ◽  
Hui Hong ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Protein Domain ◽  
Carrier Protein ◽  
Protein Surface ◽  
Prosthetic Group ◽  
Polyketide Biosynthesis ◽  
Covalently Linked

When covalently linked to an acyl carrier protein (ACP) and loaded with acyl substrate-mimics, some 4′-phosphopantetheine prosthetic group arms swing freely, whereas others stick to the protein surface, suggesting a possible mode of interaction with enzyme domains during polyketide biosynthesis.

A Constraint Based Fuzzy Object Oriented Database Model

2011 ◽  
pp. 1-45 ◽  
Author(s):  
Guy de Tre ◽  
Rita de Caluwe
Keyword(s):  
Type System ◽  
Object Oriented ◽  
Building Blocks ◽  
Constraint System ◽  
Database Model ◽  
Database Models ◽  
Data Definition ◽  
Definition Of ◽  
Object Oriented Database ◽  
Adequate Data

The objective of this chapter is to define a fuzzy object-oriented formal database model that allows us to model and manipulate information in a (true to nature) natural way. Not all the elements (data) that occur in the real world are fully known or defined in a perfect way. Classical database models only allow the manipulation of accurately defined data in an adequate way. The presented model was built upon an object-oriented type system and an elaborated constraint system, which, respectively, support the definitions of types and constraints. Types and constraints are the basic building blocks of object schemes, which, in turn, are used for defining database schemes. Finally, the definition of the database model was obtained by providing adequate data definition operators and data manipulation operators. Novelties in the approach are the incorporation of generalized constraints and of extended possibilistic truth values, which allow for a better representation of data(base) semantics.

Analysis of Service Compatibility

2010 ◽  
pp. 136-155
Author(s):  
Ken Q. Pu
Keyword(s):  
Relational Database ◽  
Type System ◽  
Large Family ◽  
Service Description ◽  
Worst Case ◽  
Database Queries ◽  
Unification Algorithm ◽  
Data Services ◽  
Wide Range ◽  
Unification Problem

In this chapter, the authors apply type-theoretic techniques to the service description and composition verification. A flexible type system is introduced for modeling instances and mappings of semi-structured data, and is demonstrated to be effective in modeling a wide range of data services, ranging from relational database queries to web services for XML. Type-theoretic analysis and verification are then reduced to the problem of type unification. Some (in)tractability results of the unification problem and the expressiveness of their proposed type system are presented in this chapter. Finally, the auhtors construct a complete unification algorithm which runs in EXP-TIME in the worst case, but runs in polynomial time for a large family of unification problems rising from practical type analysis of service compositions.

scholarly journals Modular type I polyketide synthase acyl carrier protein domains share a common N-terminally extended fold

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Luisa Moretto ◽  
Rachel Heylen ◽  
Natalie Holroyd ◽  
Steven Vance ◽  
R. William Broadhurst
Keyword(s):  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Protein Domains ◽  
Carrier Protein ◽  
Type I

scholarly journals Genetic Determinants of Reutericyclin Biosynthesis in Lactobacillus reuteri

2015 ◽  
Vol 81 (6) ◽  
pp. 2032-2041 ◽  
Author(s):  
Xiaoxi B. Lin ◽  
Christopher T. Lohans ◽  
Rebbeca Duar ◽  
Jinshui Zheng ◽  
John C. Vederas ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Genomic Island ◽  
Lactobacillus Reuteri ◽  
Acyl Carrier Protein ◽  
Open Reading Frames ◽  
Protein Domain ◽  
Regulatory Proteins ◽  
Tetramic Acid ◽  
Genetic Determinants ◽  
Content Type

ABSTRACTReutericyclin is a unique antimicrobial tetramic acid produced by some strains ofLactobacillus reuteri. This study aimed to identify the genetic determinants of reutericyclin biosynthesis. Comparisons of the genomes of reutericyclin-producingL. reuteristrains with those of non-reutericyclin-producing strains identified a genomic island of 14 open reading frames (ORFs) including genes coding for a nonribosomal peptide synthetase (NRPS), a polyketide synthase (PKS), homologues of PhlA, PhlB, and PhlC, and putative transport and regulatory proteins. The protein encoded byrtcNis composed of a condensation domain, an adenylation domain likely specific ford-leucine, and a thiolation domain.rtcKcodes for a PKS that is composed of a ketosynthase domain, an acyl-carrier protein domain, and a thioesterase domain. The products ofrtcA,rtcB, andrtcCare homologous to the diacetylphloroglucinol-biosynthetic proteins PhlABC and may acetylate the tetramic acid moiety produced by RtcN and RtcK, forming reutericyclin. Deletion ofrtcNorrtcABCinL. reuteriTMW1.656 abrogated reutericyclin production but did not affect resistance to reutericyclin. Genes coding for transport and regulatory proteins could be deleted only in the reutericyclin-negativeL. reuteristrain TMW1.656ΔrtcN, and these deletions eliminated reutericyclin resistance. The genomic analyses suggest that the reutericyclin genomic island was horizontally acquired from an unknown source during a unique event. The combination of PhlABC homologues with both an NRPS and a PKS has also been identified in the lactic acid bacteriaStreptococcus mutansandLactobacillus plantarum, suggesting that the genes in these organisms and those inL. reuterishare an evolutionary origin.

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