Combinatorial Chemistry & High
Throughput Screening, Vol. 6, No. 6, 2003
Contents
Combinatorial
Biosynthesis of Polyketides and Bioactive Peptides
Guest
Editor: Joachim Vater
Strategies
for Combinatorial Biosynthesis with Modular Polyketide Synthases Pp.489-500
S. Donadio and M. Sosio
Expanding
the Scope of Aromatic Polyketides by Combinatorial Biosynthesis Pp.501-512
J.
Kantola, T. Kunnari, P. Mantsala and K. Ylihonko
On
the Generation of Novel Anticancer Drugs by Recombinant DNA Technology: The Use
of Combinatorial Biosynthesis to Produce Novel Drugs Pp.513-526
Carmen
Mendez and Jose A. Salas
Combinatorial
Biosynthesis of Non-Ribosomal Peptides Pp.527-540
Ullrich
Keller and Florian Schauwecker
Diversity
Among Microbial Cyclic Lipopeptides: Iturins and Surfactins. Activity-Structure
Relationships to Design New
Jean-Marc
Bonmatin , Olivier Laprevote and
Francoise Peypoux
"Whole
Cell" – Matrix-Assisted Laser Desorption Ionization-Time of Flight- Mass
Spectrometry, an Emerging Technique for Efficient Screening of Biocombinatorial
Libraries of Natural Compounds Present State of Research Pp.557-567
Joachim
Vater, Xuewen Gao, Gabriele Hitzeroth, Christopher Wilde and Peter Franke
Combinatorial
Chemistry in Food Research Pp.569-574
Jeffrey
A. Khan and Evgeny N. Vulfson
Abstracts
[Back to top] Strategies
for Combinatorial Biosynthesis with Modular Polyketide Synthases
S.
Donadio and M. Sosio
Polyketides are assembled by the polyketide synthases (PKS) through a common mechanism, the condensation of small carboxylic acids. However, a large structural variety exists within these molecules, paralleled by their different bioactivities. Structural differences in polyketides mostly stem from variations in the number of elongation cycles, in the extender unit incorporated and the extent of processing occurring during each cycle. A significant fraction of polyketides is made in bacteria by modular PKSs, which direct polyketide synthesis on a protein template, where each module is responsible for selecting, incorporating and processing the appropriate carboxylate unit. Since their discovery in the early nineties, the architecture of modular PKSs and their modus operandi have attracted efforts by several laboratories to reprogram PKSs to produce tailor-made polyketides. The availability of a growing number of modular PKSs of defined sequence, and of well-developed model systems for the in vitro and in vivo analysis of these enzymes, has led to the successful production of many novel polyketides after genetic manipulation of the appropriate PKS. We discuss the different strategies that are followed for the construction of functional “hybrid” systems, with particular emphasis on what can be done in terms of generating chemical diversity, highlighting also the limitations of our current understanding. The prospects of generating novel useful polyketides by genetic engineering are also discussed.
[Back to top] Expanding the Scope of Aromatic
Polyketides by Combinatorial Biosynthesis
J.
Kantola, T. Kunnari, P. Mantsala and K. Ylihonko
Combinatorial biosynthesis is a technology for mixing genes responsible for the biosynthesis of secondary metabolites, in order to generate products for compound libraries serendipitously or to cause desired modifications to natural products. Both of these approaches are extremely useful in drug discovery. Streptomyces and related species are abundant in bioactive secondary metabolites and were therefore the first microbes to be used for combinatorial biosynthesis.
Polyketides are the most abundant medicinal agents among natural products. Structural diversity and a wide scope of bioactivities are typical of the group. However, the common feature of polyketides is a biosynthetic process from simple carboxylic acid residues. In molecular genetics, polyketides are sub-classified as types I and II, called modular and aromatic polyketides respectively. The best-known bioactivities of aromatic polyketides are their antibacterial and antitumor effects. Genetic analysis of aromatic polyketides has resulted in almost 30 cloned and identified biosynthetic gene clusters. Several biosynthetic enzymes are flexible enough to allow their use in combinatorial biosynthesis to create high diversity compound libraries.
This review describes the state of the art of combinatorial biosynthesis, giving anthracyclines as examples. Contiguous DNA sequences for antibiotics, cloned from four different anthracycline producers, provide tools for rapid lead optimization or other structural modification processes, and not only for anthracyclines. Two gene cassettes enabling fast and flexible structural modification of polyketides are introduced in this paper.
[Back to top] On the
Generation of Novel Anticancer Drugs by Recombinant DNA Technology: The Use of
Combinatorial Biosynthesis to Produce Novel Drugs
Carmen
Mendez and Jose A. Salas
Chemotherapeutic drugs for cancer treatment have been traditionally originated by the isolation of natural products from differet environmental niches, by chemical synthesis or by a combination of both approaches thus generating semisynthetic drugs. In the last years, a number of gene clusters from several antitumor biosynthetic pathways, mainly produced by actinomycetes and belonging to the polyketides family, are being characterized. Genetic manipulation of these antitumor biosynthetic pathways will offer in the near future an alternative for the generation of novel antitumor derivatives and thus complementing current methods for obtaining novel anticancer drugs. Novel antitumor derivatives have been produced by targeted gene disruption and heterologous expression of single (or a few) gene(s) in another hosts or by combining genes from different, but structurally related, biosynthetic pathways (“combinatorial biosynthesis”). These strategies take advantage from the “relaxed substrate specificity” that characterize secondary metabolism enzymes.
[Back to top] Combinatorial Biosynthesis of Non-Ribosomal Peptides
Ullrich Keller and Florian Schauwecker
Non-ribosomal peptide synthetases (NRPS) are modular assembly lines catalysing the synthesis of many small peptides in microbes. Genetic replacements of domains or modules in NRPS encoded by gene clusters in Bacillus sp. with corresponding domains or modules from foreign NRPS have led in several cases to the in vivo synthesis of peptides with predicted amino acid substitutions. Fusion points were in variable regions between C- and A- or between T- and C-domains. Successful insertions of whole modules using fusion points in conserved regions internal to functional domains have also been reported. For studying the role of C- , A-, T- and TE (thioesterase)-domains in NRPS, several bi- and trimodular model-NRPS derived from natural NRPS systems were constructed and obtained after expression in E. coli with coexpression of a 4’- phosphopantetheine transferase or in suitable hosts such as the Streptomyces. Such enzymes were shown to catalyse in vitro synthesis of di- and tripeptides, respectively, with and without turnover depending on the presence of Te-domains. The enzymatic analysis revealed the mechanisms of the domains and proved their functional autonomy suggesting the possibility to use any NRPS interdomain region for fusions. Nevertheless, recombinant synthesis of longer and more complex peptides will still be restricted to alteration of existing structures by manipulations of NRPS gene clusters located on chromosomes or artificial chromosomes. Besides targeted replacements of domains and modules, reprogramming of NRPS by altering the substrate specificities of A-domains is a promising tool for the future to get novel peptides.
[Back to top] Diversity Among Microbial Cyclic Lipopeptides: Iturins and
Surfactins. Activity-Structure Relationships to Design New Bioactive Agents
Jean-Marc Bonmatin , Olivier Laprevote and Francoise Peypoux
A prominent group of bioactive lipopeptides produced by Bacillus species is constituted by iturins, surfactins and lichenysins. Interest in such substances results in their exceptional surfactant power, and their valuable antifungal, antibacterial, antitumoral and anti-Mycoplasma properties. As is typical for peptidic secondary-metabolites synthesized by the polyenzymic pathway, they are produced as mixtures of components varying in the peptidic and/or in the lipidic structure. In the context of structure-activity relationships, it is possible to take advantage of the adaptability of the biosynthesis system by systematically adding selected amino acids in the culture medium of the producing bacterium. When an amino acid is used as the sole nitrogen source, it is inserted direcly into selected positions of the peptide sequence, thus amplifying the original structural microheterogeneity via a production of variants. This method revealed very efficient for increasing the amounts of preexisting variants and for building new variants of surfactins and lichenysins but totally inefficient with iturins. In this group, the peptidic diversity strictly depends on the selected strain. So far the screening remained the only method to discover new iturins. Another interesting peculiarity is the common occurrence in a single strain of two lipopeptides with different core structures such as surfactins and iturins. Taken together, these features led to an extensive metabolite pattern. Besides, engineered variants and chemical derivatives enlarged the array of available molecules. Despite the high degree of chemical similarity, the separation of variants and/or homologues was successfully achieved by reversed-phase HPLC leading to well-separated compounds ideally suited to investigation of structure-activity relationships. Improved physical techniques such as 2D-NMR and mass spectrometry allowed to describe efficiently and rapidly the composition of cyclic lipopeptides even in mixtures containing several variants. From NMR, the 3D structure and dynamics gave crucial data for fine structure-activity relationships as well as for understanding of the properties at the membrane and/or at the air/water interface. Here the role of residues was identified in the context of hydrophobic and electrostatic interactions that play a leader role. Such a comprehensive approach, based on both structural and biosynthesis knowledge, opened the way to rational design for enhanced properties and its validity was confirmed with 10 fold higher surfactant efficacy.
[Back to top] "Whole Cell" – Matrix-Assisted Laser Desorption
Ionization-Time of Flight- Mass Spectrometry, an Emerging Technique for
Efficient Screening of Biocombinatorial Libraries of Natural Compounds –
Present State of Research
Joachim Vater, Xuewen Gao, Gabriele Hitzeroth, Christopher Wilde and Peter Franke
Whole Cell-matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDITOF- MS) is an emerging sensitive technique for rapid typing of microorganisms, efficient screening of biocombinatorial libraries of natural compounds and the analysis of complex biological samples, as whole cells, subcellular particles, cell extracts and culture filtrates. It is unique to detect metabolites in-situ without the need to isolate and purify the investigated compounds. In favourite cases it enables in –situ structure analysis on the basis of the fragment pattern generated by postsource MALDI-TOF-mass spectrometry. The state of research of this methodology which has mainly been obtained by investigation of lipopeptides from bacilli and the large spectrum of bioactive peptides produced by cyanobacteria is reviewed. The potential of this innovative technique is demonstrated for the lipopeptides produced by various Bacillus subtilis strains.
[Back to top] Combinatorial Chemistry in Food Research
Jeffrey A. Khan and Evgeny N. Vulfson
This mini-review is concerned with emerging applications of combinatorial chemistry relevant to the needs of the food industry. More specifically, recent advances in the use of combinatorial methods for the identification and analysis of flavours, “food chemical” libraries as a potential source of enzyme inhibitors for the food industry and the utility of biocatalysis for the generation of molecular diversity are discussed.