Combinatorial Chemistry & High Throughput Screening - Volume 6, Issue 6, 2003

One of the most exciting development in modern combinatorics is the utilization of nature's immense genetic potential for taking profit of the biosynthetic productivity of living cells. In this context this special issue of CCHTS is focussed on the present knowledge in combinatorial biosynthesis of polyketides and bioactive peptides, two of the most important classes of natural compounds with a high potential for manifold biotechnological and biopharmaceutical applications. These compounds are produced by microorganisms involving large multienzyme systems of modular structure, the polyketide synthases (PKS) and the nonribosomal peptide synthetases (NRPS). Both biosynthetic pathways resemble assembly lines and proceed according to a multiple carrier mechanism, involving 4'-phosphopantetheine-cofactors as the mobile carriers at the reaction centers for the growing polyketide and peptide chain. During the last decade numerous gene structures for the biosynthesis of such compounds have been elucidated. Polyketides and nonribosomal peptides are predestinated for biocombinatorial variation because of the great diversity of the modules and domains found in the structure of their producer enzymes. In this issue are reviewed the methodology and the tool boxes for combinatorial biosyntheses of these attractive compounds which include reprogramming genes to generate recombinant enzyme structures and the creation of compound libraries for selection of metabolites with desired modifications. S. Donadio and M. Sosio present a concise overview on the fundamental aspects of the structure and function of polyketide synthases of type I and the genetic engineering of such integrated multienzymes. This is demonstrated for 6-deoxyerythronolide B synthase as a prototype of modular PKSs. The availability of numerous PKS-genes and the progress in molecular genetic techniques enabled the design of appropriate recombinant PKS for the production of novel polyketide compounds with specific activities. Various strategies to create tailor-made polyketides, as altering length and extent of reduction of the polyketide chain, changing of the starter unit, reprogramming of acyl tansferase specificity, post-polyketide modification and the design of PKSs by simultaneous engineering in different modules are discussed. Future perspectives for combinatorial biosynthesis of polyketides and the generation of large libraries of “unnatural” natural products are outlined. The review of Kantola et al. is focussed on polyketide synthases of type II which appear as aggregates of separate enzymes organised in multienzyme complexes. In particular, PKSs were addressed which form aromatic polyketide products showing potent antibacterial and antitumor activities. Genetic analysis of aromatic polyketide formation led to the identification and cloning of almost thirty gene clusters. In this review specifically the state of the art of biocombinatorial biosynthesis of anthracycline polyketides is described. Contiguous DNA-sequences for antibiotic production cloned from different anthracycline producers provided the tools for rapid lead optimization by structural modification of the producer enzymes. Two gene cassettes enabling fast and flexible structural modification of polyketides were introduced for biocombinatorial variation of anthracylines. Mendez and Salas review specific aspects of biopharmaceutical applications of polyketides. The use of such compounds in chemotherapy for cancer treatment is demonstrated. Numerous polyketides mainly from actinomycetes show potent antitumor activities. Several gene clusters coding for biosynthetic pathways producing polyketides of high importance in cancer therapy have been characterized. Their genetic manipulation afford a great potential for the generation of novel antitumor derivatives which can be produced by targetted gene disruption and expression of the modified genes in a heterologous host. Keller and Schauwecker present a comprehensive review on combinatorial biosynthesis of nonribosomal peptides. They give an overview on the architecture and functional organization of nonribosomal peptide synthetases (NRPS) which are multienzyme systems of modular structure. Their amino acid activating modules which are arranged in a tandem-like fashion display a high similarity among each other. Every NRPS-module is organized into three core domains (A, T and C) which are responsible for the two-step activation of the amino acid substrates as aminoacyl adenylates in the A-domain and thioesters in the T-domain and for peptide bond formation in the elongation process in the condensation domain (C-domain). The know-how of combinatorial biosyntheses with NRPS-multienzymes is discussed in detail. In particular, the replacement of domainencoding regions in NRPS-genes, in vitro construction of recombinant peptide synthetases by domain and module fusion as well as site-directed mutagenesis at the reaction centers are demonstrated. Targetted changing of the substrate specificity of the A-domains by exchange of specificity-conferring residues at the amino acid recognition and binding sites seems to be of particular attraction, because reprogramming of a module can be achieved without severe disturbing of the enzyme architecture. Bonmatin et al. discuss the large diversity among lipopeptides produced by Bacillus subtilis. The present state of knowledge on two of such lipopeptide families, the iturins and the surfactins / lichenysins synthesized by the nonribosomal pathway involving NRPSs is reviewed. All these agents appear in a large variety of isoforms which differ by variation of the length and branching of their fatty acid components as well as by amino acid replacements in their peptide ring. In this way pools of closely related structural variants are available for studies of structure-activity -relationships concerning their manifold interesting properties as biosurfactants, antibiotics and antiviral agents. Specific lipopeptide isoforms can be selected by bioconversion adding specific amino acids to the cultivation medium. The variety of these compounds can further be increased by the creation of bioengineered variants and by chemical modification. The characterization of surfactins and iturins by modern techniques of structure analysis such as multidimensional NMR-spectroscopy and mass spectrometry is discussed in detail. The obtained data form the basis to understand their surface-, interface- and membrane-active properties as well as the mechanisms of action of their biological activities. Vater et al. update the present state of research of “Whole Cell” matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS), a novel emerging technique for efficient screening of biocombinatorial libraries of natural compounds. This method allows the detection of metabolites directly at the cellular surface without the need to isolate and separate them. In favourite cases it even enables in-situ structure elucidation by analysis of the fragment ion spectra obtained using MALDI TOF MS with postsource decay. Representative samples of secondary metabolites that have already been thoroughly investigated by this technique are the lipopeptides formed by numerous strains of B. subtilis and other bacilli as well as the large diversity of bioactive peptides from cyanobacteria. Their production are prominent examples of natural biocombinatorial processes. The potential of this innovative technique is demonstrated for the three lipopeptide families from B. subtilis, the surfactins, iturins and fengycins. In the article of Khan and Vulfson recent advances and new ideas relating emerging applications of (bio)combinatorial chemistry in food research are highlighted. The utilization of peptide- and polyketide libraries in food biotechnology is discussed. Combinatorial methods for the creation and analysis of flavour compounds and the design of “food-chemical” libraries as potential sources of enzyme inhibitors for the food industry are addressed. Libraries of dipeptides containing Smethyl- thioesters as precursors of a variety of cheese aroma compounds and of Maillard products formed in the reaction of amines with poly-ols were generated and screened for hit compounds. Combinatorial biocatalysis using enzymes and whole cells as catalysts were introduced as a complement of the existing arsenal of combinatorial methods to generate wide molecular diversity quickly and at relatively low cost.

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.

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.

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 targetted 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.

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.

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.

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.

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.

Joachim Vater obtained his diploma (1965) and his Doctor's degrees (Dr. rer. nat., 1972) from the Technical University Berlin working under the supervision of Professor Dr. H. T. Witt at the Max-Volmer-Institute in the field of primary processes in photosynthesis of higher plants. From 1972 - 1976 he was research assistant (postdoc) of Professor Dr. H. Kleinkauf at the Institute of Biochemistry and Molecular Biology of the Technical University Berlin in the field of nonribosomal biosynthesis of bioactive peptides. Since 1976 he is a research associate at this institute. He was the leader of numerous research projects on the biosynthesis and biotechnological utilization of bioactive peptides. From 1994-1998 he was the coordinator of interdisciplinary research projects on microbial cell factories in Biotechnology programmes of the European Commission. His interests include: Nonribosomal peptide biosynthesis; peptide synthetases; lipopeptides; surfactin; antibiotic and antiviral agents; combinatorial biosynthesis of peptides; peptide design; biotechnological exploitation of bioactive peptides; functional genomics of microbial secondary metabolites; metabolite screening; advanced mass spectrometric methodology for analysis of conplex biological samples (whole cells, subcellular particles, organelles, cellular extracts); protein chemistry; surface chemistry. SELECTED PUBLICATIONS Stein, T.; Vater. J.; Kruft, V.; Otto, A.; Wittmann-Liebold, B.; Franke, P.; Panico, M.; McDowell, R.; Morris, H.R. The multiple carrier model of nonribosomal peptide biosynthesis at modular multienzymatic templates. J. Biol. Chem. 1996, 271, 15428-15435. Vollenbroich, D.; Pauli, G.; Özel, M.; Vater, J. Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis. Appl. Environ. Microbiol. 1997, 63, 44-49. Vollenbroich, D.; Ozel, M.; Vater, J.; Kamp, R.M.; Pauli, G. Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis. Biologicals 1997, 25, 289-297. Duitman, E.H.; Hamoen, L.W.; Rembold, M.; Venema, G.; Seitz, H.; Saenger, W.; Bernhard, W.; Reinhardt, R.; Schmidt, M.; Ullrich, C.; Stein, T.; Leenders, F.; Vater, J. The mycosubtilin synthetase of Bacillus subtilis ATCC6633: A multifunctional hybrid between a peptide synthetase, an aminotransferase, and a fatty acid synthase. Proc. Natl. Acad. Sci. USA 1999, 96, 13294-13299. Leenders, F.; Stein, T.H.; Kablitz, B.; Franke, P.; Vater, J. Rapid typing of Bacillus subtilis strains by their secondary metabolites using matrix-assisted laser desorption / ionization mass spectrometry of intact cells. Rapid Commun. Mass Spectrom. 1999, 13, 943-949. Vater, J.; Kablitz, B.; Wilde, C.; Franke, P.; Mehta, N.; Cameotra, S.S. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge. Appl. Environ. Microbiol. 2002, 68, 6210-6219.