Preface [Hot topic: Combinatorial Biosynthesis of Polyketides and Bioactive Peptides (Guest Editor: Joachim Vater)]

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.