Emerging concepts in the semisynthetic and mutasynthetic production of natural products.

Natural products have greatly influenced the development of drugs to combat infectious diseases, cancer, and other disorders affecting human well-being. Only rarely, a natural product is used in an unmodified form for therapeutic purposes. More often, natural product derivatives are preferred due to improved activity or toxicity profiles. These compounds are usually produced using 'hybrid' processes that integrate organic synthesis and biosynthesis. Either a natural product is isolated from a biological source and then converted into the final drug by semisynthesis or a synthetically prepared precursor is introduced into the engineered biosynthesis of a living cell in a procedure called mutasynthesis. In this review, we will present recent developments in these two research areas, which take advantage of heterologous biosynthesis.

Discovery, Biosynthetic Origin, and Heterologous Production of Massinidine, an Antiplasmodial Alkaloid.

Bacteria of the genus Massilia represent an underexplored source of bioactive natural products. Here, we report the discovery of massinidine (1), a guanidine alkaloid with antiplasmodial activity, from these microbes. The unusual scaffold of massinidine is shown to originate from l-phenylalanine, acetate, and l-arginine. Massinidine biosynthesis genes were identified in the native producer and validated through heterologous expression in Myxococcus xanthus. Bioinformatic analyses indicate that the potential for massinidine biosynthesis is distributed in various proteobacteria.

Saccharomyces cerevisiae as host for the recombinant production of polyketides and nonribosomal peptides.

As a robust, fast growing and genetically tractable organism, the budding yeast Saccharomyces cerevisiae is one of the most widely used hosts in biotechnology. Its applications range from the manufacturing of vaccines and hormones to bulk chemicals and biofuels. In recent years, major efforts have been undertaken to expand this portfolio to include structurally complex natural products, such as polyketides and nonribosomally synthesized peptides. These compounds often have useful pharmacological properties, which make them valuable drugs for the treatment of infectious diseases, cancer, or autoimmune disorders. In nature, polyketides and nonribosomal peptides are generated by consecutive condensation reactions of short chain acyl-CoAs or amino acids, respectively, with the substrates and reaction intermediates being bound to large, multidomain enzymes. For the reconstitution of these multistep catalytic processes, the enzymatic assembly lines need to be functionally expressed and the required substrates must be supplied in reasonable quantities. Furthermore, the production hosts need to be protected from the toxicity of the biosynthetic products. In this review, we will summarize and evaluate the status quo regarding the heterologous production of polyketides and nonribosomal peptides in S. cerevisiae. Based on a comprehensive literature analysis, prerequisites for a successful pathway reconstitution could be deduced, as well as recurring bottlenecks in this microbial host.

Bioengineering of Anti-Inflammatory Natural Products.

Inflammatory processes occur as a generic response of the immune system and can be triggered by various factors, such as infection with pathogenic microorganisms or damaged tissue. Due to the complexity of the inflammation process and its role in common diseases like asthma, cancer, skin disorders or Alzheimer's disease, anti-inflammatory drugs are of high pharmaceutical interest. Nature is a rich source for compounds with anti-inflammatory properties. Several studies have focused on the structural optimization of natural products to improve their pharmacological properties. As derivatization through total synthesis is often laborious with low yields and limited stereoselectivity, the use of biosynthetic, enzyme-driven reactions is an attractive alternative for synthesizing and modifying complex bioactive molecules. In this minireview, we present an outline of the biotechnological methods used to derivatize anti-inflammatory natural products, including precursor-directed biosynthesis, mutasynthesis, combinatorial biosynthesis, as well as whole-cell and in vitro biotransformation.

Complete Genome Sequence of the Cryptophycin-Producing Cyanobacterium Nostoc sp. Strain ATCC 53789.

Nostoc sp. strain ATCC 53789 is a producer of cryptophycins, which are promising anticancer agents. Here, we report the completely sequenced 8.7-Mb genome of Nostoc sp. strain ATCC 53789. The sequence provides insights into the metabolic network of this cyanobacterial strain and illuminates its potential for the biosynthesis of secondary metabolites.

Microbial synthesis of the type I polyketide 6-methylsalicylate with Corynebacterium glutamicum.

Type I polyketide synthases (PKSs) are large multi-domain proteins converting simple acyl-CoA thioesters such as acetyl-CoA and malonyl-CoA to a large diversity of biotechnologically interesting molecules. Such multi-step reaction cascades are of particular interest for applications in engineered microbial cell factories, as the introduction of a single protein with many enzymatic activities does not require balancing of several individual enzymatic activities. However, functional introduction of type I PKSs into heterologous hosts is very challenging as the large polypeptide chains often do not fold properly. In addition, PKS usually require post-translational activation by dedicated 4'-phosphopantetheinyl transferases (PPTases). Here, we introduce an engineered Corynebacterium glutamicum strain as a novel microbial cell factory for type I PKS-derived products. Suitability of C. glutamicum for polyketide synthesis could be demonstrated by the functional introduction of the 6-methylsalicylic acid synthase ChlB1 from Streptomyces antibioticus. Challenges related to protein folding could be overcome by translation fusion of ChlB1Sa to the C-terminus of the maltose-binding protein MalE from Escherichia coli. Surprisingly, ChlB1Sa was also active in the absence of a heterologous PPTase, which finally led to the discovery that the endogenous PPTase PptACg of C. glutamicum can also activate ChlB1Sa. The best strain, engineered to provide increased levels of acetyl-CoA and malonyl-CoA, accumulated up to 41 mg/L (0.27 mM) 6-methylsalicylic acid within 48 h of cultivation. Further experiments showed that PptACg of C. glutamicum can also activate nonribosomal peptide synthetases (NRPSs), rendering C. glutamicum a promising microbial cell factory for the production of several fine chemicals and medicinal drugs.

Cross-Chemistry Leads to Product Diversity from Atromentin Synthetases in Aspergilli from Section Nigri.

Non-ribosomal peptide synthetase (NRPS)-like enzymes catalyze the non-oxidative homodimerization of aromatic α-keto acids, but the exact reaction mechanism is unknown. The furanone-forming thioesterase domain of the Aspergillus terreus aspulvinone E synthetase MelA displays a predicted quinone-forming motif, whereby its catalytic triad contains an essential cysteine indicating an unusual thioester intermediate. To convert MelA into a quinone-forming atromentin synthetase its thioesterase domain was replaced with that from a Paxillus involutus or A. terreus atromentin synthetase. Phylogenetic proximity of donor and acceptor seems important, as only replacement with the A. terreus thioesterase was functional. Heterologous expression of atromentin synthetases in Aspergillus niger and Aspergillus oryzae revealed host-dependent product formation whereby cross-chemistry directed atromentin biosynthesis in A. niger toward atrofuranic acid. Screening of aspergilli from section Nigri identified an atromentin synthetase in Aspergillus brasiliensis that produced atrofuranic acid in the homologous host. Therefore, cross-chemistry on quinone cores appears common to section Nigri.

An Iterative O-Methyltransferase Catalyzes 1,11-Dimethylation of Aspergillus fumigatus Fumaric Acid Amides.

S-adenosyl-l-methionine (SAM)-dependent methyltransfer is a common biosynthetic strategy to modify natural products. We investigated the previously uncharacterized Aspergillus fumigatus methyltransferase FtpM, which is encoded next to the bimodular fumaric acid amide synthetase FtpA. Structure elucidation of two new A. fumigatus natural products, the 1,11-dimethyl esters of fumaryl-l-tyrosine and fumaryl-l-phenylalanine, together with ftpM gene disruption suggested that FtpM catalyzes iterative methylation. Final evidence that a single enzyme repeatedly acts on fumaric acid amides came from an in vitro biochemical investigation with recombinantly produced FtpM. Size-exclusion chromatography indicated that this methyltransferase is active as a dimer. As ftpA and ftpM homologues are found clustered in other fungi, we expect our work will help to identify and annotate natural product biosynthesis genes in various species.

Dandamycin and chandrananimycin E, benzoxazines from Streptomyces griseus.

Two new benzoxazines were isolated from Streptomyces griseus (HKI 0545) and assigned as chandrananimycin E (1) and dandamycin (2). Although a number of phenoxazinone-type compounds have been reported from nature, phenoxazines are rarer, and carbon substitution at N-10 such as in 1 is unprecedented. The cyclopentene-containing ring structure of dandamycin (2) is also unique. Chandrananimycin E (1) was found to possess moderate antiproliferative activity against HUVEC cells (GI50 35.3 µM) and weak cytotoxic activity towards HeLa cells (CC50 56.9 µM). Dandamycin showed neither antiproliferative activity nor cytotoxicity towards these cell lines. Structure activity comparisons with phenoxazinones isolated from S. griseus HKI 0545 suggested that the alteration of the core ring systems in 1 and 2 diminishes their activity. Natural products 1 and 2 are interesting additions to the rich secondary metabolome of S. griseus and constitute an important addition to the body of knowledge on phenoxazinone-derived metabolites.

Injury-induced biosynthesis of methyl-branched polyene pigments in a white-rotting basidiomycete.

A stereaceous basidiomycete was investigated with regard to its capacity to produce yellow pigments after physical injury of the mycelium. Two pigments were isolated from mycelial extracts, and their structures were elucidated by ESIMS and one- and two-dimensional NMR methods. The structures were identified as the previously undescribed polyenes (3Z,5E,7E,9E,11E,13Z,15E,17E)-18-methyl-19-oxoicosa-3,5,7,9,11,13,15,17-octaenoic acid (1) and (3E,5Z,7E,9E,11E,13E,15Z,17E,19E)-20-methyl-21-oxodocosa-3,5,7,9,11,13,15,17,19-nonaenoic acid (2). Stable-isotope feeding with [1-(13)C]acetate and l-[methyl-(13)C]methionine demonstrated a polyketide backbone and that the introduction of the sole methyl branch is most likely S-adenosyl-l-methionine-dependent. Dose-dependent inhibition of Drosophila melanogaster larval development was observed with both polyenes in concentrations between 12.5 and 100 µM. GI50 values for 1 and 2 against HUVEC (K-562 cells) were 71.6 and 17.4 µM (15.4 and 1.1 µM), respectively, whereas CC50 values for HeLa cells were virtually identical (44.1 and 45.1 µM).

Micromonospora schwarzwaldensis sp. nov., a producer of telomycin, isolated from soil.

A Gram-stain-positive, spore-forming actinomycete strain (HKI0641(T)) was isolated from a soil sample collected in the Black Forest, Germany. During screening for antimicrobial natural products this bacterium was identified as a producer of the antibiotic telomycin. Morphological characteristics and chemotaxonomic data indicated that the strain belonged to the genus Micromonospora. The peptidoglycan of strain HKI0641(T) contained meso-diaminopimelic acid, and the fatty acid profile consisted predominantly of anteiso-C15 : 0, iso-C15 : 0, iso-C16 : 0 and C16 : 0. MK-10(H4), MK-10(H2) and MK-10 were identified as the major menaquinones. To determine the taxonomic positioning of strain HKI0641(T), we computed a binary tanglegram of two rooted phylogenetic trees that were based upon 16S rRNA and gyrB gene sequences. The comparative analysis of the two common classification methods strongly supported the phylogenetic affiliation with the genus Micromonospora, but it also revealed discrepancies in the assignment at the level of the genomic species. 16S rRNA gene sequence analysis identified Micromonospora coxensis DSM 45161(T) (99.1 % sequence similarity) and Micromonospora marina DSM 45555(T) (99.0 %) as the nearest taxonomic neighbours, whereas the gyrB sequence of strain HKI0641(T) indicated a closer relationship to Micromonospora aurantiaca DSM 43813(T) (95.1 %). By means of DNA-DNA hybridization experiments, it was possible to resolve this issue and to clearly differentiate strain HKI0641(T) from other species of the genus Micromonospora. The type strains of the aforementioned species of the genus Micromonospora could be further distinguished from strain HKI0641(T) by several phenotypic properties, such as colony colour, NaCl tolerance and the utilization of carbon sources. The isolate was therefore assigned to a novel species of the genus Micromonospora, for which the name Micromonospora schwarzwaldensis sp. nov. is proposed. The type strain is HKI0641(T) ( = DSM 45708(T) = CIP 110415(T)).

Farinamycin, a quinazoline from Streptomyces griseus.

The metabolic potential of a Streptomyces griseus strain was investigated under various cultivation conditions. After fermentation in a flour-based medium, a new quinazoline metabolite, farinamycin (1), could be isolated from the bacterium, which was previously known only for the production of phenoxazinone antibiotics. The structure of 1 illuminates the biosynthetic versatility of S. griseus, which assembles a defined set of building blocks into structurally diverse natural products.

In vitro cytotoxicity of melleolide antibiotics: structural and mechanistic aspects.

Melleolide sesquiterpene aryl esters are secondary products of the mushroom genus Armillaria. We compared the cytotoxicity of eleven melleolides--five thereof are new natural products--against four human cancer cell lines. Armillaridin, 4-O-methylarmillaridin, and dehydroarmillylorsellinate were most active, at IC(50) = 3.0, 4.1 and 5.0 µM, respectively, against Jurkat T cells for the former two compounds, and K-562 cells for the latter. Dehydroarmillylorsellinate did not inhibit respiration and RNA-synthesis of K-562 cells at 5 µM. However, replication of DNA dropped to 35% after 120 min at this concentration, and translational activity also decreased.

Pitucamycin: structural merger of a phenoxazinone with an epoxyquinone antibiotic.

Chemical profiling of a Streptomyces griseus strain isolated from an old building with moisture damage led to the discovery of two novel phenoxazinones, chandrananimycin D and pitucamycin , along with the known grixazone B. Pitucamycin represents an unprecedented hybrid molecule composed of a phenoxazinone and an enaminomycin-like epoxyquinone moiety.

Function-oriented biosynthesis of beta-lactone proteasome inhibitors in Salinispora tropica.

The natural proteasome inhibitor salinosporamide A from the marine bacterium Salinispora tropica is a promising drug candidate for the treatment of multiple myeloma and mantle cell lymphoma. Using a comprehensive approach that combined chemical synthesis with metabolic engineering, we generated a series of salinosporamide analogues with altered proteasome binding affinity. One of the engineered compounds is equipotent to salinosporamide A in inhibition of the chymotrypsin-like activity of the proteasome yet exhibits superior activity in the cell-based HCT-116 assay.

New cytotoxic cembrane based diterpenes from the soft corals Sarcophyton cherbonnieri and Nephthea sp.

Chemical investigation of the two soft corals Nephthea sp. and Sarcophyton cherbonnieri, collected from the Fiji Islands and the Great Barrier Reef, respectively, led to the isolation of three new furano-cembranoids (1-3), two seco-cembranoid acetates (5, 6), along with the known compounds sarcoglaucol (4) and decaryiol (7). The structures of the new compounds were elucidated by employing spectroscopic techniques (NMR, MS, UV, IR and CD). Seco-cembranoids are extremely rare structures. Compounds 1, 3, and 7 were found to be cytotoxic towards several tumor cell lines (GI50 values ranged from 0.15 to 8.6 micrograms mL-1). Compound 7 arrests the cell cycle in the G2/M phase.