Combinatorial biosynthesis of reduced polyketides

The bacterial multienzyme polyketide synthases (PKSs) produce a diverse array of products that have been developed into medicines, including antibiotics and anticancer agents. The modular genetic architecture of these PKSs suggests that it might be possible to engineer the enzymes to produce novel drug candidates, a strategy known as 'combinatorial biosynthesis'. So far, directed engineering of modular PKSs has resulted in the production of more than 200 new polyketides, but key challenges remain before the potential of combinatorial biosynthesis can be fully realized.     

Combinatorial biosynthesis of polyketides and nonribosomal peptides

The engineering of polyketide biosynthesis has begun to provide robust targeted libraries for screening against pharmaceutically relevant targets. New technologies that offer methodology for the rapid generation of more structurally diverse libraries have now been demonstrated.     

Probing biosynthesis of plant polyketides with synthetic N-acetylcysteamine thioesters

Recombinant chalcone synthase (CHS) from Scutellaria baicalensis accepted cinnamoyl diketide-NAC and cinnamoyl-NAC as a substrate, and carried out sequential condensations with malonyl-CoA to produce 2',4',6'-trihydroxychalcone. Steady-state kinetic analysis revealed that the CHS accepted the diketide-NAC with less efficiency, while cinnamoyl-NAC primed the enzyme reaction almost as efficiently as cinnamoyl-CoA. On the other hand, it was for the first time demonstrated that the diketide-NAC was also a substrate for recombinant polyketide reductase (PKR) from Glycyrrhiza echinata, and converted to the corresponding beta-ketohemithioester. Furthermore, by co-action of the CHS and the PKR, the NAC-thioesters were converted to 6'-deoxychalcone in the presence of NADPH and malonyl-CoA.     

Nature's assembly line: biosynthesis of simple phenylpropanoids and polyketides

Plants produce large amounts of phenylpropanoids, both in terms of molecular diversity and absolute quantity of these compounds. The phenylpropanoids, and the related plant polyketides, have multiple biological functions. They serve to attract pollinators, support secondary cell-wall growth, provide protection against various plant diseases, and interact with beneficial soil microbes. Their basic chemical properties also make them useful in the biofuel and biomaterial industries. Phenylpropanoid metabolism begins with the amino acid phenylalanine, which feeds into various biosynthetic pathways that generate a wide range of structurally related polyphenolic compounds. This review focuses on four sub-groups of these polyphenolic compounds – polyketides, stilbenes, isoflavones and catechins. We discuss the biosynthesis of these molecules, their physiological role in plants, and their striking pharmacological and physiological effects on humans. This review also highlights metabolic engineering efforts aimed at increasing or decreasing the amounts of each class of compound in various model plants and crops.     

Engineered biosynthesis of regioselectively modified aromatic polyketides using bimodular polyketide synthases

Bacterial aromatic polyketides such as tetracycline and doxorubicin are a medicinally important class of natural products produced as secondary metabolites by actinomyces bacteria. Their backbones are derived from malonyl-CoA units by polyketide synthases (PKSs). The nascent polyketide chain is synthesized by the minimal PKS, a module consisting of four dissociated enzymes. Although the biosynthesis of most aromatic polyketide backbones is initiated through decarboxylation of a malonyl building block (which results in an acetate group), some polyketides, such as the estrogen receptor antagonist R1128, are derived from nonacetate primers. Understanding the mechanism of nonacetate priming can lead to biosynthesis of novel polyketides that have improved pharmacological properties. Recent biochemical analysis has shown that nonacetate priming is the result of stepwise activity of two dissociated PKS modules with orthogonal molecular recognition features. In these PKSs, an initiation module that synthesizes a starter unit is present in addition to the minimal PKS module. Here we describe a general method for the engineered biosynthesis of regioselectively modified aromatic polyketides. When coexpressed with the R1128 initiation module, the actinorhodin minimal PKS produced novel hexaketides with propionyl and isobutyryl primer units. Analogous octaketides could be synthesized by combining the tetracenomycin minimal PKS with the R1128 initiation module. Tailoring enzymes such as ketoreductases and cyclases were able to process the unnatural polyketides efficiently. Based upon these findings, hybrid PKSs were engineered to synthesize new anthraquinone antibiotics with predictable functional group modifications. Our results demonstrate that (i) bimodular aromatic PKSs present a general mechanism for priming aromatic polyketide backbones with nonacetate precursors; (ii) the minimal PKS controls polyketide chain length by counting the number of atoms incorporated into the backbone rather than the number of elongation cycles; and (iii) in contrast, auxiliary PKS enzymes such as ketoreductases, aromatases, and cyclases recognize specific functional groups in the backbone rather than overall chain length. Among the anthracyclines engineered in this study were compounds with (i) more superior activity than R1128 against the breast cancer cell line MCF-7 and (ii) inhibitory activity against glucose-6-phosphate translocase, an attractive target for the treatment of Type II diabetes.     

Aspects of the biosynthesis of non-aromatic fungal polyketides by iterative polyketide synthases

Lovastatin biosynthesis in Aspergillus terreus involves two unusual type I multifunctional polyketide syntheses (PKSs). Lovastatin nonaketide synthase (LNKS), the product of the lovB gene, is an iterative PKS that interacts with LovC, a putative enoyl reductase, to catalyze the 35 separate reactions in the biosynthesis of dihydromonacolin L, a lovastatin precursor. LNKS also displays Diels-Alderase activity in vitro. Lovastatin diketide synthase (LDKS) made by lovF, in contrast, acts non-iteratively like the bacterial modular PKSs to make (2R)-2–methylbutyric acid. Then, like LNKS, LDKS interacts closely with another protein, the LovD transesterase enzyme that catalyzes attachment of the 2–methylbutyric acid to monacolin J in the final step of the lovastatin pathway. Key features of the genes for these four enzymes and others, plus the regulatory and self-resistance factors involved in lovastatin production, are also described.     

The role of 4'-phosphopantetheine in t' biosynthesis of fatty acids, polyketides and peptides

The peptide part of CoA, 4'-phosphopantetheine, has been identified as an essential cofactor in in the biosynthesis of fatty acids, polyketides, depsipeptides, peptides, and compounds derived from both carboxylic and amino acid precursors, like rapamycin. The cofactor is attached to a unique protein moiety, referred to as acyl carrier protein, aminoacyl carrier protein, or peptidyl carrier protein. These carrier proteins are either associated to enzyme complexes (type II) or integrated within multifunctional polypeptide chains (type I). The cofactor is added in a post-translational modification reaction by highly specific transferases, acting on CoA. The functions of carrier proteins in directed condensation reactions are: (1) the selection of substrates to be attached as thioesters, (2) the stabilization of intermediates, (3) the presentation of intermediates for modification by associated enzyme activities, (4) facilitation of the directed condensation reactions of two adjacent intermediates, and (5) assistance in the termination reaction(s) leading to product release.     

海绵体动物中trans-AT聚酮合成酶来源的聚酮化合物的生物合成

海绵体动物分离到的聚酮类化合物很多是由其共生或附生微生物体内的trans-AT聚酮合成酶催化产生的。利用宏基因组技术克隆具有生物活性的聚酮化合物的生物合成基因簇,不但能阐明活性化合物的生物合成路径,而且可以通过异源表达获得目标化合物。本文综述了海绵体动物来源的trans-AT聚酮合成酶产生的聚酮化合物生物合成及其基因簇的研究进展。     

Enzymatic total synthesis of enterocin polyketides

Polyketides are clinically important natural products that often require elaborate organic syntheses owing to their complex chemical structures. Here we report the multienzyme total synthesis of the Streptomyces maritimus enterocin and wailupemycin bacteriostatic agents in a single reaction vessel from simple benzoate and malonate substrates. To our knowledge, our results represent the first in vitro assembly of a complete type II polyketide synthase enzymatic pathway to natural products.     

Functional replacement of the ketosynthase domain of FUM1 for the biosynthesis of fumonisins, a group of fungal reduced polyketides

The genetic manipulation of the biosynthesis of fungal reduced polyketides has been challenging due to the lack of knowledge on the biosynthetic mechanism, the difficulties in the detection of the acyclic, non-aromatic metabolites, and the complexity in genetically manipulating filamentous fungi. Fumonisins are a group of economically important mycotoxins that contaminate maize-based food and feed products worldwide. Fumonisins contain a linear dimethylated C₁₈ chain that is synthesized by Fum1p, which is a single module polyketide synthase (PKS). Using a genetic system that allows the specific manipulation of PKS domains in filamentous fungus Fusarium verticillioides, we replaced the KS domain of fumonisin FUM1 with the KS domain of T-toxin PKS1 from Cochliobolus heterostrophus. Although PKS1 synthesizes different polyketides, the F. verticillioides strain carrying the chimeric PKS produced fumonisins. This represents the first successful domain swapping in PKSs for fungal reduced polyketides and suggests that KS domain alone may not be sufficient to control the product’s structure. To further test if the whole fumonisin PKS could be functionally replaced by a PKS that has a similar domain architecture, we replaced entire FUM1 with PKS1. This strain did not produce any fumonisin or new metabolites, suggesting that the intrinsic interactions between the intact PKS and downstream enzymes in the biosynthetic pathway may play a role in the control of fungal reduced polyketides.     

Biosynthesis of polyketides in heterologous hosts.

Polyketide natural products show great promise as medicinal agents. Typically the products of microbial secondary biosynthesis, polyketides are synthesized by an evolutionarily related but architecturally diverse family of multifunctional enzymes called polyketide synthases. A principal limitation for fundamental biochemical studies of these modular megasynthases, as well as for their applications in biotechnology, is the challenge associated with manipulating the natural microorganism that produces a polyketide of interest. To ameliorate this limitation, over the past decade several genetically amenable microbes have been developed as heterologous hosts for polyketide biosynthesis. Here we review the state of the art as well as the difficulties associated with heterologous polyketide production. In particular, we focus on two model hosts, Streptomyces coelicolor and Escherichia coli. Future directions for this relatively new but growing technological opportunity are also discussed.     

Applications of microbial co-cultures in polyketides production

Polyketides are a large group of natural biomolecules that are normally produced by bacteria, fungi and plants. These molecules have clinical importance due to their anti-cancer, anti-microbial, anti-oxidant and anti-inflammatory properties. Polyketides are biosynthesized from units of acyl-CoA by different polyketide synthases (PKSs), which display wide diversity of functional domains and mechanisms of action between fungi and bacteria. Co-culture of different micro-organisms can produce novel products distinctive from those produced during single cultures. This study compared the new polyketides produced in such co-culture systems and discusses aspects of the cultivation systems, product structures and identification techniques. Current results indicate that the formation of new polyketides may be the result of activation of previously silent PKSs genes induced during co-culture. This review indicated a potential way to produce pure therapeutic polyketides by microbial fermentation and a potential way to develop functional foods and agricultural products using co-co-culture of different micro-organisms. It also pointed out a new perspective for studies on the process of functional foods, especially those involving multiple micro-organisms.     

Computer-aided design of polyketides with the required properties

An approach to rational design of new polyketides with the required spectrum of biological activity has been proposed. We have developed the BioGenPharm software, which generates combinatorial libraries of polyketides based on the user-defined input parameters, performs prediction of biological activity spectra for the generated structures and selection of molecuels with the required properties. PASS algorithm has been applied for prediction of polyketide activity spectra (http://www.ibmc.msk.ru/PASS). Validation of PASS prediction ability for polyketides was performed vs. the evaluation set containing 242 natural macrolides from the Dictionary of Natural Products. The mean prediction accuracy was 75.5%. The problem of choice of the cutting points for probability of the presence of activity (Pa), which provides optimal combination of such parameters as sensitivity, specificity, concordance has been considered. Applicability of the described method has been illustrated by generation of a virtual library of the erythromycin analogues and selection of substances with low probability of the hepatotoxic effect.     

Synthetic biology enabling access to designer polyketides

The full potential of polyketide discovery has yet to be reached owing to a lack of suitable technologies and knowledge required to advance engineering of polyketide biosynthesis. Recent investigations on the discovery, enhancement, and non-natural use of these biosynthetic gene clusters via computational biology, metabolic engineering, structural biology, and enzymology-guided approaches have facilitated improved access to designer polyketides. Here, we discuss recent successes in gene cluster discovery, host strain engineering, precursor-directed biosynthesis, combinatorial biosynthesis, polyketide tailoring, and high-throughput synthetic biology, as well as challenges and outlooks for rapidly generating useful target polyketides.     

Enzymatic formation of unnatural aromatic polyketides by chalcone synthase

Substrate specificity of recombinant chalcone synthase (CHS) from Scutellaria baicalensis (Labiatae) was investigated using chemically synthesized aromatic and aliphatic CoA esters. It was demonstrated for the first time that CHS converted benzoyl-CoA to phlorobenzophenone (2,4,6-trihydroxybenzophenone) along with pyrone by-products. On the other hand, phenylacetyl-CoA was enzymatically converted to an unnatural aromatic polyketide, phlorobenzylketone (2, 4,6-trihydroxyphenylbenzylketone), whose structure was finally confirmed by chemical synthesis. Furthermore, in agreement with earlier reports, S. baicalensis CHS also accepted aliphatic CoA esters, isovaleryl-CoA and isobutyryl-CoA, to produce phloroacylphenones. In contrast, hexanoyl-CoA only afforded pyrone derivatives without formation of a new aromatic ring. It was noteworthy that both aromatic and aliphatic CoA esters were accepted in the active site of the enzyme as a starter substrate for the complex condensation reaction. The low substrate specificity of CHS thus provided further insight into the structure and function of the enzyme.