Genetic engineering of macrolide biosynthesis: past advances, current state, and future prospects

Polyketides comprise one of the major families of natural products. They are found in a wide variety of bacteria, fungi, and plants and include a large number of medically important compounds. Polyketides are biosynthesized by polyketide synthases (PKSs). One of the major groups of polyketides are the macrolides, the activities of which are derived from the presence of a macrolactone ring to which one or more 6-deoxysugars are attached. The core macrocyclic ring is biosynthesized from acyl-CoA precursors by PKS. Genetic manipulation of PKS-encoding genes can result in predictable changes in the structure of the macrolactone component, many of which are not easily achieved through standard chemical derivatization or total synthesis. Furthermore, many of the changes, including post-PKS modifications such as glycosylation and oxidation, can be combined for further structural diversification. This review highlights the current state of novel macrolide production with a focus on the genetic engineering of PKS and post-PKS tailoring genes. Such engineering of the metabolic pathways for macrolide biosynthesis provides attractive alternatives for the production of diverse non-natural compounds. Other issues of importance, including the engineering of precursor pathways and heterologous expression of macrolide biosynthetic genes, are also considered.     

Branched-chain amino acid catabolism provides precursors for the Type II polyketide antibiotic,actinorhodin, via pathways that are nutrient dependent

Polyketide antibiotics are among the most important therapeutics used in human and animal health care. Type II polyketides are composed primarily of acetate-derived thioesters, and the subunits for the PKS are contained in a single module that includes a ketosynthase, acyl carrier protein, chain-length factor and sometimes a keto-reductase, aromatase, cyclase and modifying enzymes, such as glycosylases or hydroxylases. While the enzyme complexes that make up the PKS have been the focus of intense study (Khosla in Chem Rev 7:2577–2590, 1997), the pathways for precursor synthesis have not been established and predictions are complicated by the fact that acetate may be derived from a number of metabolic pathways. Here we show that 50% of the acetate for synthesis of the Type II polyketide, actinorhodin, in Streptomyces coelicolor, is derived from the catabolism of the branched amino acids by pathways that are nutrient dependent. The streptomycetes are apparently unique in that they contain two BCDH gene clusters, each of which is potentially capable of converting leucine, valine and isoleucine to the corresponding thioesters, and contain at least three different pathways for valine catabolism that are differentially used in response to nutrient availability.     

A comprehensive catalogue of polyketide synthase gene clusters in lichenizing fungi

Lichens are fungi that form symbiotic partnerships with algae. Although lichens produce diverse polyketides, difficulties in establishing and maintaining lichen cultures have prohibited detailed studies of their biosynthetic pathways. Creative, albeit non-definitive, methods have been developed to assign function to biosynthetic gene clusters in lieu of techniques such as gene knockout and heterologous expressions that are commonly applied to easily cultivatable organisms. We review a total of 81 completely sequenced polyketide synthase (PKS) genes from lichenizing fungi, comprising to our best efforts all complete and reported PKS genes in lichenizing fungi to date. This review provides an overview of the approaches used to locate and sequence PKS genes in lichen genomes, current approaches to assign function to lichen PKS gene clusters, and what polyketides are proposed to be biosynthesized by these PKS. We conclude with remarks on prospects for genomics-based natural products discovery in lichens. We hope that this review will serve as a guide to ongoing research efforts on polyketide biosynthesis in lichenizing fungi.     

植物类型III聚酮合酶超家族基因结构、功能及代谢产物

植物聚酮类化合物主要包括酚类、芪类及类黄酮化合物等,在植物花色、防止紫外线伤害、预防病原菌、昆虫危害以及作为植物与环境互作信号分子方面行使着重要的生物学功能。该类化合物具有显著多样的生物学活性,对人体保健及疾病治疗有显著意义。植物类型III 聚酮化合物合酶 (PKS) 在该类化合物生物合成起始反应中行使着关键作用,决定该类化合物基本分子骨架建成和代谢途径碳硫走向,为合成途径关键酶和限速酶。以查尔酮合酶为原型酶的植物类型III PKS超家族是研究系统进化和蛋白结构与功能关系的模式分子家族,目前已经分离得到14种植物类型III PKS基因,这些同祖同源基因及其表达产物既有共性,也表现出许多独特个性,这些个性赋予此类次生代谢产物结构上的多样性。以下综述了植物类型III PKS超家族基因结构、功能及代谢产物研究进展。     

Inter-domain movements in polyketide synthases: a molecular dynamics study

Insights into the structure and dynamics of modular polyketide synthases (PKS) are essential for understanding the mechanistic details of the biosynthesis of a large number of pharmaceutically important secondary metabolites. The crystal structures of the KS-AT di-domain from erythromycin synthase have revealed the relative orientation of various catalytic domains in a minimal PKS module. However, the relatively large distance between catalytic centers of KS and AT domains in the static structure has posed certain intriguing questions regarding mechanistic details of substrate transfer during polyketide biosynthesis. In order to investigate the role of inter-domain movements in substrate channeling, we have carried out a series of explicit solvent MD simulations for time periods ranging from 10 to 15 ns on the KS-AT di-domain and its sub-fragments. Analyses of these MD trajectories have revealed that both the catalytic domains and the structured inter-domain linker region remain close to their starting structures. Inter-domain movements at KS-linker and linker-AT interfaces occur around hinge regions which connect the structured linker region to the catalytic domains. The KS-linker interface was found to be more flexible compared to the linker-AT interface. However, inter-domain movements observed during the timescale of our simulations do not significantly reduce the distance between catalytic centers of KS and AT domains for facilitating substrate channeling. Based on these studies and prediction of intrinsic disorder we propose that the intrinsically unstructured linker stretch preceding the ACP domain might be facilitating movement of ACP domains to various catalytic centers.     

Evolutionary implications of bacterial polyketide synthases

Polyketide synthases (PKS) perform a stepwise biosynthesis of diverse carbon skeletons from simple activated carboxylic acid units. The products of the complex pathways possess a wide range of pharmaceutical properties, including antibiotic, antitumor, antifungal, and immunosuppressive activities. We have performed a comprehensive phylogenetic analysis of multimodular and iterative PKS of bacteria and fungi and of the distinct types of fatty acid synthases (FAS) from different groups of organisms based on the highly conserved ketoacyl synthase (KS) domains. Apart from enzymes that meet the classification standards we have included enzymes involved in the biosynthesis of mycolic acids, polyunsaturated fatty acids (PUFA), and glycolipids in bacteria. This study has revealed that PKS and FAS have passed through a long joint evolution process, in which modular PKS have a central position. They appear to have derived from bacterial FAS and primary iterative PKS and, in addition, share a common ancestor with animal FAS and secondary iterative PKS. Furthermore, we have carried out a phylogenomic analysis of all modular PKS that are encoded by the complete eubacterial genomes currently available in the database. The phylogenetic distribution of acyltransferase and KS domain sequences revealed that multiple gene duplications, gene losses, as well as horizontal gene transfer (HGT) have contributed to the evolution of PKS I in bacteria. The impact of these factors seems to vary considerably between the bacterial groups. Whereas in actinobacteria and cyanobacteria the majority of PKS I genes may have evolved from a common ancestor, several lines of evidence indicate that HGT has strongly contributed to the evolution of PKS I in proteobacteria. Discovery of new evolutionary links between PKS and FAS and between the different PKS pathways in bacteria may help us in understanding the selective advantage that has led to the evolution of multiple secondary metabolite biosyntheses within individual bacteria.     

On the temperature and pressure dependence of a range of properties of a type of water model commonly used in high-temperature protein unfolding simulations

Molecular dynamics simulations of protein folding and unfolding are often carried out at temperatures (400-600 K) that are much higher than physiological or room temperature to speed up the (un)folding process. Use of such high temperatures changes both the protein and solvent properties considerably, compared to physiological or room temperature. Water models designed for use in conjunction with biomolecules, such as the simple point charge (SPC) model, have generally been calibrated at room temperature and pressure. To determine the distortive effect of high simulation temperatures on the behavior of such "room temperature" water models, the structural, dynamic, and thermodynamic properties of the much-used SPC water model are investigated in the temperature range from 300 to 500 K. Both constant pressure and constant volume conditions, as used in protein simulations, were analyzed. We found that all properties analyzed change markedly with increasing temperature, but no phase transition in this temperature range was observed.     

Accessing natural product biosynthetic processes by mass spectrometry

Two important classes of natural products are made by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). With most biosynthetic intermediates covalently tethered during biogenesis, protein mass spectrometry (MS) has proven invaluable for their interrogation. New mass spectrometric assay formats (such as selective cofactor ejection and proteomics style LC-MS) are showcased here in the context of functional insights into new breeds of NRPS/PKS enzymes, including the first characterization of an 'iterative' PKS, the biosynthesis of the enediyne antitumor antibiotics, the study of a new strategy for PKS initiation via a GNAT-like mechanism, and the analysis of branching strategies in the so-called 'AT-less' NRPS/PKS hybrid systems. The future of MS analysis of NRPS and PKS biosynthetic pathways lies in adoption and development of methods that continue bridging enzymology with proteomics as both fields continue their post-genomic acceleration.     

Molecular Simulations of Membrane Based Separations of Supercritical Electrolyte Solutions

Abstract

Computer simulations of solutions of electrolytes (NaCl and KCl) in supercritical water undergoing membrane based separations have been carried out. These studies used a technique developed recently, in which the system is maintained at steady state by periodically recycling the solvent molecules that permeated the membrane. Our results showed that ionic clusters, formed as a result of water molecules surrounding the ions, play a significant role in these separations. The effect of the main osmotic driving forces, such as pressure, temperature, concentration, and electric fields on the rate of permeation across the membrane was studied. In addition, we also looked at the effect of changes in the pore size and the attractive force between the membrane and solvent/solute. Finally, we examined the stability of the ionic clusters in these simulations.     

Molecular Dynamics Study on Protein and it's Water Structure at High Pressure

Abstract

We have performed NPT molecular dynamics simulations (Langevin Piston Method) on two types of solvated proteins-‘denaturation-unfavorable’ protein (insulin) and ‘denaturation-favorable protein’ (ribonuclease A) at high pressure (from 1 bar up to 20 kbar). The method is based on the extended system formalism introduced by Andersen, where the deterministic equations of motion for the piston degree of freedom are replaced by Langevin equation. We report the structural changes of proteins (ribonuclease A and insulin) and water molecules through radius of gyration, solvent accessible surface area, hydrogen bond pattern, and the topology of water clusters connected by the hydrogen bonded circular network. The solvent accessibility of ribonuclease A is mainly decreased by hydrophilic residues rather than hydrophobic residues under high pressure. From the results of hydrogen bond analysis, we have found that α-helix is more stable than β-sheet under high pressure. In addition, from the analysis of the water cluster, we have observed that for ribonuclease A, 5-membered ring structure is more favorable than 6-membered ring at higher pressure. However, for insulin, the ratio of 5 to 6-ring is constant over the pressure ranges for which we have performed MD simulation. This indicates that the water structure around insulin does not change under high pressure.     

Hinge Twists and Population Shifts Deliver Regulated Catalysis for ATP-PRT in Histidine Biosynthesis

Allosteric regulation plays an important role in the control of metabolic flux in biosynthetic pathways. In microorganisms, many enzymes in these pathways adopt different strategies of allostery to allow the tuning of their activities in response to metabolic demand. Thus, it is important to uncover the mechanism of allosteric signal transmission to fully comprehend the complex control of enzyme function and its evolution. ATP-phosphoribosyltransferase (ATP-PRT), as the first enzyme in the histidine biosynthetic pathway, is allosterically regulated by histidine and offers a good platform for the study of allostery. Two forms of ATP-PRT, namely long and short forms, were discovered that show different arrangements of their regulatory machinery. Crystal structures of the long-form ATP-PRT have revealed overall conformational changes in the inhibited state, but the observed changes in the active state are quite subtle, making the elucidation of its allosteric mechanism difficult. Here, we combine computational methods (ligand docking, quantum mechanics/molecular mechanics optimization, and molecular dynamic simulations) with experimental studies to probe the signal transmission between remote allosteric and active sites. Our results reveal that distinct conformational ensembles of the catalytic domain with different dynamic properties exist in the ligand-free and histidine-bound enzymes. These ensembles display different capabilities in supporting the catalytic and allosteric function of ATP-PRT. The findings give insight into the underlying mechanism of allostery and allow us to propose that the hinge twisting within the catalytic domain is the key for both enhancement of catalysis and provision of regulation in ATP-PRT enzymes.     

Towards Prediction of Metabolic Products of Polyketide Synthases: An In Silico Analysis

Sequence data arising from an increasing number of partial and complete genome projects is revealing the presence of the polyketide synthase (PKS) family of genes not only in microbes and fungi but also in plants and other eukaryotes. PKSs are huge multifunctional megasynthases that use a variety of biosynthetic paradigms to generate enormously diverse arrays of polyketide products that posses several pharmaceutically important properties. The remarkable conservation of these gene clusters across organisms offers abundant scope for obtaining novel insights into PKS biosynthetic code by computational analysis. We have carried out a comprehensive in silico analysis of modular and iterative gene clusters to test whether chemical structures of the secondary metabolites can be predicted from PKS protein sequences. Here, we report the success of our method and demonstrate the feasibility of deciphering the putative metabolic products of uncharacterized PKS clusters found in newly sequenced genomes. Profile Hidden Markov Model analysis has revealed distinct sequence features that can distinguish modular PKS proteins from their iterative counterparts. For iterative PKS proteins, structural models of iterative ketosynthase (KS) domains have revealed novel correlations between the size of the polyketide products and volume of the active site pocket. Furthermore, we have identified key residues in the substrate binding pocket that control the number of chain extensions in iterative PKSs. For modular PKS proteins, we describe for the first time an automated method based on crucial intermolecular contacts that can distinguish the correct biosynthetic order of substrate channeling from a large number of non-cognate combinatorial possibilities. Taken together, our in silico analysis provides valuable clues for formulating rules for predicting polyketide products of iterative as well as modular PKS clusters. These results have promising potential for discovery of novel natural products by genome mining and rational design of novel natural products.     

Chronic metabolic acidosis stimulated transcellular and solvent drag-induced calcium transport in the duodenum of female rats

Chronic metabolic acidosis results in a negative calcium balance as a result of bone resorption and renal calcium loss. However, reports on the changes in intestinal calcium transport have been controversial. The present investigation therefore aimed to study the effects of chronic metabolic acidosis induced by 1.5% NH(4)Cl administration on the three components of duodenal calcium transport, namely, solvent drag-induced, transcellular active, and passive paracellular components, in rats using an in vitro Ussing chamber technique. The relative mRNA expression of genes related to duodenal calcium transport was also determined. We found that 21-day chronic metabolic acidosis stimulated solvent drag-induced and transcellular active duodenal calcium transport but not passive paracellular calcium transport. Our results further demonstrated that an acute direct exposure to serosal acidic pH, in contrast, decreased solvent drag-induced calcium transport in a pH-dependent fashion but had no effect on transcellular active calcium transport. Neither the transepithelial resistance nor duodenal permeability to Na(+), Cl(-), and Ca(2+) via the passive paracellular pathway were altered by chronic metabolic acidosis, suggesting that widening of the tight junction and changes in the charge-selective property of the tight junction did not occur. Thus the enhanced duodenal calcium transport observed in chronic metabolic acidosis could have resulted from a long-term adaptation, possibly at the molecular level. RT-PCR study revealed that chronic metabolic acidosis significantly increased the relative mRNA expression of duodenal genes associated with solvent drag-induced transport, i.e., the beta(1)-subunit of Na(+)-K(+)-ATPase, zonula occludens-1, occludin, and claudin-3, and with transcellular active transport, i.e., transient receptor potential vanilloid family Ca(2+) channels 5 and 6 and plasma membrane Ca(2+)-ATPase isoform 1b. Total plasma calcium and free ionized calcium and magnesium concentrations were also increased, whereas serum parathyroid hormone and 1alpha,25-dihydroxyvitamin D(3) levels were not changed. The results indicated that 21-day chronic metabolic acidosis affected the calcium metabolism in rats partly through enhancing the mRNA expression of crucial duodenal genes involved in calcium absorption, thereby stimulating solvent drag-induced and transcellular active calcium transport in the duodenum.     

Experimental and mathematical approaches to modeling plant metabolic networks

To support their sessile and autotrophic lifestyle higher plants have evolved elaborate networks of metabolic pathways. Dynamic changes in these metabolic networks are among the developmental forces underlying the functional differentiation of organs, tissues and specialized cell types. They are also important in the various interactions of a plant with its environment. Further complexity is added by the extensive compartmentation of the various interconnected metabolic pathways in plants. Thus, although being used widely for assessing the control of metabolic flux in microbes, mathematical modeling approaches that require steady-state approximations are of limited utility for understanding complex plant metabolic networks. However, considerable progress has been made when manageable metabolic subsystems were studied. In this article, we will explain in general terms and using simple examples the concepts underlying stoichiometric modeling (metabolic flux analysis and metabolic pathway analysis) and kinetic approaches to modeling (including metabolic control analysis as a special case). Selected studies demonstrating the prospects of these approaches, or combinations of them, for understanding the control of flux through particular plant pathways are discussed. We argue that iterative cycles of (dry) mathematical modeling and (wet) laboratory testing will become increasingly important for simulating the distribution of flux in plant metabolic networks and deriving rational experimental designs for metabolic engineering efforts.     

Metabolic Changes Precede the Development of Pulmonary Hypertension in the Monocrotaline Exposed Rat Lung

There is increasing interest in the potential for metabolic profiling to evaluate the progression of pulmonary hypertension (PH). However, a detailed analysis of the metabolic changes in lungs at the early stage of PH, characterized by increased pulmonary artery pressure but prior to the development of right ventricle hypertrophy and failure, is lacking in a preclinical animal model of PH. Thus, we undertook a study using rats 14 days after exposure to monocrotaline (MCT), to determine whether we could identify early stage metabolic changes prior to the manifestation of developed PH. We observed changes in multiple pathways associated with the development of PH, including activated glycolysis, increased markers of proliferation, disruptions in carnitine homeostasis, increased inflammatory and fibrosis biomarkers, and a reduction in glutathione biosynthesis. Further, our global metabolic profile data compare favorably with prior work carried out in humans with PH. We conclude that despite the MCT-model not recapitulating all the structural changes associated with humans with advanced PH, including endothelial cell proliferation and the formation of plexiform lesions, it is very similar at a metabolic level. Thus, we suggest that despite its limitations it can still serve as a useful preclinical model for the study of PH.