Evolution of the clusters of genes for beta-lactam antibiotics: a model for evolutive combinatorial assembly of new beta-lactams.

beta-Lactam antibiotics are produced by prokaryotic and eukaryotic organisms. The genes for beta-lactam biosynthesis are organized in clusters but the location of the different genes is not identical. Biosynthesis genes are clustered with genes for resistance (bla, pbp) and for the efflux of the antibiotic (cmcT) in prokaryotes. Comparison of proteins reveals much larger differences for primary metabolism enzymes than for beta-lactam biosynthesis enzymes in producing organisms. This suggests a horizontal transfer of the beta-lactam antibiotic biosynthesis genes.     

The expression of antibiotic resistance genes in antibiotic‐producing bacteria

Antibiotic‐producing bacteria encode antibiotic resistance genes that protect them from the biologically active molecules that they produce. The expression of these genes needs to occur in a timely manner: either in advance of or concomitantly with biosynthesis. It appears that there have been at least two general solutions to this problem. In many cases, the expression of resistance genes is tightly linked to that of antibiotic biosynthetic genes. In others, the resistance genes can be induced by their cognate antibiotics or by intermediate molecules from their biosynthetic pathways. The regulatory mechanisms that couple resistance to antibiotic biosynthesis are mechanistically diverse and potentially relevant to the origins of clinical antibiotic resistance.     

On the evolution of functional secondary metabolites (natural products)

It is argued that organisms have evolved the ability to biosynthesize secondary metabolites (natural products) because of the selectional advantages they obtain as a result of the functions of the compounds. The clustering together of antibiotic biosynthesis, regulation, and resistance genes implies that these genes have been selected as a group and that the antibiotics function in antagonistic capacities in nature. Pleiotropic switching, the simultaneous expression of sporulation and antibiotic biosynthesis genes, is interpreted in terms of the defence roles of antibiotics. We suggest a general mechanism for the evolution of secondary metabolite biosynthesis pathways, and argue against the hypothesis that modern antibiotics had prebiotic effector functions, on the basis that it does not account for modern biosynthetic pathways.     

Hybrid antibiotics — the contribution of the new gene combinations

Genes coding for entire pathways of antibiotic biosynthesis have been cloned in Streptomyces. Inter-species cloning of antibiotic biosynthesis genes makes it possible to express in the same cell two biosynthetic pathways, which normally operate in different organisms, resulting in the formation of new hybrid structures different from those produced by either parent organism.     

Homology modeling of phosphoryl thymidine kinase of enterohemorrhagic Escherichia coli OH: 157

Enterohemorrhagic Escherichia coli (EHEC) are source of emerging infectious disease in India. Escherichia coli O157:H7 is an EHEC strain showing multiple antibiotic resistances and the cause of infantile diarrhea and hemolytic uremic syndrome worldwide. A novel strategy to counteract multiple antibiotic resistant organisms is to design drugs which specifically target metabolic pathways such as thiamine biosynthetic pathways found exclusively in prokaryotes. Homology modeling was used for model building of a terminal thiamine biosynthesis enzyme phosphoryl thymidine kinase (Thi E) using Geno3D, Swiss Model and Modeller. The best model was selected based on overall stereochemical quality. The potential ligand binding sites in the model were identified by CASTp server. The validated theoretical model of the 3D structure of the thiE protein of E. coli O157:H7 was predicted using a thiamine phosphate pyrophosphatase from Pyrococcus furiosus (PDB ID: 1X13_A) as template. The active pockets of ligand binding sites in the enzyme were identified. In this study, phosphoryl thymidine kinase (thi E), a terminal enzyme in the thiamine biosynthesis pathway in the pathogen has been modeled to be used in future as a potential drug target by the design of suitable inhibitors.     

The evolutionary history of the first three enzymes in pyrimidine biosynthesis

Some metabolic pathways are nearly ubiquitous among organisms: the genes encoding the enzymes for such pathways must therefore be ancient and essential. De novo pyrimidine biosynthesis is an example of one such metabolic pathway. In animals a single protein called CAD

1 Abbreviations: CAD, trifunctional protein catalyzing the first three steps of de novo pyrimidine biosynthesis in higher eukaryotes; CPS, carbamyl phosphate synthetase domain; CPSase, carbamyl phosphate synthetase activity; ATC, aspartate transcarbamylase domain; ATCase, aspartate transcarbamylase activity; DHO, dihydroorotase domain; DHOase, dihydroorotase activity; GLN, glutaminase subdomain or subunit of carbamyl phosphate synthetase, GL Nase, glutaminase activity; SYN, synthetase subdomain or subunit of carbamyl phosphate synthetase; SYNase, synthetase activity.

carries the first three steps of this pathway. The same three enzymes in prokaryotes are associated with separate proteins. The CAD gene appears to have evolved through a process of gene duplication and DNA rearrangement, leading to an in-frame gene fusion encoding a chimeric protein. A driving force for the creation of eukaryotic genes encoding multienzymatic proteins such as CAD may be the advantage of coordinate expression of enzymes catalyzing steps in a biosynthetic pathway. The analogous structure in bacteria is the operon. Differences in the translational mechanisms of eukaryotes and prokaryotes may have dictated the different strategies used by organisms to evolve coordinately regulated genes.     

Three unlinked gene clusters are involved in clavam metabolite biosynthesis in Streptomyces clavuligerus

In Streptomyces clavuligerus, three groups of genes are known to be involved in the biosynthesis of the clavam metabolites. Since antibiotic biosynthetic genes are invariably clustered on the chromosome in prokaryotes, chromosome walking was undertaken in an attempt to show that the three groups of clavam genes would resolve into a single super-cluster when analyzed at larger scale. However, no evidence of linkage between the three groups was obtained. Furthermore, Southern analysis of macro-restriction fragments of genomic DNA separated by pulsed-field gel electrophoresis also indicated that the three groups of genes are not linked. Despite the structural and biosynthetic relatedness of the clavam metabolites, our results suggest that the genes involved in their production lie in three unlinked gene clusters. We believe that this represents the first instance in bacteria of genes involved in the biosynthesis of a single family of antibiotics sharing a common biosynthetic pathway and yet residing in three separate locations on the chromosome.     

Constructing and deconstructing the bacterial cell wall

The history of modern medicine cannot be written apart from the history of the antibiotics. Antibiotics are cytotoxic secondary metabolites that are isolated from Nature. The antibacterial antibiotics disproportionately target bacterial protein structure that is distinct from eukaryotic protein structure, notably within the ribosome and within the pathways for bacterial cell‐wall biosynthesis (for which there is not a eukaryotic counterpart). This review focuses on a pre‐eminent class of antibiotics—the β‐lactams, exemplified by the penicillins and cephalosporins—from the perspective of the evolving mechanisms for bacterial resistance. The mechanism of action of the β‐lactams is bacterial cell‐wall destruction. In the monoderm (single membrane, Gram‐positive staining) pathogen Staphylococcus aureus the dominant resistance mechanism is expression of a β‐lactam‐unreactive transpeptidase enzyme that functions in cell‐wall construction. In the diderm (dual membrane, Gram‐negative staining) pathogen Pseudomonas aeruginosa a dominant resistance mechanism (among several) is expression of a hydrolytic enzyme that destroys the critical β‐lactam ring of the antibiotic. The key sensing mechanism used by P. aeruginosa is monitoring the molecular difference between cell‐wall construction and cell‐wall deconstruction. In both bacteria, the resistance pathways are manifested only when the bacteria detect the presence of β‐lactams. This review summarizes how the β‐lactams are sensed and how the resistance mechanisms are manifested, with the expectation that preventing these processes will be critical to future chemotherapeutic control of multidrug resistant bacteria.     

Enzymes of triacylglycerol synthesis and their regulation

Since the pathways of glycerolipid biosynthesis were elucidated in the 1950's, considerable knowledge has been gained about the enzymes that catalyze the lipid biosynthetic reactions and the factors that regulate triacylglycerol biosynthesis. In the last few decades, in part due to advances in technology and the wide availability of nucleotide and amino acid sequences, we have made enormous strides in our understanding of these enzymes at the molecular level. In many cases, sequence information obtained from lipid biosynthetic enzymes of prokaryotes and yeast has provided the means to search the genomic and expressed sequence tag databases for mammalian homologs and most of the genes have now been identified. Surprisingly, multiple isoforms appear to catalyze the same chemical reactions, suggesting that each isoform may play a distinct functional role in the pathway of triacylglycerol and phospholipid biosynthesis. This review focuses on the de novo biosynthesis of triacylglycerol in eukaryotic cells, the isoenzymes that are involved, their subcellular locations, how they are regulated, and their putative individual roles in glycerolipid biosynthesis.     

The evolutionary role of secondary metabolites--a review.

It is argued that organisms have evolved the ability to biosynthesise secondary metabolites ('natural products') due to the selectional advantages they obtain as a result of the functions of the compounds. Pleiotropic switching, the simultaneous expression of sporulation and antibiotic biosynthesis genes in Streptomyces, is interpreted in terms of the defense roles of antibiotics. The clustering together of antibiotic biosynthesis, regulation, and resistance genes, and in particular the staggering complexity shown in the case of the gene cluster for erythromycin A biosynthesis, implies that these genes have been selected as a group and that the antibiotics function in antagonistic capacities in nature.     

Nutrient stress is a target for new antibiotics

Novel approaches are required to address the looming threat of pan-resistant Gram-negative pathogens and forestall the rise of untreatable infections. Unconventional targets that are uniquely important during infection and tractable to high-throughput drug discovery methods hold high potential for innovation in antibiotic discovery programs. In this context, inhibitors of bacterial nutrient stress are particularly exciting candidates for future antibiotic development. Amino acid, nucleotide, and vitamin biosynthesis pathways are critical for bacterial growth in nutrient-limiting conditions in the laboratory and the host. Although historically dismissed as dispensable for pathogens, a wealth of transposon mutagenesis and single-mutant studies have emerged which demonstrate that several such pathways are critical for infection. Indeed, high-throughput screens of diverse synthetic compounds and natural products have uncovered inhibitors of nutrient biosynthesis. Herein, we review bacterial nutrient biosynthesis and its role during host infection. Further, we explore screening platforms developed to search for inhibitors of these targets and highlight successes among these. Finally, we feature important and sometimes surprising connections between bacterial nutrient biosynthesis, antibiotic activity, and antibiotic resistance.     

Mycobacterium Sulfur Metabolism and Implications for Novel Drug Targets

New antibiotic targets are urgently needed to tackle the multidrug resistant and latent Mycobacterium tuberculosis, the causative agent of the most formidable infectious disease tuberculosis. Sulfur metabolism is essential for the survival and virulence of many pathogens including M. tuberculosis. The absence of most genes involved in microbial sulfur metabolism in human beings suggests abundant novel potential antibiotic targets in pathogen sulfur metabolism. In this article, a comparative genomic landscape of Mycobacterium sulfur metabolism, such as the uptake, activation, and reduction of sulfate and allied enzymes, the biosynthesis pathway of some sulfated metabolites, and the enzymes involved in these pathways were presented. Novel clues for antibiotic targets are put forward.     

Genetic engineering of beta-lactam antibiotic biosynthetic pathways in filamentous fungi.

Recombinant DNA technology has facilitated a rapid increase in our knowledge of beta-lactam antibiotic biosynthesis. Using the tools of this technology, beta-lactam biosynthetic genes and proteins have been characterized at the molecular level, cephalosporin-C production has been improved, new beta-lactams produced, and novel beta-lactam biosynthetic pathways have been constructed.     

Mechanisms of enzymatic CbondO bond cleavages in deoxyhexose biosynthesis

In the past few years, significant progress has been made in our understanding of the biosynthesis of deoxyhexoses. Mechanistic studies have revealed how enzymes can cleave CbondO bonds of a hexose substrate to make unusual sugars. The increasing amount of knowledge about the biosynthesis of deoxysugars may allow the assembly of a repertoire of novel sugar structures through recruitment and collaborative action of genes from a variety of biosynthetic pathways to create diverse secondary metabolites in our search for novel antibiotic/antitumour agents.     

达托霉素生物合成研究进展

达托霉素是由玫瑰孢链霉菌(Streptomyces roseosporus)生产的一种环脂肽类抗生素, 具有强大的抗革兰氏阳性致病细菌的作用, 是继“抗生素最后一道防线”万古霉素后的新型抗生素。本文主要对达托霉素的结构、作用机制、合成基因簇及合成机制等当前的研究成果进行综述, 并且总结了利用组合生物学对达托霉素进行结构改造的策略, 以此来研究结构与活性之间的关系, 并寻找更广谱高效的抗生素。最后, 本文总结了提高达托霉素产量的策略, 为工业上降低达托霉素生产成本提供理论参考。     

林可霉素生物合成的研究进展

林可霉素是林可链霉菌(Streptomyces lincolnensis)产生的林可酰胺类抗生素,它抑制细菌细胞的蛋白质合成,临床上主要用于治疗革兰氏阳性菌引起的感染性疾病。林可霉素生物合成基因簇已被克隆和测序。近年来,围绕林可酰胺和丙基脯氨酸的生物合成、调控等进行了深入研究,其硫化反应取得了突破性成果,本文综述了林可霉素生物合成的新进展。     

Metabolic and pathway engineering to influence native and altered erythromycin production through E. coli

The heterologous production of the complex antibiotic erythromycin through Escherichia coli provides a unique challenge in metabolic engineering. In addition to introducing the 19 foreign genes needed for heterologous biosynthesis, E. coli metabolism must be engineered to provide the propionyl-CoA and (2S)-methylmalonyl-CoA substrates required to allow erythromycin formation. In this work, three different pathways to propionyl-CoA were compared in the context of supporting E. coli erythromycin biosynthesis. The comparison revealed that alternative citramalate and threonine metabolic pathways (both starting from exogenous glycerol) were capable of supporting final compound formation equal to a proven pathway reliant upon exogenous propionate. Furthermore, two pathways to (2S)-methylmalonyl-CoA were compared in the production of a novel benzyl-erythromycin analog. A pathway dependent upon exogenous methylmalonate improved selectivity and facilitated antibiotic assessment of this new analog.