Ribosomally synthesized peptides with antimicrobial properties: biosynthesis, structure, function, and applications

Ribosomally synthesized peptides with antimicrobial properties (antimicrobial peptides-AMPs) are produced by eukaryotes and prokaryotes and represent crucial components of their defense systems against microorganisms. Although they differ in structure, they are nearly all cationic and very often amphiphilic, which reflects the fact that many of them attack their target cells by permeabilizing the cell membrane. They can be roughly categorized into those that have a high content of a certain amino acid, most often proline, those that contain intramolecular disulfide bridges, and those with an amphiphilic region in their molecule if they assume an alpha-helical structure. Most of the known ribosomally synthesized peptides with antimicrobial functions have been identified and studied during the last 20 years. As a result of these studies, new knowledge has been acquired into biology and biochemistry. It has become evident that these peptides may be developed into useful antimicrobial additives and drugs. The use of two-peptide antimicrobial peptides as replacement for clinical antibiotics is promising, though their applications in preservation of foods (safe and effective for use in meat, vegetables, and dairy products), in veterinary medicine, and in dentistry are more immediate. This review focuses on the current status of some of the main types of ribosomally synthesized AMPs produced by eucaryotes and procaryotes and discusses the novel antimicrobial functions, new developments, e.g. heterologous production of bacteriocins by lactic acid bacteria, or construction of multibacteriocinogenic strains, novel applications related to these peptides, and future research paradigms.     

Lantibiotics produced by lactic acid bacteria: structure,function and applications

Lantibiotics are a diverse group of heavily modified antimicrobial and/or signalling peptides produced by a wide range of bacteria, including a variety of lactic acid bacteria. Based on their diverse structures and mode of action, at least six separate lantibiotic subgroups can be suggested, but all subgroups are characterized by significant post-translational modifications, which include the formation of (-methyl)lanthionines, among other unusual alterations. These small peptides are produced, modified, exported, sensed and combated by a complex set of proteins encoded by (usually) co-ordinately regulated operons. In some instances, the production and immunity have been shown to be auto-regulated by the mature lantibiotic. Since their discovery, interest in lantibiotics has been fuelled by their obvious potential as food-grade antimicrobials to improve food safety and quality; a potential which, to date, has been realised only by the longest characterised molecule, nisin. In addition, these peptides are often mooted as alternatives to antibiotics for some biomedical applications. The purpose of this paper is to review recent developments in our understanding of lantibiotic structure, molecular genetics and applications for this unusual class of bacteriocins.     

Metabolic engineering of beta-lactam production

Metabolic engineering has become a rational alternative to classical strain improvement in optimisation of beta-lactam production. In metabolic engineering directed genetic modification are introduced to improve the cellular properties of the production strains. This has resulted in substantial increases in the existing beta-lactam production processes. Furthermore, pathway extension, by heterologous expression of novel genes in well-characterised strains, has led to introduction of new fermentation processes that replace environmentally damaging chemical methods. This minireview discusses the recent developments in metabolic engineering and the applications of this approach for improving beta-lactam production.     

Current status in biotechnological production and applications of glycolipid biosurfactants

Biosurfactants are natural compounds with surface activity and emulsifying properties produced by several types of microorganisms and have been considered an interesting alternative to synthetic surfactants. Glycolipids are promising biosurfactants, due to low toxicity, biodegradability, and chemical stability in different conditions and also because they have many biological activities, allowing wide applications in different fields. In this review, we addressed general information about families of glycolipids, rhamnolipids, sophorolipids, mannosylerythritol lipids, and trehalose lipids, describing their chemical and surface characteristics, recent studies using alternative substrates, and new strategies to improve of production, beyond their specificities. We focus in providing recent developments and trends in biotechnological process and medical and industrial applications.     

Synthesis of alkoxylipids

Improved and new methods for the chemical synthesis of alkoxylipids are described. In addition, some applications of synthetic compounds in biological and physicochemical studies as well as recent developments in the biosynthesis of alkyl and alk-1-enyl glycerolipids are discussed briefly.     

Interpretation of chemical shifts and coupling constants in macromolecules

Recent developments in NMR spectroscopy, along with advances in computational techniques, have produced new approaches to the interpretation of chemical shifts and spin-spin coupling constants in biomolecules. Quantum chemical studies of useful accuracy are now becoming more routine and are increasingly being used in conjunction with experimental studies to map out expected structural patterns for peptides and oligonucleotides. Topics of recent special interest include spin couplings across hydrogen bonds and patterns of chemical shift anisotropies, in both diamagnetic and paramagnetic proteins.     

Advances in laboratory evolution of enzymes

We address recent developments in the area of laboratory, or directed evolution, with a focus on enzymes and on new methodologies of generic potential. We survey three main areas: (i) library making techniques, including the application of computational and rational methods for library design; (ii) screening and selection techniques, including recent applications of enzyme screening by FACS (fluorescence activated cell sorter); (iii) new approaches for performing directed evolution, and in particular, the application of 'neutral drifts' (libraries generated by rounds of mutation and selection for the enzyme's original function) and of consensus mutations to generate highly evolvable starting points for directed evolution.     

A comprehensive review of signal peptides: Structure,roles, and applications

Signal peptides (SP) are short peptides located in the N-terminal of proteins, carrying information for protein secretion. They are ubiquitous to all prokaryotes and eukaryotes. SPs have been of special interest in several scientific and industrial fields, including recombinant protein production, disease diagnosis, immunization, and laboratory techniques. Recently, the role of SPs in recombinant protein production has gained too much attention. Herein, several studies have been reviewed to elucidate the precise structure and function of SPs, particularly the optimized ones for recombinant protein production. However, some features of SPs still have remained obscure. In this review, some approaches concerning elucidation and optimization of SPs are discussed, and pragmatic conclusions and suggestions for future studies are also proposed. Moreover, a summary of secretory pathways, evolutionary changes, functions, applications, and different types of SPs is mentioned. At last, current limitations and prospects are discussed.     

A review of fish-derived antioxidant and antimicrobial peptides: their production, assessment, and applications

Fishes are rich sources of structurally diverse bioactive compounds. In recent years, much attention has been paid to the existence of peptides with biological activities and proteins derived from foods that might have beneficial effects for humans. Antioxidant and antimicrobial peptides isolated from fish sources may be used as functional ingredients in food formulations to promote consumer health and improve the shelf life of food products. This paper presents an overview of the antioxidant and antimicrobial peptides derived from various fishes. In addition, we discuss the extraction of fish proteins, enzymatic production, and the techniques used to isolate and characterize these compounds. Furthermore, we review the methods used to assay the bioactivities and their applications in food and nutraceuticals.     

Flow cytometric analysis of microorganisms

The application of flow cytometry to microorganisms is as old as the technique itself, but it has historically been underexploited for microbial applications. This is now being reversed and microbiologists are ideally placed to benefit from recent technological advances. While earlier papers demonstrated the use of flow cytometry for studies of viability and taxonomy, recent developments in bioinformatics and reporter gene technologies are leading to novel applications in microbiology. Variants of green fluorescent protein have been used for the study of conditional microbial gene regulation in medically important host-pathogen interactions and fluorescence-activated cell sorting is being applied to the isolation of novel mutants in directed evolution studies. This paper reviews the reasons for the delay in the application of flow cytometry to microbial problems, the range of applications, and their limitations and considers the progress made in developing new strategies for use in microbiological investigations.     

Synthesis of new 3-alkoxy-7-amino-4-chloro-isocoumarin derivatives as new beta-amyloid peptide production inhibitors and their activities on various classes of protease

A series of new 7-substituted-4-chloro-3-alkoxy isocoumarin derivatives were synthesized and evaluated as inhibitors of representative classes of proteases: serine protease (alpha-chymotrypsin, trypsin), cysteine protease (Caspase-3), and aspartyl protease (HIV-protease), 20S proteasome and also as inhibitors of amyloid peptide gamma-secretase-mediated production. Protease inhibition selectivity is directly related to the structure of the substituent at the 7-position of the isocoumarin nucleus. 7-Nitro-isocoumarin derivatives (4c, 4d, 4f) are potent alpha-chymotrypsin inhibitors but slightly active or inactive on HIV-protease, as well as on cysteine protease. In contrast, only derivatives bearing a free amino (5d, 5f) or a substituted amino group (6f) at the 7-position of the isocoumarin nucleus, were found weakly active or inactive on alpha-chymotrypsin, trypsin, Caspase-3 and HIV-protease, but prevent gamma-secretase-mediated production of Abeta 40/42 amyloid peptides, which is known to be involved in Alzheimer's disease. Moreover, the most active compounds on beta-amyloid peptide production [JLK6 (5d), JLK2 (5f) and JLK7 (6f)] show only weak or moderate inhibitory activity on the 20S proteasome. The obtained results suggest that the described new isocoumarin analogues could be of interest, since compounds like JLK6 (5d), JLK2 (5f) and JLK7 (6f) can be considered as possible hits for the development of new agents directed towards Alzheimer's disease.     

Peptides for immunological purposes: design, strategies and applications

The development of new vaccines remains an attractive goal for disease prevention and therapy, in combination or alternative to drug-based treatment. In parallel, a growing awareness of the importance of early diagnosis in successful disease management is driving the demand for new reliable diagnostic tools. As a consequence, over the last decades an impressive amount of work has been directed toward the search for new solutions to address vaccine design and biomarker discovery. In this context, peptides have generated considerable interest thanks to their general accessibility and ease of manipulation. The aim of this review is to provide the reader a general picture of the traditional peptide-based strategies adopted in immunology and to report on recent advances made in this field, highlighting advantages and limitations of classic versus innovative approaches. Case studies are described to provide illustrative examples, and cross references to more topic-focused and exhaustive reviews are proposed throughout the text.     

Toxins from cone snails: properties,applications and biotechnological production

Cone snails are marine predators that use venoms to immobilize their prey. The venoms of these mollusks contain a cocktail of peptides that mainly target different voltage- and ligand-gated ion channels. Typically, conopeptides consist of ten to 30 amino acids but conopeptides with more than 60 amino acids have also been described. Due to their extraordinary pharmacological properties, conopeptides gained increasing interest in recent years. There are several conopeptides used in clinical trials and one peptide has received approval for the treatment of pain. Accordingly, there is an increasing need for the production of these peptides. So far, most individual conopeptides are synthesized using solid phase peptide synthesis. Here, we describe that at least some of these peptides can be obtained using prokaryotic or eukaryotic expression systems. This opens the possibility for biotechnological production of also larger amounts of long chain conopeptides for the use of these peptides in research and medical applications.     

Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to beta-lactam antibiotics in Staphylococcus aureus.

A novel penicillin-binding protein, PBP-2' (Mr about 75,000), is known to be induced in excessively large amount by most beta-lactam compounds in cells of a clinically isolated strain of Staphylococcus aureus, TK784, that is highly resistant to beta-lactams and also most other antibiotics. This protein has very low affinities to most beta-lactam compounds and has been supposed to be the cause of the resistance of the cells to beta-lactams. A 14-kilobase DNA fragment was isolated from the cells that carried the gene encoding this penicillin-binding protein and also a genetically linked marker that is responsible for the resistance to tobramycin. This DNA was cloned on plasmid pACYC184 and was shown to cause both production of PBP-2' and resistance to tobramycin in Escherichia coli cells. However, the formation of PBP-2' in E. coli was only moderate and was independent of normal inducer beta-lactams. The PBP-2' formed in the E. coli cells showed slow kinetics of binding to beta-lactams similar to that of PBP-2' formed in the original S. aureus cells and gave a similar pattern of peptides to the latter when digested with the proteolytic V8 enzyme of S. aureus.     

Improvement of hairy root cultures and plants by changing biosynthetic pathways leading to pharmaceutical metabolites: Strategies and applications

A plethora of bioactive plant metabolites has been explored for pharmaceutical, food chemistry and agricultural applications. The chemical synthesis of these structures is often difficult, so plants are favorably used as producers. While whole plants can serve as a source for secondary metabolites and can be also improved by metabolic engineering, more often cell or organ cultures of relevant plant species are of interest. It should be noted that only in few cases the production for commercial application in such cultures has been achieved. Their genetic manipulation is sometimes faster and the production of a specific metabolite is more reliable, because of less environmental influences. In addition, upscaling in bioreactors is nowadays possible for many of these cultures, so some are already used in industry. There are approaches to alter the profile of metabolites not only by using plant genes, but also by using bacterial genes encoding modifying enzymes. Also, strategies to cope with unwanted or even toxic compounds are available. The need for metabolic engineering of plant secondary metabolite pathways is increasing with the rising demand for (novel) compounds with new bioactive properties. Here, we give some examples of recent developments for the metabolic engineering of plants and organ cultures, which can be used in the production of metabolites with interesting properties.     

Xenon for NMR biosensing – Inert but alert

NMR studies with hyperpolarized xenon as functionalized sensor or contrast agent recently made notable progress in developing a new approach for detecting molecular markers and parameters of biomedical interest. Combining spin polarization enhancement with novel indirect detection schemes easily enables a 10⁷-fold signal gain, thus having promising potential to solve the NMR sensitivity problem in many applications. Though an inert element, ¹²⁹Xe has exquisite NMR properties to sense molecular environments. This review summarizes recent developments in the production of hyperpolarized xenon and the design and detection schemes of xenon biosensors.     

Selection of novel ligands from phage display libraries: an alternative approach to drug and vaccine discovery?

Phage display involves the production and screening of large numbers of random peptide sequences of a specific length expressed on the surface of phage particles. This approach provides a powerful tool to probe the molecular basis of many biological processes, including host-parasite interactions. Phage display libraries have been used to study the binding specificity of numerous peptides and protein domains. Practical applications include the identification of peptide sequences that bind with high affinity to antibodies, enzymes or receptors, and that may serve as diagnostics and vaccine or drug candidates. Here, David Jefferies outlines the concept of phage display and summarizes recent developments in the field, with emphasis on those that may be of interest to parasitologists.     

Directed evolution of fungal laccases

Fungal laccases are generalists biocatalysts with potential applications that range from bioremediation to novel green processes. Fuelled by molecular oxygen, these enzymes can act on dozens of molecules of different chemical nature, and with the help of redox mediators, their spectrum of oxidizable substrates is further pushed towards xenobiotic compounds (pesticides, industrial dyes, PAHs), biopolymers (lignin, starch, cellulose) and other complex molecules. In recent years, extraordinary efforts have been made to engineer fungal laccases by directed evolution and semi-rational approaches to improve their functional expression or stability. All these studies have taken advantage of Saccharomyces cerevisiae as a heterologous host, not only to secrete the enzyme but also, to emulate the introduction of genetic diversity through in vivo DNA recombination. Here, we discuss all these endeavours to convert fungal laccases into valuable biomolecular platforms on which new functions can be tailored by directed evolution.     

生产聚羟基脂肪酸酯的微生物细胞工厂

聚羟基脂肪酸酯(PHA)是一类由微生物合成的、生物可再生、生物可降解、具有多种材料学性能的高分子聚合物,在很多领域有着广泛的应用前景。以下从辅酶工程、代谢工程、微氧生产等方面综述了微生物法生产PHA的研究进展,并对利用PHA合成基因提高基因工程菌的代谢潜能进行了讨论。