Recent Advances in Function-based Metagenomic Screening

Metagenomes from uncultured microorganisms are rich resources for novel enzyme genes. The methods used to screen the metagenomic libraries fall into two categories, which are based on sequence or function of the enzymes. The sequence-based approaches rely on the known sequences of the target gene families. In contrast, the function-based approaches do not involve the incorporation of metagenomic sequencing data and, therefore, may lead to the discovery of novel gene sequences with desired functions. In this review, we discuss the function-based screening strategies that have been used in the identification of enzymes from metagenomes. Because of its simplicity, agar plate screening is most commonly used in the identification of novel enzymes with diverse functions. Other screening methods with higher sensitivity are also employed, such as microtiter plate screening. Furthermore, several ultra-high-throughput methods were developed to deal with large metagenomic libraries. Among these are the FACS-based screening, droplet-based screening, and the in vivo reporter-based screening methods. The application of these novel screening strategies has increased the chance for the discovery of novel enzyme genes.     

Signaling advances from immunogenetics to immunogenomics

Recent studies describe new genome-wide mutagenesis strategies, coupled with phenotypic screening, and demonstrate the power of such approaches to provide new insights into the genetics of the immune response.     

ENU诱导点突变——大规模基因突变和功能研究

ENU诱导点突变正在成为一种大规模基因功能研究的有效手段,这里介绍了ENU诱变的机制、影响诱效变效率的因素,ENU诱变的策略,表现的筛选、点突变的鉴定以及相关的研究和取得的一些最新进展。     

Towards high-throughput characterization of small molecule mechanisms of action

Drug discovery is hampered by the lack of general strategies to characterize the mechanisms of action and intracellular targets of bioactive small molecules. Genomics and proteomics promise to aid in this process. Genome-wide approaches in yeast have proven useful to infer the targets and target pathways of small molecules. These approaches are being systematically transferred into mammalian cell culture systems in order to interrogate more complex pathways in a more relevant setting. Advances in proteomics and in vivo genetic screening in multicellular model organism systems are also becoming increasingly powerful and amenable to high-throughput. Current methodologies and technologies are discussed, including how these global approaches complement affinity-based target identification strategies.     

Fragment screening: an introduction

There are clearly many different philosophies associated with adapting fragment screening into mainstream Drug Discovery Lead Generation strategies. Scientists at Astex, for instance, focus entirely on strategies involving use of X-ray crystallography and NMR. However, AstraZeneca uses a number of different fragment screening strategies. One approach is to screen a 2000 compound fragment set (with close to "lead-like" complexity) at 100 microM in parallel with every HTS such that the data are obtained on the entire screening collection at 10 microM plus the extra samples at 100 microM; this provides valuable compound potency data in a concentration range that is usually unexplored. The fragments are then screen-specific "privileged structures" that can be searched for in the rest of the HTS output and other databases as well as having synthesis follow-up. A typical workflow for a fragment screen within AstraZeneca is shown below (Figure 24) and highlights the desirability (particularly when screening >100 microM) for NMR and X-ray information to validate weak hits and give information on how to optimise them. In this chapter, we have provided an introduction to the theoretical and practical issues associated with the use of fragment methods and lead-likeness. Fragment-based approaches are still in an early stage of development and are just one of many interrelated techniques that are now used to identify novel lead compounds for drug development. Fragment based screening has some advantages, but like every other drug hunting strategy will not be universally applicable. There are in particular some practical challenges associated with fragment screening that relate to the generally lower level of potency that such compounds initially possess. Considerable synthetic effort has to be applied for post-fragment screening to build the sort of potency that would be expected to be found from a traditional HTS. However, if there are no low-hanging fruit in a screening collection to be found by HTS then the use of fragment screening can help find novelty that may lead to a target not being discarded as intractable. As such, the approach offers some significant advantages by providing less complex molecules, which may have better potential for novel drug optimisation and by enabling new chemical space to be more effectively explored. Many literature examples that cover examples of fragment screening approaches are still at the "proof of concept" stage and although delivering inhibitors or ligands, may still prove to be unsuitable when further ADMET and toxicity profiling is done. The next few years should see a maturing of the area, and as our understanding of how the concepts can be best applied, there are likely to be many more examples of attractive, small molecule hits, leads and candidate drugs derived from the approaches described.     

Chemical genetics: tailoring tools for cell biology

Chemical genetics is a research approach that uses small molecules as probes to study protein functions in cells or whole organisms. Here, I review the parallels between classical genetic and chemical-genetic approaches and discuss the merits of small molecules to dissect dynamic cellular processes. I then consider the pros and cons of different screening approaches and specify strategies aimed at identifying and validating cellular target proteins. Finally, I highlight the impact of chemical genetics on our current understanding of cell biology and its potential for the future.     

Approaches to library screening

Fuelled by the drive to complete the Human Genome Project, many laboratories have developed new methods of screening clone libraries. From PCR-based strategies to pooling schemes and increased automation, the tedious task of library screening has become less labour-intensive and more cost-efficient. Currently, two main screening methods dominate: hybridization and polymerase chain reaction (PCR). In the following article, we present a brief overview of hybridization and PCR-based screening of yeast and bacterial libraries. Multi-faceted approaches combining different techniques, as well as less frequently employed methods such as fingerprinting are also described.     

Strategies for improving heterologous protein production from filamentous fungi

Despite the naturally high capacity for protein secretion by many species of filamentous fungi, secteted yields of many heterologous proteins have been comparatively low. The strategies for yield improvement have included the use of strong homologous promoters, increased gene copy number, gene fusions with a gene encoding a naturally well-secreted protein, protease-deficient host strains and screening for high yields following random mutagenesis. Such approaches have been effective with some target heterologous proteins but not others.Approaches used in heterologous protein production from filamentous fungi are discussed and a perspective on emerging strategies is presented.     

Dynamic metabolic engineering: New strategies for developing responsive cell factories

Metabolic engineering strategies have enabled improvements in yield and titer for a variety of valuable small molecules produced naturally in microorganisms, as well as those produced via heterologous pathways. Typically, the approaches have been focused on up‐ and downregulation of genes to redistribute steady‐state pathway fluxes, but more recently a number of groups have developed strategies for dynamic regulation, which allows rebalancing of fluxes according to changing conditions in the cell or the fermentation medium. This review highlights some of the recently published work related to dynamic metabolic engineering strategies and explores how advances in high‐throughput screening and synthetic biology can support development of new dynamic systems. Dynamic gene expression profiles allow trade‐offs between growth and production to be better managed and can help avoid build‐up of undesired intermediates. The implementation is more complex relative to static control, but advances in screening techniques and DNA synthesis will continue to drive innovation in this field.     

β-Lactam and glycopeptide antibiotics: first and last line of defense?

Most infections are caused by bacteria, many of which are ever-evolving and resistant to nearly all available antibiotics. β-Lactams and glycopeptides are used to combat these infections by inhibiting bacterial cell-wall synthesis. This mechanism remains an interesting target in the search for new antibiotics in light of failed genomic approaches and the limited input of major pharmaceutical companies. Several strategies have enriched the pipeline of bacterial cell-wall inhibitors; examples include combining screening strategies with lesser-explored microbial diversity, or reinventing known scaffolds based on structure-function relationships. Drugs developed using novel strategies will contribute to the arsenal in fight against the continued emergence of bacterial resistance.     

Mapping protein post-translational modifications with mass spectrometry

Post-translational modifications of proteins control many biological processes, and examining their diversity is critical for understanding mechanisms of cell regulation. Mass spectrometry is a fundamental tool for detecting and mapping covalent modifications and quantifying their changes. Modern approaches have made large-scale experiments possible, screening complex mixtures of proteins for alterations in chemical modifications. By profiling protein chemistries, biologists can gain deeper insight into biological control. The aim of this review is introduce biologists to current strategies in mass spectrometry-based proteomics that are used to characterize protein post-translational modifications, noting strengths and shortcomings of various approaches.     

Advancing chemistry and biology through diversity-oriented synthesis of natural product-like libraries

Natural products provide the inspiration for a variety of strategies used in the diversity-oriented synthesis of novel small-molecule libraries. These libraries can be based on core scaffolds from individual natural products, specific substructures found across a class of natural products, or general structural characteristics of natural products. An increasing body of evidence supports the effectiveness of these strategies for identifying new biologically active molecules. Moreover, these efforts have led to significant advances in synthetic organic chemistry. Larger-scale evaluation of these approaches is on the horizon, using screening data that will be made publicly available in the new PubChem database.     

Target-based discovery of novel herbicides

In the past 10 years, strategies for the first steps of herbicide discovery have switched from the testing of chemicals for efficacy on whole plants towards the use of in-vitro assays against molecular targets. Many different approaches have been developed to identify bona fide targets for in-vitro screening. Developments in functional genomics and in pharmaceutical research could aid the development of assay systems for the evaluation of chemicals for their suitability as lead structures in herbicide discovery.     

Designing specific protein-protein interactions using computation, experimental library screening, or integrated methods

Given the importance of protein-protein interactions for nearly all biological processes, the design of protein affinity reagents for use in research, diagnosis or therapy is an important endeavor. Engineered proteins would ideally have high specificities for their intended targets, but achieving interaction specificity by design can be challenging. There are two major approaches to protein design or redesign. Most commonly, proteins and peptides are engineered using experimental library screening and/or in vitro evolution. An alternative approach involves using protein structure and computational modeling to rationally choose sequences predicted to have desirable properties. Computational design has successfully produced novel proteins with enhanced stability, desired interactions and enzymatic function. Here we review the strengths and limitations of experimental library screening and computational structure-based design, giving examples where these methods have been applied to designing protein interaction specificity. We highlight recent studies that demonstrate strategies for combining computational modeling with library screening. The computational methods provide focused libraries predicted to be enriched in sequences with the properties of interest. Such integrated approaches represent a promising way to increase the efficiency of protein design and to engineer complex functionality such as interaction specificity.     

Target deconvolution from phenotype-based drug discovery by using chemical proteomics approaches

Phenotype-based drug discovery is a key strategy for small molecule drug screening, and the molecular target identification of small molecules, termed “target deconvolution,” is critical albeit challenging. In this review, we classify approaches for target deconvolution, including both direct and indirect approaches, summarize their underlying principles, and provide examples of current chemical proteomics strategies including affinity purification using compound-immobilized beads, photoaffinity labeling (PAL), cellular thermal shift assay (CETSA), and activity-based protein profiling (ABPP). Because there is no single best target deconvolution strategy, it is important to carefully select a strategy on the basis of the test compound characteristics.     

High-throughput RNAi screening to dissect cellular pathways: A how-to guide

RNA interference (RNAi) has become a powerful tool to dissect cellular pathways and characterize gene functions. The availability of genome-wide RNAi libraries for various model organisms and mammalian cells has enabled high-throughput RNAi screenings. These RNAi screens successfully identified key components that had previously been missed in classical forward genetic screening approaches and allowed the assessment of combined loss-of-function phenotypes. Crucially, the quality of RNAi screening results depends on quantitative assays and the choice of the right biological context. In this review, we provide an overview on the design and application of high-throughput RNAi screens as well as data analysis and candidate validation strategies.     

高通量筛选系统在定向改造中的新进展

酶和细胞工厂是工业生物技术的核心,在医药、化工、食品、农业、能源等诸多领域发挥重要作用。一般天然酶和细胞均需通过分子改造提高其催化效率、稳定性及立体选择性等。定向改造为快速改善酶和细胞工厂的性能提供了可能性,其中灵敏可靠的高通量筛选方法是决定酶和细胞工厂成功高效定向改造的关键。文中阐述并分析讨论了各种筛选方法的优缺点、适用范围以及信号产生策略,并总结了近3年超高通量筛选技术在酶和细胞工厂定向改造中的最新研究进展。在此基础上,讨论了高通量筛选系统目前面临的限制性因素,并对高通量筛选方法未来的发展趋势作出了展望。希望生物技术和仪器开发等各领域的研究者能够紧密合作,实现协同发展,进一步提升高通量筛选技术的可靠性和适用性。     

Using display technologies to identify macrocyclic peptide antibiotics

Antibiotic resistant bacterial infections are now a leading cause of global mortality. While drug resistance continues to spread, the clinical antibiotic pipeline has become bare. This discord has focused attention on developing new strategies for antimicrobial discovery. Natural macrocyclic peptide-based products have provided novel antibiotics and antibiotic scaffolds targeting several essential bacterial cell envelope processes, but discovery of such natural products remains a slow and inefficient process. Synthetic strategies employing peptide display technologies can quickly screen large libraries of macrocyclic sequences for specific target binding and general antibacterial potential providing alternative approaches for new antibiotic discovery. Here we review cell envelope processes that can be targeted with macrocyclic peptide therapeutics, outline important macrocyclic peptide display technologies, and discuss future strategies for both library design and screening.     

Illuminating the host – How RNAi screens shed light on host-pathogen interactions

Over millions of years pathogens have coevolved with their respective hosts utilizing host cell functions for survival and replication. Despite remarkable progress in developing antibiotics and vaccination strategies in the last century, infectious diseases still remain a severe threat to human health. Meanwhile, genomic research offers a new era of data-generating platforms that will dramatically enhance our knowledge of pathogens and the diseases they cause. Improvements in gene knockdown studies by RNA interference (RNAi) combined with recent developments in instrumentation and image analysis enable the use of high-throughput screening approaches to elucidate host gene functions exploited by pathogens. Although only a few RNAi-based screens focusing on host genes have been reported so far, these studies have already uncovered hundreds of genes not previously known to be involved in pathogen infection. This review describes recent progress in RNAi screening approaches, highlighting both the limitations and the tremendous potential of RNAi-based screens for the identification of essential host cell factors during infection.     

Directed evolution for enzyme development in biocatalysis

As an important sector of the chemical industry, biocatalysis requires the continuous development of enzymes with tailor-made activity, selectivity, stability, or tolerance to unnatural environments. This is now routinely achieved by directed evolution based on iterative cycles of genetic diversification and activity screening. Here, we highlight its recent developments. First, the design of “smarter” libraries by focused mutagenesis may be a crucial start-up for a fast and successful outcome. Then library assembly and expression are also key steps that benefits from modern molecular biology progresses. Finally, various strategies may be considered for library screening depending on the final objective: while low-throughput direct assays have been very successful in generating enzymes for important biocatalytic processes, even in bringing completely new chemistries to the enzyme world, ultrahigh-throughput screening methods are emerging as powerful approaches for engineering the next generation of industrial enzymes.