应用代谢网络模型解析工业微生物胞内代谢

为了快速、高效地理解工业微生物胞内代谢特征,寻找潜在的代谢工程改造靶点,基因组规模代谢网络模型(GSMM)作为一种系统生物学工具越来越受到人们的关注。文中在回顾GSMM 20年发展历程的基础上,分析了当前GSMM的研究现状,总结了GSMM的构建及分析方法,从预测细胞表型和指导代谢工程两个方面阐述了GSMM在解析工业微生物胞内代谢中的应用,并展望了GSMM未来的发展趋势。     

稳定性同位素探测技术在微生物生态学研究中的应用

稳定性同位素标记技术同分子生物学技术相结合而发展起来的稳定性同位素探测技术（stableisotope probing,SIP）,在对各种环境中微生物群落组成进行遗传分类学鉴定的同时,可确定其在环境过程中的功能,提供复杂群落中微生物相互作用及其代谢功能的大量信息,具有广阔的应用前景.其基本原理是：将原位或微宇宙（microcosm）的环境样品暴露于稳定性同位素富集的基质中,这些样品中存在的某些微生物能够以基质中的稳定（性同位素为碳源或氮源进行物质代谢并满足其自身生长需要,基质中的稳定性同位素被吸收同化进入微生物体内,参与各类物质如核酸（DNA和RNA）及磷脂脂肪酸（PLFA）等的生物合成,通过提取、分离、纯化、分析这些微生物体内稳定性同位素标记的生物标志物,从而将微生物的组成与其功能联系起来.在介绍稳定性同位素培养基质的选择及标记方法、合适的生物标志物的选择及提取分离方法的基础上,举例阐述了此项技术在甲基营养菌、有机污染物降解菌、根际微生物生态、互营微生物、宏基因组学等方面的应用.     

Captured metagenomics: large-scale targeting of genes based on ‘sequence capture’ reveals functional diversity in soils

Microbial enzyme diversity is a key to understand many ecosystem processes. Whole metagenome sequencing (WMG) obtains information on functional genes, but it is costly and inefficient due to large amount of sequencing that is required. In this study, we have applied a captured metagenomics technique for functional genes in soil microorganisms, as an alternative to WMG. Large-scale targeting of functional genes, coding for enzymes related to organic matter degradation, was applied to two agricultural soil communities through captured metagenomics. Captured metagenomics uses custom-designed, hybridization-based oligonucleotide probes that enrich functional genes of interest in metagenomic libraries where only probe-bound DNA fragments are sequenced. The captured metagenomes were highly enriched with targeted genes while maintaining their target diversity and their taxonomic distribution correlated well with the traditional ribosomal sequencing. The captured metagenomes were highly enriched with genes related to organic matter degradation; at least five times more than similar, publicly available soil WMG projects. This target enrichment technique also preserves the functional representation of the soils, thereby facilitating comparative metagenomics projects. Here, we present the first study that applies the captured metagenomics approach in large scale, and this novel method allows deep investigations of central ecosystem processes by studying functional gene abundances.     

宏基因组学及其在口腔微生物研究中的应用

宏基因组是指环境中所有微生物的遗传物质总和。宏基因组学技术可以最大限度地利用环境中的微生物资源,受到了国内外微生物研究者的重点关注。口腔中寄居着大量的微生物群落,以往对口腔疾病微生物的研究大多局限于单纯的细菌培养技术,然而,由于培养技术的局限性,部分微生物很难或根本不能培养,宏基因组学技术打破了这一局限性,帮助人类发掘更丰富的口腔微生物资源。最近,以宏基因组学测序为基础的研究描绘出了口腔生态系统的图谱,越来越多的实验证明口腔微生物组在各种口腔疾病甚至全身系统性疾病中的重要作用。同时,这也为基于人类微生物组的诊断和治疗开辟了新的途径。本综述旨在说明宏基因组学是研究人类口腔疾病及全身疾病相关微生物的得力工具,而且具有广阔的发展前景,同时也讨论了宏基因组学在应用中有待克服的局限性。     

高通量测序技术在食品微生物研究中的应用

高通量测序技术的快速发展对食品微生物发酵过程和机制研究产生了深刻的影响,主要体现在食品微生物生理功能、代谢能力和进化的研究以及食品微生物群落结构、动态变化及其对环境的响应机制等方面。另外,通过对食品微生物基因组和元基因组进行数据分析,也对食品发酵过程优化、微生物功能改造、食源性微生物疾病预防和控制等提供了重要的依据。本文总结了近年来利用高通量测序技术对食品微生物基因组和元基因组进行测序的研究,并探讨了测序技术的发展对食品微生物研究的影响及发展趋势。     

Exploring mixed microbial community functioning: recent advances in metaproteomics

System approaches to elucidate ecosystem functioning constitute an emerging area of research within microbial ecology. Such approaches aim at investigating all levels of biological information (DNA, RNA, proteins and metabolites) to capture the functional interactions occurring in a given ecosystem and track down characteristics that could not be accessed by the study of isolated components. In this context, the study of the proteins collectively expressed by all the microorganisms present within an ecosystem (metaproteomics) is not only crucial but can also provide insights into microbial functionality. Overall, the success of metaproteomics is closely linked to metagenomics, and with the exponential increase in the availability of metagenome sequences, this field of research is starting to experience generation of an overwhelming amount of data, which requires systematic analysis. Metaproteomics has been employed in very diverse environments, and this review discusses the recent advances achieved in the context of human biology, soil, marine and freshwater environments as well as natural and bioengineered systems.     

Systems approaches to succinic acid-producing microorganisms

Succinic acid is a cellular metabolite belonging to the C4-dicarboxylic acid family, and the fermentative production of succinic acid via the use of recombinant microorganisms has recently become the focus of an increasing amount of attention. Considering the difficulty inherent to the direct application of natural succinic acid producers to the industrial process, a variety of systems biology studies have been conducted regarding the development of enhanced succinic acid production systems. This review shows how the metabolic processes of microorganisms, includingEscherichia coli andMannheimia succiniciproducens, have been optimized in order to achieve enhanced succinic acid production. First, their metabolic networks were constructed on the basis of complete genome sequences, after which their metabolic characteristics were estimated viain silico computer modeling. Metabolic engineering strategies were designed in accordance with the results ofin silico modeling and metabolically engineered versions of bothE. coli andM. succiniciproducens have been constructed. The succinic acid productivity and yield obtained using metabolically engineered bacteria was significantly higher than that obtained using wild-type bacteria.     

Systemic approaches to biodegradation

Biodegradation, the ability of microorganisms to remove complex chemicals from the environment, is a multifaceted process in which many biotic and abiotic factors are implicated. The recent accumulation of knowledge about the biochemistry and genetics of the biodegradation process, and its categorization and formalization in structured databases, has recently opened the door to systems biology approaches, where the interactions of the involved parts are the main subject of study, and the system is analysed as a whole. The global analysis of the biodegradation metabolic network is beginning to produce knowledge about its structure, behaviour and evolution, such as its free-scale structure or its intrinsic robustness. Moreover, these approaches are also developing into useful tools such as predictors for compounds' degradability or the assisted design of artificial pathways. However, it is the environmental application of high-throughput technologies from the genomics, metagenomics, proteomics and metabolomics that harbours the most promising opportunities to understand the biodegradation process, and at the same time poses tremendous challenges from the data management and data mining point of view.     

How mutualisms arise in phytoplankton communities: building eco‐evolutionary principles for aquatic microbes

Extensive sampling and metagenomics analyses of plankton communities across all aquatic environments are beginning to provide insights into the ecology of microbial communities. In particular, the importance of metabolic exchanges that provide a foundation for ecological interactions between microorganisms has emerged as a key factor in forging such communities. Here we show how both studies of environmental samples and physiological experimentation in the laboratory with defined microbial co‐cultures are being used to decipher the metabolic and molecular underpinnings of such exchanges. In addition, we explain how metabolic modelling may be used to conduct investigations in reverse, deducing novel molecular exchanges from analysis of large‐scale data sets, which can identify persistently co‐occurring species. Finally, we consider how knowledge of microbial community ecology can be built into evolutionary theories tailored to these species’ unique lifestyles. We propose a novel model for the evolution of metabolic auxotrophy in microorganisms that arises as a result of symbiosis, termed the Foraging‐to‐Farming hypothesis. The model has testable predictions, fits several known examples of mutualism in the aquatic world, and sheds light on how interactions, which cement dependencies within communities of microorganisms, might be initiated.     

Flux control at the ecosystem level

The analysis of metabolic control has reached a high level of understanding of the regulation in cellular metabolic pathways. However, as soon as we leave the realm of cell compartments and enter into the demise of coordination at the organism or ecosystem level, control theory enters unstable ground. Organisms act as individuals. Here, I compare control features at different levels of organization (cell to ecosystem), to indicate how we may approach understanding of control in complex and multiple-species systems.     

Proteome profiling and its use in metabolic and cellular engineering

Proteome profiling of microorganisms makes it possible to generate valuable knowledge that can be used for the development of metabolic and cellular engineering strategies, which consequently are used to enhance the yield and productivity of native or foreign bioproducts and to modify cellular properties to improve mid-stream and down-stream processes. Advances in two-dimensional gel electrophoresis technology combined with mass spectrometry allow the creation of global scale proteome contents which can be used to elucidate valuable information on the dynamics of the metabolic, signaling and regulatory networks apart from understanding the physiological changes. In this paper, we review the approaches of exploiting the proteome profiling results to the development of the strategies for the metabolic and cellular engineering of microorganisms.     

Bacterial amino acid transport proteins: occurrence, functions, and significance for biotechnological applications

Transport processes play a pivotal role in cellular metabolism, e.g. for the uptake of nutrients or the excretion of metabolic waste products. Moreover, they are also important in biotechnological processes such as the production of various amino acids by the use of microorganisms. The focus of this review is on bacterial amino acid transport systems, in particular those of Corynebacterium glutamicum and Escherichia coli, with respect to their function and biotechnological significance.     

Understanding and engineering of microbial cells based on proteomics and its conjunction with other omics studies

The abilities of microorganisms to produce a wide variety of products ranging from human therapeutics to chemicals and to tolerate or detoxify exogenous stresses such as toxic compounds and pollutants are of great importance in fundamental and applied research. Proteomics has become an indispensable tool for large-scale protein analyses and can be used to understand the resulting physiological changes and uncover the mechanisms responsible for the cellular processes under various genetic and environmental conditions. Recent development of a multi-omic approach that combines proteomics with one or more of other omics is allowing us to better understand cellular physiology and metabolism at the systems-wide level, and consequently paving a way toward more efficient metabolic engineering. In this review, we describe the use of proteomics and its combination with other omics to broaden our knowledge on microorganisms in the field of bioscience and biotechnology. With the increasing interest in practical applications, the strategies of employing proteomics for the successful metabolic engineering of microorganisms toward the enhanced production of desired products as well as the approaches taken to identify novel bacterial components are reviewed with corresponding examples.     

DNA稳定同位素探针技术在宏基因组学中的应用及前景

DNA稳定同位素探针 (DNA-SIP) 是一种新兴的技术,通过将同位素稳定结合到特定的底物来确定环境中微生物的作用。DNA-SIP与宏基因组学结合可以让某些微生物的特性与其特殊新陈代谢联系在一起,不仅可以从宏基因组库里检测到低含量的微生物,而且加速了对新的酶类和其他生物活性物质的发现。以下总结了SIP-宏基因组学技术的原理、应用及研究进展,并讨论了其在环境微生物学和生物技术的应用前景。     

Plant Metabolic Modeling: Achieving New Insight into Metabolism and Metabolic Engineering

Models are used to represent aspects of the real world for specific purposes, and mathematical models have opened up new approaches in studying the behavior and complexity of biological systems. However, modeling is often time-consuming and requires significant computational resources for data development, data analysis, and simulation. Computational modeling has been successfully applied as an aid for metabolic engineering in microorganisms. But such model-based approaches have only recently been extended to plant metabolic engineering, mainly due to greater pathway complexity in plants and their highly compartmentalized cellular structure. Recent progress in plant systems biology and bioinformatics has begun to disentangle this complexity and facilitate the creation of efficient plant metabolic models. This review highlights several aspects of plant metabolic modeling in the context of understanding, predicting and modifying complex plant metabolism. We discuss opportunities for engineering photosynthetic carbon metabolism, sucrose synthesis, and the tricarboxylic acid cycle in leaves and oil synthesis in seeds and the application of metabolic modeling to the study of plant acclimation to the environment. The aim of the review is to offer a current perspective for plant biologists without requiring specialized knowledge of bioinformatics or systems biology.     

Bioactive compounds synthesized by non-ribosomal peptide synthetases and type-I polyketide synthases discovered through genome-mining and metagenomics

Non-ribosomal peptide synthetases (NRPS) and type-I polyketide synthases (PKS-I) are multimodular enzymes involved in biosynthesis of oligopeptide and polyketide secondary metabolites produced by microorganisms such as bacteria and fungi. New findings regarding the mechanisms underlying NRPS and PKS-I evolution illustrate how microorganisms expand their metabolic potential. During the last decade rapid development of bioinformatics tools as well as improved sequencing and annotation of microbial genomes led to discovery of novel bioactive compounds synthesized by NRPS and PKS-I through genome-mining. Taking advantage of these technological developments metagenomics is a fast growing research field which directly studies microbial genomes or specific gene groups and their products. Discovery of novel bioactive compounds synthesized by NRPS and PKS-I will certainly be accelerated through metagenomics, allowing the exploitation of so far untapped microbial resources in biotechnology and medicine.     

Metabolic engineering based on systems biology for chemicals production

Microorganisms have been the main sources for the production of chemicals. Production of chemicals requires the development of low-cost and higher-yield processes. Towards this goal, microbial strains with higher levels of production should be first considered. Metabolic engineering has been used extensively over the past two to three decades to increase production of these chemicals. Advances in omics technology and computational simulation are allowing us to perform metabolic engineering at the systems level. By combining the results of omics analyses and computational simulation, systems biology allows us to understand cellular physiology and characteristics, which can subsequently be used for designing strategies. Here, we review the current status of metabolic engineering based on systems biology for chemical production and discuss future prospects.     

Unraveling uncultivable pesticide degraders via stable isotope probing (SIP)

Uncultivable microorganisms account for over 99% of all species on earth, playing essential roles in ecological processes such as carbon/nitrogen cycle and chemical mineralization. Their functions remain unclear in ecosystems and natural habitats, requiring cutting-edge biotechnologies for a deeper understanding. Stable isotope probing (SIP) incorporates isotope-labeled elements, e.g. ^13?C, ^18?O or ^15?N, into the cellular components of active microorganisms, serving as a powerful tool to link phylogenetic identities to their ecological functions in situ. Pesticides raise increasing attention for their persistence in the environment, leading to severe damage and risks to the ecosystem and human health. Cultivation and metagenomics help to identify either cultivable pesticide degraders or potential pesticide metabolisms within microbial communities, from various environmental media including the soil, groundwater, activated sludge, plant rhizosphere, etc. However, the application of SIP in characterizing pesticide degraders is limited, leaving considerable space in understanding the natural pesticide mineralization process. In this review, we try to comprehensively summarize the fundamental principles, successful cases and technical protocols of SIP in unraveling functional-yet-uncultivable pesticide degraders, by raising its shining lights and shadows. Particularly, this study provides deeper insights into various feasible isotope-labeled substrates in SIP studies, including pesticides, pesticide metabolites, and similar compounds. Coupled with other techniques, such as next-generation sequencing, nanoscale secondary ion mass spectrometry (NanoSIMS), single cell genomics, magnetic-nanoparticle-mediated isolation (MMI) and compound-specific isotope analysis (CSIA), SIP will significantly broaden our understanding of pesticide biodegradation process in situ.     

Metabolic engineering based on systems biology for chemicals production

Microorganisms have been the main sources for the production of chemicals. Production of chemicals requires the development of low-cost and higher-yield processes. Towards this goal, microbial strains with higher levels of production should be first considered. Metabolic engineering has been used extensively over the past two to three decades to increase production of these chemicals. Advances in omics technology and computational simulation are allowing us to perform metabolic engineering at the systems level. By combining the results of omics analyses and computational simulation, systems biology allows us to understand cellular physiology and characteristics, which can subsequently be used for designing strategies. Here, we review the current status of metabolic engineering based on systems biology for chemical production and discuss future prospects.