Towards systems metabolic engineering of microorganisms for amino acid production

Microorganisms capable of efficient production of amino acids have traditionally been developed by random mutation and selection method, which might cause unwanted physiological changes in cellular metabolism. Rational genome-wide metabolic engineering based on systems and synthetic biology tools, which is termed 'systems metabolic engineering', is rising as an alternative to overcome these problems. Recently, several amino acid producers have been successfully developed by systems metabolic engineering, where the metabolic engineering procedures were performed within a systems biology framework, and entire metabolic networks, including complex regulatory circuits, were engineered in an integrated manner. Here we review the current status of systems metabolic engineering successfully applied for developing amino acid producing strains and discuss future prospects.     

Characterization of physiological responses to 22 gene knockouts in Escherichia coli central carbon metabolism

Understanding the impact of gene knockouts on cellular physiology, and metabolism in particular, is centrally important to quantitative systems biology and metabolic engineering. Here, we present a comprehensive physiological characterization of wild-type Escherichia coli and 22 knockouts of enzymes in the upper part of central carbon metabolism, including the PTS system, glycolysis, pentose phosphate pathway and Entner–Doudoroff pathway. Our results reveal significant metabolic changes that are affected by specific gene knockouts. Analysis of collective trends and correlations in the data using principal component analysis (PCA) provide new, and sometimes surprising, insights into E. coli physiology. Additionally, by comparing the data-to-model predictions from constraint-based approaches such as FBA, MOMA and RELATCH we demonstrate the important role of less well-understood kinetic and regulatory effects in central carbon metabolism.     

Database and tools for metabolic network analysis

Metabolic network analysis has attracted much attention in the area of systems biology. It has a profound role in understanding the key features of organism metabolic networks and has been successfully applied in several fields of systems biology, including in silico gene knockouts, production yield improvement using engineered microbial strains, drug target identification, and phenotype prediction. A variety of metabolic network databases and tools have been developed in order to assist research in these fields. Databases that comprise biochemical data are normally integrated with the use of metabolic network analysis tools in order to give a more comprehensive result. This paper reviews and compares eight databases as well as twenty one recent tools. The aim of this review is to study the different types of tools in terms of the features and usability, as well as the databases in terms of the scope and data provided. These tools can be categorised into three main types: standalone tools; toolbox-based tools; and web-based tools. Furthermore, comparisons of the databases as well as the tools are also provided to help software developers and users gain a clearer insight and a better understanding of metabolic network analysis. Additionally, this review also helps to provide useful information that can be used as guidance in choosing tools and databases for a particular research interest.     

Systems biology strategy to study lipotoxicity and the metabolic syndrome

Systems biology views and studies the biological systems in the context of complex interactions between their building blocks and processes. Given its multi-level complexity, metabolic syndrome (MetS) makes a strong case for adopting the systems biology approach. Despite many MetS traits being highly heritable, it is becoming evident that the genetic contribution to these traits is mediated via gene–gene and gene–environment interactions across several spatial and temporal scales, and that some of these traits such as lipotoxicity may even be a product of long-term dynamic changes of the underlying genetic and molecular networks. This presents several conceptual as well as methodological challenges and may demand a paradigm shift in how we study the undeniably strong genetic component of complex diseases such as MetS. The argument is made here that for adopting systems biology approaches to MetS an integrative framework is needed which glues the biological processes of MetS with specific physiological mechanisms and principles and that lipotoxicity is one such framework. The metabolic phenotypes, molecular and genetic networks can be modeled within the context of such integrative framework and the underlying physiology.     

Metabolic flux analysis and visualization

One of the ultimate goals of systems biology research is to obtain a comprehensive understanding of the control mechanisms of complex cellular metabolisms. Metabolic Flux Analysis (MFA) is a important method for the quantitative estimation of intracellular metabolic flows through metabolic pathways and the elucidation of cellular physiology. The primary challenge in the use of MFA is that many biological networks are underdetermined systems; it is therefore difficult to narrow down the solution space from the stoichiometric constraints alone. In this tutorial, we present an overview of Flux Balance Analysis (FBA) and (13)C-Metabolic Flux Analysis ((13)C-MFA), both of which are frequently used to solve such underdetermined systems, and we demonstrate FBA and (13)C-MFA using the genome-scale model and the central carbon metabolism model, respectively. Furthermore, because such comprehensive study of intracellular fluxes is inherently complex, we subsequently introduce various pathway mapping and visualization tools to facilitate understanding of these data in the context of the pathways. Specific visualization of MFA results using the BioCyc Omics Viewer and Pathway Projector are shown as illustrative examples.     

Generating orthogonal glycosyltransferase and nucleotide sugar pairs as next-generation glycobiology tools

Protein glycosylation fundamentally impacts biological processes. Nontemplated biosynthesis introduces unparalleled complexity into glycans that needs tools to understand their roles in physiology. The era of quantitative biology is a great opportunity to unravel these roles, especially by mass spectrometry glycoproteomics. However, with high sensitivity come stringent requirements on tool specificity. Bioorthogonal metabolic labeling reagents have been fundamental to studying the cell surface glycoproteome but typically enter a range of different glycans and are thus of limited specificity. Here, we discuss the generation of metabolic ‘precision tools’ to study particular subtypes of the glycome. A chemical biology tactic termed bump-and-hole engineering generates mutant glycosyltransferases that specifically accommodate bioorthogonal monosaccharides as an enabling technique of glycobiology. We review the groundbreaking discoveries that have led to applying the tactic in the living cell and the implications in the context of current developments in mass spectrometry glycoproteomics.     

生理学与基因组学:走向系统生物学

近十年来,生理学与基因组学达到了空前的融合。尽管生理基因组学还是一个非常年轻的研究领域,系统生物学概念的引入必将推进生理基因组学达到全新的水平。本文概要地叙述了这个令人振奋的生理科学的新时代给生理学家带来的机遇和挑战,并以我们自己近十年来的经验为例,讨论了怎样通过扩展和延伸生理学与基因组学的结合,从而对生物学得到系统的理解。     

Physiology of microbial cells and metabolic engineering

This review is devoted to the problems of the physiology and cell biology of microorganisms in relation to metabolic engineering. The latter is considered as a branch of fundamental and applied biotechnology aimed at controlling microbial metabolism by methods of genetic engineering and classical genetics and based on intimate knowledge of cell metabolism. Attention is also given to the problems associated with the metabolic limitation of microbial biosyntheses, analysis and control of metabolic fluxes, rigidity of metabolic pathways, the role of pleiotropic (global) regulatory systems in the control of metabolic fluxes, and prospects of physiological and evolutionary approaches in metabolic engineering.     

Physiology of microbial cell and metabolic engineering

This review is devoted to the problems of the physiology and cell biology of microorganisms in relation to metabolic engineering. The latter is considered as a branch of fundamental and applied biotechnology aimed at controlling microbial metabolism by methods of genetic engineering and classical genetics and based on intimate knowledge of cell metabolism. Attention is also given to the problems associated with the metabolic limitation of microbial biosyntheses, analysis and control of metabolic fluxes, rigidity of metabolic pathways, the role of pleiotropic (global) regulatory systems in the control of metabolic fluxes, and prospects of physiological and evolutionary approaches in metabolic engineering.     

Can yeast systems biology contribute to the understanding of human disease?

Saccharomyces cerevisiae is a unicellular eukaryal microorganism that has traditionally been regarded either as a model system for investigating cellular physiology or as a cell factory for biotechnological use, for example for the production of fuels and commodity chemicals such as lactate or pharmaceuticals, including human insulin and HPV vaccines. Systems biology has recently gained momentum and has successfully been used for mapping complex regulatory networks and resolving the dynamics of signal transduction pathways. So far, yeast systems biology has mainly focused on the development of new methods and concepts. There are also some examples of the application of yeast systems biology for improving biotechnological processes. We discuss here how yeast systems biology could be used in elucidating fundamental cellular principles such as those relevant for the study of molecular mechanisms underlying complex human diseases, including the metabolic syndrome and ageing.     

Toward systems metabolic engineering of Aspergillus and Pichia species for the production of chemicals and biofuels

Recently genome sequence data have become available for Aspergillus and Pichia species of industrial interest. This has stimulated the use of systems biology approaches for large-scale analysis of the molecular and metabolic responses of Aspergillus and Pichia under defined conditions, which has resulted in much new biological information. Case-specific contextualization of this information has been performed using comparative and functional genomic tools. Genomics data are also the basis for constructing genome-scale metabolic models, and these models have helped in the contextualization of knowledge on the fundamental biology of Aspergillus and Pichia species. Furthermore, with the availability of these models, the engineering of Aspergillus and Pichia is moving from traditional approaches, such as random mutagenesis, to a systems metabolic engineering approach. Here we review the recent trends in systems biology of Aspergillus and Pichia species, highlighting the relevance of these developments for systems metabolic engineering of these organisms for the production of hydrolytic enzymes, biofuels and chemicals from biomass.     

Software for systems biology: from tools to integrated platforms

Understanding complex biological systems requires extensive support from software tools. Such tools are needed at each step of a systems biology computational workflow, which typically consists of data handling, network inference, deep curation, dynamical simulation and model analysis. In addition, there are now efforts to develop integrated software platforms, so that tools that are used at different stages of the workflow and by different researchers can easily be used together. This Review describes the types of software tools that are required at different stages of systems biology research and the current options that are available for systems biology researchers. We also discuss the challenges and prospects for modelling the effects of genetic changes on physiology and the concept of an integrated platform.     

Industrial biotechnology: Tools and applications

Industrial biotechnology involves the use of enzymes and microorganisms to produce value-added chemicals from renewable sources. Because of its association with reduced energy consumption, greenhouse gas emissions, and waste generation, industrial biotechnology is a rapidly growing field. Here we highlight a variety of important tools for industrial biotechnology, including protein engineering, metabolic engineering, synthetic biology, systems biology, and downstream processing. In addition, we show how these tools have been successfully applied in several case studies, including the production of 1, 3-propanediol, lactic acid, and biofuels. It is expected that industrial biotechnology will be increasingly adopted by chemical, pharmaceutical, food, and agricultural industries.     

Recent advances on proteins of plant terminal membranes.

Since the beginning of the 1990s, our knowledge of the protein equipment of plant membranes progresses at an accelerating pace, owing to the irruption of molecular biology tools and genetics strategies in plant biology. Map-based cloning strategies and exploration of EST databases rapidly enrich the catalog of cDNA or gene sequences expected to code for membrane proteins. The accumulation of 'putative' membrane proteins reinforces the need for structural, functional and physiological information. Indeed, ambiguities often exist concerning the association to a membrane, the membrane identity and the topology of the protein inserted in the membrane. The combination of directed mutagenesis and heterologous expression of plant genes in various systems and plant reverse genetics has opened the possibility to study molecular and physiological functions. This review will emphasize how these tools have been essential for the exciting recent discoveries on plant terminal membrane proteins. These discoveries concern a variety of transport systems for ions, organic solutes including auxin, water channels, a large collection of systems suspected to act as receptors of chemical signals, proteins thought to control vesicle trafficking and enzymatic systems.