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.     

利用代谢工程技术提高工业微生物对胁迫的抗性

代谢工程是工业微生物菌种改造的平台技术,不仅可用于改变微生物细胞内的代谢流向,也可以用于改善工业微生物的生理功能。在工业生产过程中,微生物细胞会面临多种胁迫作用,这些胁迫诱导的基因调节作用,都有可能影响细胞的许多重要生理功能,从而影响生物转化过程的效率。从工业应用的观点出发,选择生产性能良好、对发酵过程中的主要胁迫因素有较强耐受性的菌株至关重要。以下评述了借鉴传统代谢工程技术和反向代谢工程技术来提高工业微生物对胁迫抗性的若干研究策略,提出了该领域目前存在的问题,以及利用代谢工程技术改善微生物胁迫抗性——即微生物生理功能工程的发展方向。     

微生物细胞工厂中多基因表达的控制策略

微生物代谢工程和合成生物学是当今微生物技术领域研究的热点,微生物的生长速度快、容易进行大规模培养;遗传背景清楚、遗传操作简便可靠等性质使其在与人类生活相关的多个领域中起到重要的作用。微生物细胞工厂是指人工设计的能够进行物质生产的微生物代谢体系。许多微生物细胞工厂的构建由于引入多个基因或整条代谢途径,而可能导致代谢失衡、部分代谢中间产物积累等问题,需要使用一定的调控策略加以控制。以下对涉及多个基因作用的微生物细胞工厂中所使用的调控策略,分为若干层次进行了总结和探讨,并对今后多基因控制策略的发展方向进行了预测与展望。     

ATP调控策略及其在微生物代谢产物合成中的应用

辅因子工程是代谢工程的一个新兴分支领域,主要通过直接调控细胞内关键酶的辅因子,如ATP/ADP、NADH/NAD+、NADPH/NADP+等的浓度和形式来实现代谢流的最大化,快速地将物质流导向目标代谢物。ATP作为一种重要辅因子参与微生物细胞内大量的酶催化反应,将物质代谢途径串联或并联成复杂的网络体系,最终使得物质代谢流的分配受到牵制。因此ATP调控策略有望成为微生物菌株改造的有利工具,用于提高目标代谢物的浓度和生产能力,强化微生物对于环境的耐受以及促进底物利用等。文中将重点论述目前常用的有效ATP调控策略以及ATP调控对于细胞代谢的影响,以期为微生物细胞工厂的高效构建提供参考。     

转录组平台技术及其在代谢工程中的应用

组学技术在系统水平上对细胞代谢进行全面的分析,极大地促进了代谢工程的发展和应用。全基因组水平的转录分析可以使研究者更加精确地评估细胞表型,加深对细胞代谢的理解。而且转录组分析也有助于研究者鉴定菌种改良的目标基因,加速对微生物细胞工厂的合理设计及构建。文中介绍了3种主要转录组平台技术的原理,并总结了转录组学在代谢工程领域中应用的最新进展和未来发展趋势。     

基于图论和约束优化的异源代谢途径设计方法及应用

构建高产高附加值产品的微生物细胞工厂是代谢工程的研究目标之一,设计高效的产品合成途径是实现这一目标的重要方式.不同微生物底盘因其代谢能力有所差异,故而可以利用的底物和生产的产品范围有限.为了扩大其生产能力,需要进行代谢途径从无到有的设计.传统代谢工程基于经验进行异源途径设计的方式低效且无法确保结果的全面性,而系统生物学...     

Metabolomics assisted metabolic network modeling and network wide analysis of metabolites in microbiology

Metabolomics is the science of qualitatively and quantitatively analyzing low molecular weight metabolites occur in a given biological system. It provides valuable information to elucidate the functional roles and relations of different metabolites in a metabolic pathway. In recent years, a large amount of research on microbial metabolomics has been conducted. It has become a useful tool for achieving highly efficient synthesis of target metabolites. At the same time, many studies have been conducted over the years in order to integrate metabolomics data into metabolic network modeling, which has yielded many exciting results. Additionally, metabolomics also shows great advantages in analyzing the relationship of metabolites network wide. Integrating metabolomics data into metabolic network construction and applying it in network wide analysis of cell metabolism would further improve our ability to control cellular metabolism and optimize the design of cell factories for the overproduction of valuable biochemicals. This review will examine recent progress in the application of metabolomics approaches in metabolic network modeling and network wide analysis of microbial cell metabolism.     

Meta‐Omics‐ and Metabolic Modeling‐Assisted Deciphering of Human Microbiota Metabolism

The human microbiota is a complex community of commensal, symbiotic, and pathogenic microbes that play a crucial role in maintaining the homeostasis of human health. Such a homeostasis is maintained through the collective functioning of enzymatic genes responsible for the production of metabolites, enabling the interaction and signaling within microbiota as well as between microbes and the human host. Understanding microbial genes, their associated chemistries and functions would be valuable for engineering systemic metabolic pathways within the microbiota to manage human health and diseases. Given that there are many unknown gene metabolic functions and interactions, increasing efforts have been made to gain insights into the underlying functions of microbiota metabolism. This can be achieved through culture‐independent metagenomic approaches and metabolic modeling to simulate the microenvironment of human microbiota. In this article, the recent advances in metagenome mining and functional profiling for the discovery of the genetic and biochemical links in human microbiota metabolism as well as metabolic modeling for simulation and prediction of metabolic fluxes in the human microbiota are reviewed. This review provides useful insights into the understanding, reconstruction, and modulation of the human microbiota guided by the knowledge acquired from the basic understanding of the human microbiota metabolism.     

基于比较基因组学重构细菌的代谢途径和调控网络

微生物基因组学的迅速发展为从功能基因与蛋白、网络及其调控等不同的角度,全面理解与认识微生物的代谢过程、构建细胞工厂,提供了丰富的背景信息。基于基因组序列进行代谢网络重构,有助于发现新的代谢功能基因、调控元件、甚至新的代谢途径,从而优化设计细胞内从原料到产品的生物合成路线。然而,目前公共数据库平台中代谢途径基因的功能注释,许多是错误或不完整的。以下着重介绍了近年来出现的一些用于代谢途径和调控网络重构的新型比较基因组学技术,并以近期的丙酮丁醇梭菌中木糖代谢途径的重构工作为例来说明其应用。     

Dynamic control in metabolic engineering: Theories,tools, and applications

Metabolic engineering has allowed the production of a diverse number of valuable chemicals using microbial organisms. Many biological challenges for improving bio-production exist which limit performance and slow the commercialization of metabolically engineered systems. Dynamic metabolic engineering is a rapidly developing field that seeks to address these challenges through the design of genetically encoded metabolic control systems which allow cells to autonomously adjust their flux in response to their external and internal metabolic state. This review first discusses theoretical works which provide mechanistic insights and design choices for dynamic control systems including two-stage, continuous, and population behavior control strategies. Next, we summarize molecular mechanisms for various sensors and actuators which enable dynamic metabolic control in microbial systems. Finally, important applications of dynamic control to the production of several metabolite products are highlighted, including fatty acids, aromatics, and terpene compounds. Altogether, this review provides a comprehensive overview of the progress, advances, and prospects in the design of dynamic control systems for improved titer, rate, and yield metrics in metabolic engineering.     

Temporal fluxomics reveals oscillations in TCA cycle flux throughout the mammalian cell cycle

Cellular metabolic demands change throughout the cell cycle. Nevertheless, a characterization of how metabolic fluxes adapt to the changing demands throughout the cell cycle is lacking. Here, we developed a temporal‐fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle. This is achieved by combining pulse‐chase LC‐MS‐based isotope tracing in synchronized cell populations with computational deconvolution and metabolic flux modeling. We find that TCA cycle fluxes are rewired as cells progress through the cell cycle with complementary oscillations of glucose versus glutamine‐derived fluxes: Oxidation of glucose‐derived flux peaks in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase. These complementary flux oscillations maintain a constant production rate of reducing equivalents and oxidative phosphorylation flux throughout the cell cycle. The shift from glucose to glutamine oxidation in S phase plays an important role in cell cycle progression and cell proliferation.     

A method for accounting for maintenance costs in flux balance analysis improves the prediction of plant cell metabolic phenotypes under stress conditions

Flux balance models of metabolism generally utilize synthesis of biomass as the main determinant of intracellular fluxes. However, the biomass constraint alone is not sufficient to predict realistic fluxes in central heterotrophic metabolism of plant cells because of the major demand on the energy budget due to transport costs and cell maintenance. This major limitation can be addressed by incorporating transport steps into the metabolic model and by implementing a procedure that uses Pareto optimality analysis to explore the trade‐off between ATP and NADPH production for maintenance. This leads to a method for predicting cell maintenance costs on the basis of the measured flux ratio between the oxidative steps of the oxidative pentose phosphate pathway and glycolysis. We show that accounting for transport and maintenance costs substantially improves the accuracy of fluxes predicted from a flux balance model of heterotrophic Arabidopsis cells in culture, irrespective of the objective function used in the analysis. Moreover, when the new method was applied to cells under control, elevated temperature and hyper‐osmotic conditions, only elevated temperature led to a substantial increase in cell maintenance costs. It is concluded that the hyper‐osmotic conditions tested did not impose a metabolic stress, in as much as the metabolic network is not forced to devote more resources to cell maintenance.     

Genome-scale metabolic modeling of P. thermoglucosidasius NCIMB 11955 reveals metabolic bottlenecks in anaerobic metabolism

Parageobacillus thermoglucosidasius represents a thermophilic, facultative anaerobic bacterial chassis, with several desirable traits for metabolic engineering and industrial production. To further optimize strain productivity, a systems level understanding of its metabolism is needed, which can be facilitated by a genome-scale metabolic model. Here, we present p-thermo, the most complete, curated and validated genome-scale model (to date) of Parageobacillus thermoglucosidasius NCIMB 11955. It spans a total of 890 metabolites, 1175 reactions and 917 metabolic genes, forming an extensive knowledge base for P. thermoglucosidasius NCIMB 11955 metabolism. The model accurately predicts aerobic utilization of 22 carbon sources, and the predictive quality of internal fluxes was validated with previously published ¹³C-fluxomics data. In an application case, p-thermo was used to facilitate more in-depth analysis of reported metabolic engineering efforts, giving additional insight into fermentative metabolism. Finally, p-thermo was used to resolve a previously uncharacterised bottleneck in anaerobic metabolism, by identifying the minimal required supplemented nutrients (thiamin, biotin and iron(III)) needed to sustain anaerobic growth. This highlights the usefulness of p-thermo for guiding the generation of experimental hypotheses and for facilitating data-driven metabolic engineering, expanding the use of P. thermoglucosidasius as a high yield production platform.     

代谢流量比率分析及其在代谢工程中的应用

细胞内代谢反应流量在系统理解细胞代谢特性和指导代谢工程改造等方面都起着重要的作用。由于代谢流量难以直接测量得到,在很多情况下通过跟踪稳定同位素在代谢网络中的转移并进行相应的模型计算能有效地定量代谢流量。代谢流量比率分析法能够高度体现系统的生物化学真实性、辨别细胞代谢网络的拓扑结构,并且能够相对简单快速地定量反应速率等,因此受到代谢工程研究者越来越多的重视。以下着重介绍并讨论了利用代谢物同位体分布信息分析关键代谢节点合成途径的流量比率、基于流量比率的代谢流量解析、以及应用于代谢工程等的相关原理、实验测量、数据分析、使用条件等,以期充分发挥代谢流量比率分析法的优势,并将其拓展推广至更多细胞体系的代谢特性阐明和代谢工程改造中去。     

Metabolic fluxes and metabolic engineering

Metabolic engineering is the directed improvement of cellular properties through the modification of specific biochemical reactions or the introduction of new ones, with the use of recombinant DNA technology. As such, metabolic engineering emphasizes metabolic pathway integration and relies on metabolic fluxes as determinants of cell physiology and measures of metabolic control. The combination of analytical methods to quantify fluxes and their control with molecular biological techniques to implement genetic modifications is the essence of metabolic engineering. Strategies for metabolic flux determination are reviewed in this paper and it is shown how metabolic fluxes can be used in the systematic elucidation of metabolic control in the framework of reaction grouping and top-down metabolic control analysis.     

Metabolic flux balance analysis during lactate and glucose concomitant consumption in HEK293 cell cultures

At early stages of the exponential growth phase in HEK293 cell cultures, the tricarboxylic acid cycle is unable to process all the amount of NADH generated in the glycolysis pathway, being lactate the main by-product. However, HEK293 cells are also able to metabolize lactate depending on the environmental conditions. It has been recently observed that one of the most important modes of lactate metabolization is the cometabolism of lactate and glucose, observed even during the exponential growth phase. Extracellular lactate concentration and pH appear to be the key factors triggering the metabolic shift from glucose consumption and lactate production to lactate and glucose concomitant consumption. The hypothesis proposed for triggering this metabolic shift to lactate and glucose concomitant consumption is that HEK293 cells metabolize extracellular lactate as a response to both extracellular protons and lactate accumulation, by means of cotransporting them (extracellular protons and lactate) into the cytosol. At this point, there exists a considerable controversy about how lactate reaches the mitochondrial matrix: the first hypothesis proposes that lactate is converted into pyruvate in the cytosol, and afterward, pyruvate enters into the mitochondria; the second alternative considers that lactate enters first into the mitochondria, and then, is converted into pyruvate. In this study, lactate transport and metabolization into mitochondria is shown to be feasible, as evidenced by means of respirometry tests with isolated active mitochondria, including the depletion of lactate concentration of the respirometry assay. Although the capability of lactate metabolization by isolated mitochondria is demonstrated, the possibility of lactate being converted into pyruvate in the cytosol cannot be excluded from the discussion. For this reason, the calculation of the metabolic fluxes for an HEK293 cell line was performed for the different metabolic phases observed in batch cultures under pH controlled and noncontrolled conditions, considering both hypotheses. The main objective of this study is to evaluate the redistribution of cellular metabolism and compare the differences or similarities between the phases before and after the metabolic shift of HEK293 cells (shift observed when pH is not controlled). That is from a glucose consumption/lactate production phase to a glucose-lactate coconsumption phase. Interestingly, switching to a glucose and lactate cometabolization results in a better-balanced cell metabolism, with decreased glucose and amino acids uptake rates, affecting minimally cell growth. This behavior could be applied to further develop new approaches in terms of cell engineering and to develop improved cell culture strategies in the field of animal cell technology.     

Dynamic modeling of the central carbon metabolism of Escherichia coli

Application of metabolic engineering principles to the rational design of microbial production processes crucially depends on the ability to describe quantitatively the systemic behavior of the central carbon metabolism to redirect carbon fluxes to the product-forming pathways. Despite the importance for several production processes, development of an essential dynamic model for central carbon metabolism of Escherichia coli has been severely hampered by the current lack of kinetic information on the dynamics of the metabolic reactions. Here we present the design and experimental validation of such a dynamic model, which, for the first time, links the sugar transport system (i.e., phosphotransferase system [PTS]) with the reactions of glycolysis and the pentose-phosphate pathway. Experimental observations of intracellular concentrations of metabolites and cometabolites at transient conditions are used to validate the structure of the model and to estimate the kinetic parameters. Further analysis of the detailed characteristics of the system offers the possibility of studying important questions regarding the stability and control of metabolic fluxes.