动物细胞代谢工程进展

动物细胞被越来越广泛地用于工业生产,一些现代化企业已采用分子生物学技术,将一些比较重要的基因导入动物细胞,生产具有医用价值的药物。但该技术并未成熟,主要是因为体外培养的细胞,其生长代谢及生理模式都比较复杂,而且细胞的应答机制还受外界因素的影响。因此,采用细胞代谢工程手段,提高体外培养细胞的生长率、产品产率及有效性,成为人们追求的新目标。我们从细胞代谢中心途径、抑制细胞调亡的因素、细胞生长周期的控制及其相关代谢、多基因共表达代谢工程及糖基化代谢工程等方面对代谢工程进行阐述,为动物细胞的培养提供新的思路。     

Overview of Cell Models: From Organs Cultured in a Petri Dish to Organs-on-Chips

In this review, we tried to elucidate the origin and development of different animal and human cell culture methodologies used to evaluate the effects of various factors and substances in vitro. Organ cultures and conventional two-dimensional cultures of dissociated cells of various types, such as primary, tumor, induced pluripotent, stem, etc., have their advantages and drawbacks but usually do not represent accurate models for studying biological processes that take place in living organisms. Nowadays, high-throughput cell assays on the basis of various methods of signal detection (optical utilizing colorimetric, luminescent and fluorescent methods of detection, and electrochemical) are widely used at early stages of drug development for selection of the most active compounds and evaluation of their cytotoxic effects. The use of animals as models for drug testing is being criticized because of the lack of correlation between the results obtained in studies on them and on humans, and also because of the high cost and ethical issues. Therefore, much effort is put to create models based on human cells. This is how cultures emerged that utilize a three-dimensional network to simulate the architecture of tissues in vivo, and then so-called organs-on-chips—microfluidic microfabricated devices combining several types of cells—that replicate physical and chemical parameters of the microenvironment of cells in living organisms. In summary, experimental cell models have come a long way from the whole organs cultivated in a growth medium to almost complete reconstruction of organs in vitro based on the cutting-edge engineering approach with the use of different cell types. This currently enables one to replicate complex biological processes and study the influence of different substances and factors on them more successfully.     

Inverse metabolic engineering with phosphagen kinase systems improves the cellular energy state

Inverse metabolic engineering attempts to identify or construct desired phenotypes of applied interest to endow them on appropriate host organisms. A particular desirable phenotype is the ATP homeostasis exhibited by animal cells with high and variable ATP turnover through temporal and spatial energy buffering. This buffering is achieved by phosphagen kinase systems that consist of a specific kinase and its cognate phosphagen, which functions as a large pool of 'high-energy phosphates' that are used to replenish ATP during periods of high energetic demand. This review discusses recent advances and potentials of inverse metabolic engineering of cell types that do not normally contain such systems--bacteria, yeast, plants, and liver--with creatine or arginine kinase systems. Examples are discussed that illustrate how microbial metabolism can be tailored for large-scale industrial processes with imperfect mixing and how the liver can be protected from metabolic insults or stimulated for better regeneration.     

Pathway Engineering in Secondary Metabolite-Producing Actinomycetes

Abstract

Actinomycetes represent the microbial group richest in production of variable secondary metabolites. These mostly bioactive molecules are the end products of complex multistep biosynthetic pathways. Recent progress in the molecular genetics and biochemistry of the biosynthetic capacities of actinomycetes enables first attempts to redesign these pathways in a directed fashion. However, in contrast to several examples of designed biochemical improvement of primary metabolic processes in microorganisms, none of the products or strains derived from pathway engineering in actinomycetes discussed herein have reached pilot or production scale. The main reasons for this slow progress are the complicated pathways themselves, their complex regulation during the actinomycete cell cycle, and their uniqueness, as most pathways and products are specific for a strain rather than for a given species or larger taxonomic group. However, the modular use of a minimum of very similar enzymes and their conversion of similar intermediates to form the building blocks for the production of a maximum of divergent end products gives hope for the future application of these genetic models for the redesign of complex pathways for modified or new natural products. Several strategies that can be followed to reach this aim are discussed, mainly for the variable 6-deoxyhexose metabolism as an ubiquitously applicable example.     

Hybrid metabolic flux analysis: combining stoichiometric and statistical constraints to model the formation of complex recombinant products

Background  

Stoichiometric models constitute the basic framework for fluxome quantification in the realm of metabolic engineering. A recurrent bottleneck, however, is the establishment of consistent stoichiometric models for the synthesis of recombinant proteins or viruses. Although optimization algorithms for in silico metabolic redesign have been developed in the context of genome-scale stoichiometric models for small molecule production, still rudimentary knowledge of how different cellular levels are regulated and phenotypically expressed prevents their full applicability for complex product optimization.     

Toward protein engineering for phytoremediation: possibilities and challenges

The combination of rational protein engineering and directed evolution techniques allow for the redesign of enzymes with tailored properties for use in environmental remediation. This review summarizes current molecular methods for either altering or improving protein function and highlights examples of how these methods can address bioremediation problems. Although much of the protein engineering applied to environmental clean-up employs microbial systems, there is great potential for and significant challenges to translating these approaches to plant systems for phytoremediation purposes. Protein engineering technologies combined with genomic information and metabolic engineering strategies hold promise for the design of plants and microbes to remediate organic and inorganic pollutants.     

Microbial metabolism as an evolutionary response: the cybernetic approach to modeling

The growth and metabolic capabilities of microorganisms depend on their interactions with the culture medium. Many media contain two or more key substrates, and an organism may have different preferences for the components. Microorganisms adjust their preferences according to the prevailing conditions so as to favor their own survival. Cybernetic modeling describes this evolutionary strategy by defining a goal that an organism tries to attain optimally at all times. The goal is often, but not always, maximization of growth, and it may require the cells to manipulate their metabolic processes in response to changing environmental conditions. The cybernetic approach overcomes some of the limitations of metabolic control analysis (MCA), but it does not substitute MCA. Here we review the development of the cybernetic modeling of microbial metabolism, how it may be combined with MCA, and what improvements are needed to make it a viable technique for industrial fermentation processes.     

Shining a light on metabolic vulnerabilities in non-small cell lung cancer

Metabolic reprogramming is a hallmark of cancer which contributes to essential processes required for cell survival, growth, and proliferation. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and its genomic classification has given rise to the design of therapies targeting tumors harboring specific gene alterations that cause aberrant signaling. Lung tumors are characterized with having high glucose and lactate use, and high heterogeneity in their metabolic pathways. Here we review how NSCLC cells with distinct mutations reprogram their metabolic pathways and highlight the potential metabolic vulnerabilities that might lead to the development of novel therapeutic strategies.     

Regulated multicistronic expression technology for mammalian metabolic engineering

Contemporary basic research is rapidly revealing increasingly complex molecular regulatory networks which are often interconnected via key signal integrators. These connections among regulatory and catalytic networks often frustrate bioengineers as promising metabolic engineering strategies are bypassed by compensatory metabolic responses or cause unexpected, undesired outcomes such as apoptosis, product protein degradation or inappropriate post- translational modification. Therefore, for metabolic engineering to achieve greater success in mammalian cell culture processes and to become important for future applications such as gene therapy and tissue engineering, this technology must be enhanced to allow simultaneous, in cases conditional, reshaping of metabolic pathways to access difficult-to-attain cell states. Recent advances in this new territory of multigene metabolic engineering are intimately linked to the development of multicistronic expression technology which allows the simultaneous, and in some cases, regulated expression of several genes in mammalian cells. Here we review recent achievements in multicistronic expression technology in view of multigene metabolic engineering. This revised version was published online in August 2006 with corrections to the Cover Date.     

Optimization of energy-consuming pathways towards rapid growth in HPV-transformed cells

Cancer is a complex, multi-step process characterized by misregulated signal transduction and altered metabolism. Cancer cells divide faster than normal cells and their growth rates have been reported to correlate with increased metabolic flux during cell transformation. Here we report on progressive changes in essential elements of the biochemical network, in an in vitro model of transformation, consisting of primary human keratinocytes, human keratinocytes immortalized by human papillomavirus 16 (HPV16) and passaged repeatedly in vitro, and the extensively-passaged cells subsequently treated with the carcinogen benzo[a]pyrene. We monitored changes in cell growth, cell size and energy metabolism. The more transformed cells were smaller and divided faster, but the cellular energy flux was unchanged. During cell transformation the protein synthesis network contracted, as shown by the reduction in key cap-dependent translation factors. Moreover, there was a progressive shift towards internal ribosome entry site (IRES)-dependent translation. The switch from cap to IRES-dependent translation correlated with progressive activation of c-Src, an activator of AMP-activated protein kinase (AMPK), which controls energy-consuming processes, including protein translation. As cellular protein synthesis is a major energy-consuming process, we propose that the reduction in cell size and protein amount provide energy required for cell survival and proliferation. The cap to IRES-dependent switch seems to be part of a gradual optimization of energy-consuming mechanisms that redirects cellular processes to enhance cell growth, in the course of transformation.     

CHOmpact: A reduced metabolic model of Chinese hamster ovary cells with enhanced interpretability

Metabolic modeling has emerged as a key tool for the characterization of biopharmaceutical cell culture processes. Metabolic models have also been instrumental in identifying genetic engineering targets and developing feeding strategies that optimize the growth and productivity of Chinese hamster ovary (CHO) cells. Despite their success, metabolic models of CHO cells still present considerable challenges. Genome-scale metabolic models (GeMs) of CHO cells are very large (>6000 reactions) and are difficult to constrain to yield physiologically consistent flux distributions. The large scale of GeMs also makes the interpretation of their outputs difficult. To address these challenges, we have developed CHOmpact, a reduced metabolic network that encompasses 101 metabolites linked through 144 reactions. Our compact reaction network allows us to deploy robust, nonlinear optimization and ensure that the computed flux distributions are physiologically consistent. Furthermore, our CHOmpact model delivers enhanced interpretability of simulation results and has allowed us to identify the mechanisms governing shifts in the anaplerotic consumption of asparagine and glutamate as well as an important mechanism of ammonia detoxification within mitochondria. CHOmpact, thus, addresses key challenges of large-scale metabolic models and will serve as a platform to develop dynamic metabolic models for the control and optimization of biopharmaceutical cell culture processes.     

Basement membrane remodeling guides cell migration and cell morphogenesis during development

Basement membranes (BMs) are thin, dense forms of extracellular matrix that underlie or surround most animal tissues. BMs are enormously complex and harbor numerous proteins that provide essential signaling, mechanical, and barrier support for tissues during their development and normal functioning. As BMs are found throughout animal tissues, cells frequently migrate, change shape, and extend processes along BMs. Although sometimes used only as passive surfaces by cells, studies in developmental contexts are finding that BMs are often actively modified to help guide cell motility and cell morphogenesis. Here, I provide an overview of recent work revealing how BMs are remodeled in remarkably diverse ways to direct cell migration, cell orientation, axon guidance, and dendrite branching events during animal development.     

Reflections on the scope and the future of metabolic engineering and its connections to functional genomics and drug discovery

Concepts, experience, and tools from metabolic engineering are immediately applicable to the challenge of understanding how the genome influences phenotype. However, new experimental approaches and mathematical and computational resources are needed to maximize the contributions of metabolic engineering to general questions in functional genomics. Among the priorities are systems for studying physiology on a microscale, theoretical tools for understanding biological control systems, and metabolic simulators "in silico" which provide reasonable predictions of stimulus-response relationships at engineering and medical resolution, with incomplete information on cellular mechanisms and their parameters. Approaching cells as complex systems, already a well-established principle in metabolic engineering, is essential to surmount stagnation in the rate of pharmaceutical discovery which is still based on a naive single-target paradigm.     

不同能量的培养条件对人神经母细胞瘤株SH-SY5Y细胞生长的影响

目的建立热量限制的体外模型,观察不同能量培养条件下对人神经母细胞瘤细胞株SH-SY5Y细胞生长代谢的影响。方法将人神经母细胞瘤细胞株SH-SY5Y细胞分别采用含有低浓度（2 g/L）、正常浓度（3.15g/L）或高浓度（4.5 g/L）葡萄糖的培养基进行常规传代培养,利用MTT代谢率、细胞生长曲线及LDH漏出率等指标观察各组细胞生长情况。结果与正常葡萄糖浓度培养条件下培养的对照组相比,高糖组细胞突起缩短,细胞胞体皱缩,MTT代谢率稍低（0.573±0.001）,LDH漏出率高,细胞生长状态差;与对照组相比,低糖组细胞突起伸展,MTT代谢率较低（0.428±0.003）,LDH漏出率低,细胞生长速度缓慢,但是形态良好。结论高糖培养对细胞有损伤作用,细胞代谢加速,更容易衰老死亡;而低糖培养起到保护作用,在热量限制允许范围内降低培养液的含糖量,不但不会对细胞造成损伤,反而对细胞的代谢及生长起到保护作用,延长细胞的总体寿命。     

Biomaterials meet microfluidics: building the next generation of artificial niches

Biomaterials are increasingly being developed as in vitro microenvironments mimicking in vivo stem cell niches. However, current macroscale methodologies to produce these niche models fail to recapitulate the spatial and temporal characteristics of the complex native stem cell regulatory systems. Microfluidic technology offers unprecedented control over the spatial and temporal display of biological signals and therefore promises new avenues for stem cell niche engineering. Here we discuss how the two approaches can be combined to generate more physiological models of stem cell niches that could facilitate the identification of new mechanisms of stem cell regulation, profoundly impacting drug discovery and ultimately therapeutic applications of stem cells.     

Metabolic engineering for the utilization of carbohydrate portions of lignocellulosic biomass

The petrochemical industry has grown to meet the need for massive production of energy and commodities along with an explosive population growth; however, serious side effects such as greenhouse gas emissions and global warming have negatively impacted the environment. Lignocellulosic biomass with myriad quantities on Earth is an attractive resource for the production of carbon-neutral fuels and chemicals through environmentally friendly processes of microbial fermentation. This review discusses metabolic engineering efforts to achieve economically feasible industrial production of fuels and chemicals from microbial cell factories using the carbohydrate portion of lignocellulosic biomass as substrates. The combined knowledge of systems biology and metabolic engineering has been applied to construct robust platform microorganisms with maximum conversion of monomeric sugars, such as glucose and xylose, derived from lignocellulosic biomass. By comprehensively revisiting carbon conversion pathways, we provide a rationale for engineering strategies, as well as their features, feasibility, and recent representative studies. In addition, we briefly discuss how tools in systems biology can be applied in the field of metabolic engineering to accelerate the development of microbial cell factories that convert lignocellulosic biomass into carbon-neutral fuels and chemicals with economic feasibility.     

动植物细胞或组织生物反应器悬浮培养过程中的通气方式

通气在动植物细胞或组织生物反应器培养过程中起着至关重要的作用,而同时通气过程所产生的机械损伤力亦可对细胞造成直接的伤害,因此,通气方式是动植物细胞或组织生物反应器培养过程设计与工程放大的关键技术之一。本文综述了动植物细胞或组织生物反应器悬浮培养过程中三种主要通气(异养培养时又称供氧)方式的结构特点,及其对气液传质、生物量、代谢产物量和细胞损伤的影响,以及改进的新型通气方式和几种通气方式的融合并用。     

Apoptosis in Plants

Apoptosis is a feature of animal cells that explains some aspects of programmed cell death in plants. Differences between plant and animal cell development require that concepts be reexamined to signify how plant cells have evolved the need for cell elimination in the meristematic growth habit, life cycle, and alternation of generations. Central to this theme is the regulation of divisional cycles for mitosis, meiosis, apomeiosis, and their related sexual and asexual reproductive processes. Apoptosis depends on the coordinated expression of genes regulating divisional cycles and apoptotic pathways so that irreversible nuclear and cytoplasmic elimination occurs. Cellular degradation products are salvaged to sustain adaptation, viability, structural function, and ontogeny. The cell wall is usually retained and further differentiated or eliminated. A model of factors predisposing apoptosis and comprising checkpoints in cell divisional cycles is presented for comparisons among plant and animal cells.     

Microbial Metabolism as an Evolutionary Response: The Cybernetic Approach to Modeling

ABSTRACT:?

The growth and metabolic capabilities of microorganisms depend on their interactions with the culture medium. Many media contain two or more key substrates, and an organism may have different preferences for the components. Microorganisms adjust their preferences according to the prevailing conditions so as to favor their own survival. Cybernetic modeling describes this evolutionary strategy by defining a goal that an organism tries to attain optimally at all times. The goal is often, but not always, maximization of growth, and it may require the cells to manipulate their metabolic processes in response to changing environmental conditions.

The cybernetic approach overcomes some of the limitations of metabolic control analysis (MCA), but it does not substitute MCA. Here we review the development of the cybernetic modeling of microbial metabolism, how it may be combined with MCA, and what improvements are needed to make it a viable technique for industrial fermentation processes.

IMTECH communication no.001/2001