形态工程在生物基化学品生产中的应用进展

合成生物学和代谢工程是构建微生物细胞工厂、实现化学品绿色生物制造的重要方法,目前主要集中在微生物代谢网络的改造及调控上,很少考虑到微生物细胞特性的影响。形态工程通过改造微生物细胞形态相关蛋白,有目的地对微生物细胞形态及分裂方式进行合理调控,从而优化微生物细胞的特性,是降低生物炼制成本的一种新兴生物工程技术。文中首先介绍了与微生物细胞形态相关的各类蛋白,并重点总结了形态工程在生物基化学品合成方面的应用进展,包括调控细胞体积以提高胞内产物积累量、改善细胞通透性以促进胞外产物分泌、实现高密度发酵以降低生产成本、控制产物水解程度以提高产品性能。最后,提出了形态工程面临的主要问题并展望了其未来的发展趋势。     

应用合成生物学构建蓝细菌细胞工厂的研究进展

蓝细菌是一类能进行放氧光合作用的原核微生物,具有生长速度快、光合效率高、易于基因遗传操作等特点。它们能够将捕获的光能和二氧化碳转化为生物能源分子,在解决当前社会面临的能源紧缺和环境污染等问题上有着重要的理论和应用研究价值。近年来,随着合成生物学的迅猛发展,构建以蓝细菌为底盘的“人工细胞工厂”用于合成各类生物能源和精细化学产品取得了令人瞩目的成绩。重点介绍了应用合成生物学构建蓝细菌细胞合成工厂的研究进展,并对“光合自养型细胞工厂”面临的两大问题——产物毒性问题以及细胞内氧化胁迫问题进行了重点讨论。     

微生物代谢工程的研究进展和展望

代谢工程技术是构建微生物细胞工厂的重要方法,其主要目标是通过基因工程等手段将目标代谢产物产量最大化。然而基因工程等操作往往会影响细胞生长速率,导致其生产强度降低。随着合成生物学及相关技术的发展,多种调控策略被应用于代谢工程领域以解决上述问题。通过这些调控可以有效地解决细胞生长与产物合成之间的竞争关系,平衡代谢途径,避免中间代谢产物的过量积累。对这些策略的研究及应用进行了概述和展望。     

基于模式微生物生长特性调控的食品功能因子生物制造研究进展

食品功能因子作为功能性食品制造的基础素材,是食品中真正起生理作用的有效成分,在调节人体机能,改善睡眠和促进生长发育等方面发挥着重要作用.合成生物学作为一种更安全、更健康和绿色可持续的食品获取方式,已经成为推动食品行业发展的重要技术支撑.食品合成生物技术主要通过采用合成生物学技术设计构建食品组分的合成途径,创建具有食品工业应用能力的智能化细胞工厂,大幅提升食品功能因子等高附加值产品的合成效率.目前,以大肠杆菌、枯草芽孢杆菌、谷氨酸棒杆菌和酿酒酵母等模式微生物作为合成载体,通过对其生长进行精细调控,食品功能因子的生物制造已取得重大进展.本文主要从转运蛋白工程改造提高细胞生长速率、重编程细胞能量代谢和平衡细胞生长与产物合成方面总结了基于模式微生物生长特性调控合成食品功能因子的研究策略和进展,提出了目前所面临的挑战,并对其未来发展做出展望.     

辅因子在微生物细胞工厂中的代谢调控与应用*

生物体中大部分酶催化反应都需要辅因子参与,辅因子平衡对维持正常的细胞代谢至关重要,而辅因子失衡则会导致细胞生长和生产的紊乱。在微生物细胞工厂的构建中,通过调节辅因子代谢平衡来提高产物合成途径的效率,从而调控细胞生长与产物生产,使代谢流能够最大限度地流向目标产物,已经成为代谢调控的重要手段。目前常见的用于代谢调控的辅因子有NAD(P)H/NAD(P)⁺、辅酶、ATP/ADP等。围绕这几种辅因子的代谢途径及功能分类进行了综述,并总结了微生物中不同产物利用辅因子平衡策略进行合成调控的研究,以期为各类化合物的高效生物合成提供参考。     

合成支架在代谢工程中的研究进展

代谢工程作为通过引入外源合成途径或改造优化代谢网络,进行高附加值的天然代谢产物生物合成的技术,已经得到广泛应用。但随着目标合成产物的结构日渐复杂,构建多基因的从头合成途径造成宿主生物代谢失衡与中间产物对宿主细胞产生毒害作用等一系列问题发生的可能性也随之增加。为解决这些问题合成支架策略应运而生,合成支架将途径酶共定位以提高局部酶和代谢物的浓度,来增强代谢通量并限制中间产物与宿主细胞环境间的相互作用,成为生物催化和合成生物学研究的热点之一。尽管由核酸、蛋白质构成的合成支架策略已经应用于多种代谢物的异源合成,并取得了不同程度的成功,但合成支架的精确组装仍然是一项艰巨的任务。文中详细介绍了合成支架技术的研究现状,详细阐述了合成支架技术的原理和实例,并初步探讨了其应用前景。     

合成生物学在微生物细胞工厂构建中的应用

通过微生物发酵的方法生产大宗化学品和天然产物能够部分替代石油化工炼制和植物提取。合成生物学技术的发展极大地提高了构建微生物细胞工厂生产大宗化学品和天然产物的能力。一方面综述了合成生物学在构建细胞工厂时的关键技术,包括最优合成途径的设计、合成途径的创建与优化、细胞性能的优化;另一方面,介绍了应用这些技术构建细胞工厂生产燃料化学品、大宗化学品和天然产物的典型案例。     

2013合成生物学专刊序言

合成生物学目前在全球得到迅猛发展。在此专刊中,综述了一些相关技术在合成生物学领域的进展,其中有:链霉菌无痕敲除方法、基因合成技术、DNA组装新方法、最小化基因组的方法及分析、合成生物系统的组合优化。也讨论了应用合成生物学策略优化光合蓝细菌底盘、产溶剂梭菌分子遗传操作技术、蛋白质预算(Protein budget)作为合成生物学的成本标尺。最后,用几个例子说明了合成生物学的应用,包括复杂天然产物合成人工生物系统的设计与构建、微生物木糖代谢途径改造制备生物基化学品以及构建酿酒酵母工程菌合成香紫苏醇。     

居瘤胃解纤维素菌细胞密度与纤维素酶的合成

居瘤胃解纤维素菌(Cellulosilyticum ruminicola)H1是本实验室分离自青海牦牛瘤胃的一株新的纤维素降解细菌。前期研究发现,菌株H1在滤纸纤维素上连续传代数次后无法生长,只有在纤维素降解产物纤维二糖中培养后方能继续在纤维素中生长,并恢复其纤维素降解活性。这与纤维素酶合成受"代谢产物抑制"的传统认识相悖。【目的】证明菌株H1的纤维素酶合成受细胞密度调控。【方法】检测菌株H1的纤维素酶活和转录水平在高和低密度细胞培养物中差异,并检测高密度细胞培养物中的寡肽对低密度细胞纤维素酶活和转录水平的促进作用。【结果】菌株H1的高密度细胞培养物的纤维素酶活和转录水平比低密度细胞的高3-10倍;并且高密度细胞培养液能显著提高低密度细胞纤维素酶活和转录水平。【结论】居瘤胃解纤维素菌(Cellulosilyticum ruminicola)H1纤维素酶的合成受细胞密度调控。     

合成生物学及其在生物技术中的应用进展

合成生物学是一门21世纪生物学的新兴学科,它着眼生物科学与工程科学的结合,把生物系统当作工程系统"从下往上"进行处理,由"单元"(unit)到"部件"(device)再到"系统"(system)来设计,修改和组装细胞构件及生物系统.合成生物学是分子和细胞生物学、进化系统学、生物化学、信息学、数学、计算机和工程等多学科交叉的产物.目前研究应用包括两个主要方面:一是通过对现有的、天然存在的生物系统进行重新设计和改造,修改已存在的生物系统,使该系统增添新的功能.二是通过设计和构建新的生物零件、组件和系统,创造自然界中尚不存在的人工生命系统.合成生物学作为一门建立在基因组方法之上的学科,主要强调对创造人工生命形态的计算生物学与实验生物学的协同整合.必须强调的是,用来构建生命系统新结构、产生新功能所使用的组件单元既可以是基因、核酸等生物组件,也可以是化学的、机械的和物理的元件.本文跟踪合成生物学研究及应用,对其在DNA水平编程、分子修饰、代谢途径、调控网络和工业生物技术等方面的进展进行综述.     

微生物细胞工厂的代谢调控

代谢调控是构建微生物细胞工厂的重要技术手段。随着合成生物学技术的不断突破,挖掘和人工设计的高质量调控元件大幅度提升了对细胞代谢网络的改造能力;代谢调控研究也已从单基因的静态调控发展到系统水平上的智能精确动态调控。文中简要综述了近30年来代谢途径表达调控技术在代谢工程领域的研究进展。     

Shifts in growth strategies reflect tradeoffs in cellular economics

The growth rate‐dependent regulation of cell size, ribosomal content, and metabolic efficiency follows a common pattern in unicellular organisms: with increasing growth rates, cell size and ribosomal content increase and a shift to energetically inefficient metabolism takes place. The latter two phenomena are also observed in fast growing tumour cells and cell lines. These patterns suggest a fundamental principle of design. In biology such designs can often be understood as the result of the optimization of fitness. Here we show that in basic models of self‐replicating systems these patterns are the consequence of maximizing the growth rate. Whereas most models of cellular growth consider a part of physiology, for instance only metabolism, the approach presented here integrates several subsystems to a complete self‐replicating system. Such models can yield fundamentally different optimal strategies. In particular, it is shown how the shift in metabolic efficiency originates from a tradeoff between investments in enzyme synthesis and metabolic yields for alternative catabolic pathways. The models elucidate how the optimization of growth by natural selection shapes growth strategies.     

How to make a minimal genome for synthetic minimal cell

As a key focus of synthetic biology, building a minimal artificial cell has given rise to many discussions. A synthetic minimal cell will provide an appropriate chassis to integrate functional synthetic parts, devices and systems with functions that cannot generally be found in nature. The design and construction of a functional minimal genome is a key step while building such a cell/chassis since all the cell functions can be traced back to the genome. Kinds of approaches, based on bioinformatics and molecular biology, have been developed and proceeded to derive essential genes and minimal gene sets for the synthetic minimal genome. Experiments about streamlining genomes of model bacteria revealed genome reduction led to unanticipated beneficial properties, such as high electroporation efficiency and accurate propagation of recombinant genes and plasmids that were unstable in other strains. Recent achievements in chemical synthesis technology for large DNA segments together with the rapid development of the whole-genome sequencing, have transferred synthesis of genes to assembly of the whole genomes based on oligonucleotides, and thus created strong preconditions for synthesis of artificial minimal genome. Here in this article, we review briefly the history and current state of research in this field and summarize the main methods for making a minimal genome. We also discuss the impacts of minimized genome on metabolism and regulation of artificial cell.     

Characterization of neurosphere cell phenotypes by flow cytometry

BACKGROUND: Neural stem cell research regularly utilizes neurosphere cultures as a continuous source of primitive neural cells. Results from current progenitor cell assays show that these cultures contain a low number of neural progenitors. Our goal is to characterize neurosphere cultures and define subpopulations in order to purify neural progenitor cells. METHODS: Cells from embryonic mouse neurosphere cultures were stained with Hoechst 33342 and analyzed by flow cytometry. Subpopulations were sorted based on their relative fluorescence intensity in the blue and red regions of the spectrum. Individual sorted subpopulations were reanalyzed after 7 days in culture. RESULTS: Neurosphere cultures contain a relatively high number of cells that stain weakly with Hoechst 33342. This subpopulation is present when cultured as an entire batch in the presence of epidermal growth factor (EGF). When cultured separately, this subpopulation gives rise to a neurosphere population with essentially the same characteristics as freshly isolated embryonic mouse brain cells but contains substantially fewer weakly Hoechst-stained cells. CONCLUSIONS: Similar to hemopoietic systems, neurosphere cultures contain a subpopulation that can be characterized by a low emission of Hoechst fluorescence. When cultured separately, this subpopulation gives rise to a phenotype similar to freshly isolated, uncultured neural cells.     

Kinetic evidence for a critical rate of protein synthesis in the Saccharomyces cerevisiae yeast cell cycle

The kinetics of cell cycle initiation were measured at pH 2.7 for cells that had been arrested at the "start" step of cell division with the polypeptide pheromone alpha-factor. Cell cycle initiation was induced by the removal of alpha-factor. The rate at which cells completed start was identical to the rate of subsequent bud emergence. After short times of prearrest with alpha-factor (e.g. 5.2 h), the kinetics of bud emergence were biphasic, indicative of two subpopulations of cells that differed by greater than 10-fold in their rates of cell cycle initiation. The subpopulation that exhibited a slow rate of cell cycle initiation is comprised of cells that resided in G1 prior to start at the time of removal of alpha-factor, whereas the subpopulation that initiated the cell cycle rapidly is comprised of cells that had reached and become blocked at start. A critical concentration of cycloheximide was found to reintroduce slow budding cells into a population of 100% fast budding cells, suggesting that the two subpopulations differ with respect to attainment of a critical rate of protein synthesis that is necessary for the performance of start. Cycloheximide and an increase in the time of prearrest with alpha-factor had opposite effects on both the partitioning of cells between the two subpopulations and the net rate of protein synthesis per cell, consistent with this conclusion. Cell cycle initiation by the subpopulation of fast budding cells required protein synthesis even though the critical rate of protein synthesis had been achieved during arrest. It is concluded that alpha-factor inhibits the synthesis of and/or inactivates specific proteins that are required for the performance of start, but alpha-factor does not prevent attainment of the critical rate of protein synthesis.     

非天然氨基酸细胞工厂的构建与应用

非天然氨基酸在医药、农药、材料等领域得到广泛应用,其绿色、高效合成越来越受到关注.近年来,随着合成生物学的快速发展,微生物细胞工厂为非天然氨基酸的制造提供了重要手段.文中从合成途径的重构、关键酶的设计改造及与前体的协同调控、竞争性旁路途径的敲除、辅因子循环系统的构建等方面介绍了 一系列非天然氨基酸细胞工厂构建与应用的研...     

When nutrient limitation places bacteria in the domains of slow growth: metabolics, morphologic and cell cycle behavior

Abstract Growth systems appropriate for studying mass transfer in different bacterial environments are reviewed. Fed batch and recycling fermentors are suited to modelling nutrient limitation and slow growth. Use of these two growth systems reveals the existence of three growth rate regions, or domains, defined by maintenance energy demands, nucleotide regulation, metabolism, and physiological behavior. They are exemplified in Escherichia coli by domain-dependent synthesis of attachment antigens, heat-labile toxin, and inducible enzymes. Distribution of the bacterial population among cell cycle stages changes with growth rate domain because lengths of the stages differ in their dependence on growth rate. This produces subpopulations whose ratios vary with growth rate and that are likely to differ in both molecular composition and stress resistance.     

Predicting and controlling the reactivity of immune cell populations against cancer

Heterogeneous cell populations form an interconnected network that determine their collective output. One example of such a heterogeneous immune population is tumor‐infiltrating lymphocytes (TILs), whose output can be measured in terms of its reactivity against tumors. While the degree of reactivity varies considerably between different TILs, ranging from null to a potent response, the underlying network that governs the reactivity is poorly understood. Here, we asked whether one can predict and even control this reactivity. To address this we measured the subpopulation compositions of 91 TILs surgically removed from 27 metastatic melanoma patients. Despite the large number of subpopulations compositions, we were able to computationally extract a simple set of subpopulation‐based rules that accurately predict the degree of reactivity. This raised the conjecture of whether one could control reactivity of TILs by manipulating their subpopulation composition. Remarkably, by rationally enriching and depleting selected subsets of subpopulations, we were able to restore anti‐tumor reactivity to nonreactive TILs. Altogether, this work describes a general framework for predicting and controlling the output of a cell mixture.     

Competitive coexistence in antiviral immunity

Malignant brain tumors consist of a number of distinct subclonal populations. Each of these subpopulations may be characterized by its own behaviors and properties. These subpopulations arise from the constant genetic and epigenetic alteration of existing cells in the rapidly growing tumor. However, since each single-cell mutation only leads to a small number of offspring initially, very few newly arisen subpopulations survive more than a short time. The present work quantifies "emergence", i.e. the likelihood of an isolated subpopulation surviving for an extended period of time. Only competition between clones is considered; there are no cooperative effects included. The probability that a subpopulation emerges under these conditions is found to be a sigmoidal function of the degree of change in cell division rates. This function has a non-zero value for mutations which confer no advantage in growth rate, which represents the emergence of a distinct subpopulation with an advantage that has yet to be selected for, such as hypoxia tolerance or treatment resistance. A logarithmic dependence on the size of the mutated population is also observed. A significant probability of emergence is observed for subpopulations with any growth advantage that comprise even 0.1% of the proliferative cells in a tumor. The impact of even two clonal populations within a tumor is shown to be sufficient such that a prognosis based on the assumption of a monoclonal tumor can be markedly inaccurate.