基于糖原代谢调控的蓝细菌光合细胞工厂优化研究进展

蓝细菌是重要的光合自养微生物,也是最具潜力的光合微生物底盘之一,被广泛应用于光驱固碳细胞工厂的开发.糖原是蓝细菌最重要的天然碳汇物质,糖原代谢对蓝细菌光合碳流的分配和调控具有重要意义.为了优化蓝细菌光合细胞工厂的合成效能,驱动更多的光合碳流重定向至目标代谢产物的合成,已经有多种策略和方法被成功开发用于调控蓝细菌的糖原代...     

Tailoring cyanobacterial cell factory for improved industrial properties

Photosynthetic biomanufacturing provides a promising solution for sustainable production of biofuels and biochemicals. Cyanobacteria are among the most promising microbial platforms for the construction of photosynthetic cell factories. Metabolic engineering of cyanobacteria has enabled effective photosynthetic synthesis of diverse natural or non-natural metabolites, while commercialization of photosynthetic biomanufacturing is usually restricted by process and economic feasibilities. In actual outdoor conditions, active cell growth and product synthesis is restricted to narrow light exposure windows of the day-night cycles and is threatened by diverse physical, chemical, and biological environmental stresses. For biomass harvesting and bioproduct recovery, energy and cost consuming processing and equipment is required, which further decreases the economic and environmental competitiveness of the entire process. To facilitate scaled photosynthetic biomanufacturing, lots of efforts have been made to engineer cyanobacterial cell properties required by robust & continual cultivation and convenient & efficient recovery. In this review, we specifically summarized recently reported engineering strategies on optimizing industrial properties of cyanobacterial cells. Through systematically re-editing the metabolism, morphology, mutualism interaction of cyanobacterial chassis cells, the adaptabilities and compatibilities of the cyanobacterial cell factories to the industrial process could be significantly improved. Cell growth and product synthesis of the tailored cyanobacterial cells could be expanded and maintained at night and in stressful environments, while convenient biomass harvesting could also be expected. For developing more feasible cyanobacterial photosynthetic biomanufacturing in large scale, we here propose the importance of tailoring industrial properties of cyanobacteria and outline the directions that should be exploited in the future.     

蓝细菌光驱固碳合成蔗糖技术的发展与展望

生物炼制技术体系是缓解能源和环境危机,推动社会可持续发展的重要选择,而充足的糖原料供应是生物炼制的基础。蓝细菌光驱固碳合成蔗糖是一种潜力巨大的新型糖原料供应路线。基于高效的蓝细菌光驱固碳细胞工厂,可以在单平台上以太阳能为驱动将二氧化碳和水直接转化为蔗糖,过程简单、产品明确、易于提取,而且可以同时达到固碳减排和供应糖原料的效果,具有重要的研究和应用价值。本文回顾了蓝细菌光驱固碳合成蔗糖技术的发展现状,从合成机制、代谢工程策略、技术延伸应用等层面对其最新进展和所遇到的问题进行了总结介绍,并对该技术未来发展方向进行了展望。     

Toolboxes for cyanobacteria: Recent advances and future direction

Photosynthetic cyanobacteria are important primary producers and model organisms for studying photosynthesis and elements cycling on earth. Due to the ability to absorb sunlight and utilize carbon dioxide, cyanobacteria have also been proposed as renewable chassis for carbon-neutral “microbial cell factories”. Recent progresses on cyanobacterial synthetic biology have led to the successful production of more than two dozen of fuels and fine chemicals directly from CO₂, demonstrating their potential for scale-up application in the future. However, compared with popular heterotrophic chassis like Escherichia coli and Saccharomyces cerevisiae, where abundant genetic tools are available for manipulations at levels from single gene, pathway to whole genome, limited genetic tools are accessible to cyanobacteria. Consequently, this significant technical hurdle restricts both the basic biological researches and further development and application of these renewable systems. Though still lagging the heterotrophic chassis, the vital roles of genetic tools in tuning of gene expression, carbon flux re-direction as well as genome-wide manipulations have been increasingly recognized in cyanobacteria. In recent years, significant progresses on developing and introducing new and efficient genetic tools have been made for cyanobacteria, including promoters, riboswitches, ribosome binding site engineering, clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease (CRISPR/Cas) systems, small RNA regulatory tools and genome-scale modeling strategies. In this review, we critically summarize recent advances on development and applications as well as technical limitations and future directions of the genetic tools in cyanobacteria. In addition, toolboxes feasible for using in large-scale cultivation are also briefly discussed.     

蓝细菌乙醇光合细胞工厂的发展与展望

生物乙醇是极具应用潜力和代表性的生物能源产品之一。以蓝细菌为光合平台,利用二氧化碳和太阳能直接进行乙醇合成可以同时起到降低二氧化碳排放和提供可再生能源的效果,具有重要的研究与应用价值。本文回顾了蓝细菌乙醇光合细胞工厂相关技术的发展历程和现状,从途径优化、底盘选择和代谢工程策略等层面对其最新进展和所遇到的问题进行了总结介绍,并对该技术未来发展方向进行了展望。     

Glycogen phosphorylase from Dictyostelium: a kinetic analysis by computer simulation.

A model of carbohydrate metabolism during differentiation in Dictyostelium discoideum has been used to investigate which enzyme kinetic mechanism(s) might be operative for glycogen phosphorylase in vivo. The model, which has been described previously, is capable of simulating experimentally observed changes in metabolite concentrations and fluxes during differentiation under both the standard starvation condition and in the presence of glucose (25 mM). The concentrations of saccharide end products of differentiation under these 2 conditions differ substantially.Glycogen phosphorylase is described in the model by a rapid equilibrium random bi bi mechanism and the effect of substituting 4 other kinetic mechanisms was examined. Each of these mechanisms in the model allows simulations compatible with the saccharide accumulation patterns found during differentiation in the absence of glucose. However, in the presence of glucose, only a reversible mechanism (random or ordered) is compatible with the experimental data. It is concluded that glycogen degradation in vivo is controlled by an enzyme catalyzing a reversible reaction, the rate of which is inversely related to the glucose-1-P concentration.     

Control of glycogen deposition

Traditionally, glycogen synthase (GS) has been considered to catalyze the key step of glycogen synthesis and to exercise most of the control over this metabolic pathway. However, recent advances have shown that other factors must be considered. Moreover, the control of glycogen deposition does not follow identical mechanisms in muscle and liver. Glucose must be phosphorylated to promote activation of GS. Glucose-6-phosphate (Glc-6-P) binds to GS, causing the allosteric activation of the enzyme probably through a conformational rearrangement that simultaneously converts it into a better substrate for protein phosphatases, which can then lead to the covalent activation of GS. The potency of Glc-6-P for activation of liver GS is determined by its source, since Glc-6-P arising from the catalytic action of glucokinase (GK) is much more effective in mediating the activation of the enzyme than the same metabolite produced by hexokinase I (HK I). As a result, hepatic glycogen deposition from glucose is subject to a system of control in which the 'controller', GS, is in turn controlled by GK. In contrast, in skeletal muscle, the control of glycogen synthesis is shared between glucose transport and GS. The characteristics of the two pairs of isoenzymes, liver GS/GK and muscle GS/HK I, and the relationships that they establish are tailored to suit specific metabolic roles of the tissues in which they are expressed. The key enzymes in glycogen metabolism change their intracellular localization in response to glucose. The changes in the intracellular distribution of liver GS and GK triggered by glucose correlate with stimulation of glycogen synthesis. The translocation of GS, which constitutes an additional mechanism of control, causes the orderly deposition of hepatic glycogen and probably represents a functional advantage in the metabolism of the polysaccharide.     

Development and application of CRISPR-based genetic tools in Bacillus species and Bacillus phages

Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed into a precise and efficient genome editing tool. Since its discovery as an adaptive immune system in prokaryotes, it has been applied in many different research fields including biotechnology and medical sciences. The high demand for rapid, highly efficient and versatile genetic tools to thrive in bacteria-based cell factories accelerates this process. This review mainly focuses on significant advancements of the CRISPR system in Bacillus subtilis, including the achievements in gene editing, and on problems still remaining. Next, we comprehensively summarize this genetic tool's up-to-date development and utilization in other Bacillus species, including B. licheniformis, B. methanolicus, B. anthracis, B. cereus, B. smithii and B. thuringiensis. Furthermore, we describe the current application of CRISPR tools in phages to increase Bacillus hosts' resistance to virulent phages and phage genetic modification. Finally, we suggest potential strategies to further improve this advanced technique and provide insights into future directions of CRISPR technologies for rendering Bacillus species cell factories more effective and more powerful.     

应用CRISPRi技术敲低耻垢分枝杆菌异柠檬酸脱氢酶基因

成簇的规律间隔的短回文重复序列干扰(clustered regularly interspaced short palindromic repeat interference,CRISPRi)是一种新型转录抑制技术,该系统包含RNA介导的DNA内切酶dCas9和针对目的基因的特异性单向导RNA(single guide RNA,sgRNA),通过形成DNA识别复合物特异性识别相应DNA序列以抑制目的基因的转录。异柠檬酸脱氢酶(isocitrate dehydrogenase,ICD)是三羧酸循环中的关键代谢酶,在分枝杆菌的碳代谢过程中发挥重要作用。本研究利用CRISPRi高效抑制分枝杆菌特定基因表达的方法构建耻垢分枝杆菌icd敲低(icd knockdown,ICD-KD)株。定量聚合酶链反应(quantitative polymerase chain reaction,qPCR)和蛋白免疫印迹检测结果显示,耻垢分枝杆菌中icd转录水平与ICD蛋白表达水平显著下降,表明采用CRISPRi技术成功构建了耻垢分枝杆菌ICD-KD株。进一步研究ICD-KD株的生长情况,测定其在固体培养基点板及液体培养基中的生长曲线,结果均显示ICD-KD株生长速率明显减慢,同时菌体内ICD酶活显著降低,提示ICD对分枝杆菌的生长存活起重要作用。本研究使用CRISPRi技术快速构建了分枝杆菌必需基因的敲低菌株,为后续研究分枝杆菌ICD在碳源代谢通路中的功能和碳通量流向调控机制提供了重要基础。     

Synthetic Biology Toolkits for Metabolic Engineering of Cyanobacteria

Cyanobacteria are of great importance to Earth's ecology. Due to their capability in photosynthesis and C1 metabolism, they are ideal microbial chassis that can be engineered for direct conversion of carbon dioxide and solar energy into biofuels and biochemicals. Facilitated by the elucidation of the basic biology of the photoautotrophic microbes and rapid advances in synthetic biology, genetic toolkits have been developed to enable implementation of nonnatural functionalities in engineered cyanobacteria. Hence, cyanobacteria are fast becoming an emerging platform in synthetic biology and metabolic engineering. Herein, the progress made in the synthetic biology toolkits for cyanobacteria and their utilization for transforming cyanobacteria into microbial cell factories for sustainable production of biofuels and biochemicals is outlined. Current techniques in heterologous gene expression, strategies in genome editing, and development of programmable regulatory parts and modules for engineering cyanobacteria towards biochemical production are discussed and prospected. As cyanobacteria synthetic biology is still in its infancy, apart from the achievements made, the difficulties and challenges in applying and developing genetic toolkits in cyanobacteria for biochemical production are also evaluated.     

蓝藻光驱固碳合成糖类物质的技术研究进展*

糖类物质在食品、医药、日化、发酵领域有着广泛应用,对人类健康和社会发展有着重要意义。发展新型糖类物质合成技术有利于解决传统植物生物质“采集-炼制”产糖模式所面临的高成本、长周期、时空限制等风险和问题。蓝藻是一类重要的光自养微生物,也是极具潜力的新型微生物光合平台,发展蓝藻光驱固碳产糖技术有望实现二氧化碳向特定糖类产物的一站式定向转化,实现糖类物质合成的模式变革。糖类物质本身在蓝藻天然光合代谢网络中发挥重要作用,特别是卡尔文循环、糖原代谢、相容性物质代谢等几个重要生理模块的运转都是以不同糖类物质的转化来驱动的;而合成生物技术的发展又为光合产糖网络重塑和扩展注入了新的驱动力,在产品类型、合成模式及生产效率上显著提升了蓝藻光驱固碳产糖技术的发展和应用潜力。针对蓝藻光驱固碳产糖技术的发展应用,从模式、策略、产物等不同维度总结了相关进展和风险挑战,并对其未来前景和方向进行了展望。     

蓝细菌细胞工厂合成聚合物单体的研究进展

蓝细菌是当前合成生物学研究的热门底盘生物之一,是光合自养底盘微生物的典型代表。随着化石资源的逐渐枯竭和碳排放所导致的全球变暖问题的加剧,以CO2为碳源的蓝细菌细胞工厂的研究又迎来了一次新的浪潮。长期以来,人们对于蓝细菌细胞工厂的关注点主要是在生物能源的生产,比如液体燃料及氢气等。蓝细菌细胞工厂研究的主要瓶颈之一是其低效率导致的经济性问题。这一问题对于成本异常敏感的能源产品而言尤其突出。聚合物作为人类生产生活的重要基础,属于附加值较大的大宗化学品,对克服蓝细菌细胞工厂商业化所面临的经济性问题具有优势,近来得到了越来越多的关注。本文对蓝细菌的聚合物单体生产的相关研究进行了系统综述,阐述了各类单体的增产策略,并回顾了蓝细菌细胞工厂应用的相关技术,提出了蓝细菌合成生物学的应用领域所存在的问题并对未来的研究进行了展望。     

Mapping photoautotrophic metabolism with isotopically nonstationary (13)C flux analysis

Understanding in vivo regulation of photoautotrophic metabolism is important for identifying strategies to improve photosynthetic efficiency or re-route carbon fluxes to desirable end products. We have developed an approach to reconstruct comprehensive flux maps of photoautotrophic metabolism by computational analysis of dynamic isotope labeling measurements and have applied it to determine metabolic pathway fluxes in the cyanobacterium Synechocystis sp. PCC6803. Comparison to a theoretically predicted flux map revealed inefficiencies in photosynthesis due to oxidative pentose phosphate pathway and malic enzyme activity, despite negligible photorespiration. This approach has potential to fill important gaps in our understanding of how carbon and energy flows are systemically regulated in cyanobacteria, plants, and algae.     

Biochemical and genetic engineering strategies to enhance hydrogen production in photosynthetic algae and cyanobacteria

As an energy carrier, hydrogen gas is a promising substitute to carbonaceous fuels owing to its superb conversion efficiency, non-polluting nature, and high energy content. At present, hydrogen is predominately synthesized via chemical reformation of fossil fuels. While various biological methods have been extensively explored, none of them is justified as economically feasible. A sustainable platform for biological production of hydrogen will certainly impact the biofuel market. Among a selection of biological systems, algae and cyanobacteria have garnered major interests as potential cell factories for hydrogen production. In conjunction with photosynthesis, these organisms utilize inexpensive inorganic substrates and solar energy for simultaneous biosynthesis and hydrogen evolution. However, the hydrogen yield associated with these organisms remains far too low to compete with the existing chemical systems. This article reviews recent advances of biochemical, bioprocess, and genetic engineering strategies in circumventing technological limitations to hopefully improve the applicative potential of these photosynthetic hydrogen production systems.     

基于CRISPR/Cas生物传感原理的病原菌检测技术研究进展*

病原菌的快速准确检测是实现疫情高效防控、疾病精准治疗、污染环境及时处置的关键。而现有的病原菌现场快速检测技术,主要以定性分析为主,假阳性/假阴性受到诟病,检测准确性仍有待提升,亟待发展基于新原理、新方法的病原菌快速检测技术。基于CRISPR(clustered regularly interspaced short palindromic repeats)的生物传感技术因具有高灵活性(对不同的基因靶点只需改变crRNA序列)、高特异性(单碱基分辨)、高灵敏(优于10^-18 mol/L浓度)、可编程、可模块化、低成本、可在各种体外介质中高效稳定运行等独特优势,打破了传统分子诊断与检测技术的局限性,正在成为下一代病原菌检测技术的引领者。在该技术中,Cas效应蛋白被用作高特异性的序列识别元件,结合不同的生物传感机制,即可用于病原菌的高特异性快速灵敏检测。在总结CRISPR/Cas生物传感技术原理的基础上,综述了用于病原菌检测的CRISPR/Cas12和CRISPR/Cas13生物传感技术研究进展。通过阐述CRISPR/Cas生物传感技术在实际应用中面临的挑战,展望其未来的发展前景。     

A state of the art of metabolic networks of unicellular microalgae and cyanobacteria for biofuel production

The most promising and yet challenging application of microalgae and cyanobacteria is the production of renewable energy: biodiesel from microalgae triacylglycerols and bioethanol from cyanobacteria carbohydrates. A thorough understanding of microalgal and cyanobacterial metabolism is necessary to master and optimize biofuel production yields. To this end, systems biology and metabolic modeling have proven to be very efficient tools if supported by an accurate knowledge of the metabolic network. However, unlike heterotrophic microorganisms that utilize the same substrate for energy and as carbon source, microalgae and cyanobacteria require light for energy and inorganic carbon (CO₂ or bicarbonate) as carbon source. This double specificity, together with the complex mechanisms of light capture, makes the representation of metabolic network nonstandard. Here, we review the existing metabolic networks of photoautotrophic microalgae and cyanobacteria. We highlight how these networks have been useful for gaining insight on photoautotrophic metabolism.     

Progress and challenges in engineering cyanobacteria as chassis for light-driven biotechnology

Cyanobacteria are prokaryotic phototrophs that, in addition to being excellent model organisms for studying photosynthesis, have tremendous potential for light-driven synthetic biology and biotechnology. These versatile and resilient microorganisms harness the energy of sunlight to oxidise water, generating chemical energy (ATP) and reductant (NADPH) that can be used to drive sustainable synthesis of high-value natural products in genetically modified strains. In this commentary article for the Synthetic Microbiology Caucus we discuss the great progress that has been made in engineering cyanobacterial hosts as microbial cell factories for solar-powered biosynthesis. We focus on some of the main areas where the synthetic biology and metabolic engineering tools in cyanobacteria are not as advanced as those in more widely used heterotrophic chassis, and go on to highlight key improvements that we feel are required to unlock the full power of cyanobacteria for future green biotechnology.     

CRISPR相关生物信息学基础

成簇规律间隔短回文序列（clustered regularly interspaced short palindromic repeats,CRISPR）是细菌和古细菌在不断进化的过程中获得的一种适应性免疫防御机制,该结构与一些功能相关的蛋白质（CRISPR associated, Cas）合称CRISPR Cas系统。由于其致突变效率高、操作简单及成本较低的特点,近年来对CRISPR/Cas系统的研究获得越来越广泛的关注。该系统迅速在各领域中得到广泛应用,被认为是一种具有广阔应用前景的基因组定点改造分子工具。但是,该系统存在脱靶效应、测序数据分析等挑战。为此,许多研究者开发出各种软件解决以上问题。本文着重从生物信息学的角度出发,对CRISPR/Cas系统中sgRNA的设计软件、CRISPR全基因组筛选功能基因的测序数据分析软件以及CRISPR在生物信息学中的运用作一系统综述。