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。     

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...     

蓝细菌光驱固碳细胞工厂的合成生物学开发策略

光合生物制造技术是指以光合自养生物为底盘,通过光合固碳过程,将太阳能和二氧化碳直接转化为生物燃料和生物基化学品的全新生物制造模式。发展光合生物制造技术可以同时实现固碳减排和清洁生产。蓝细菌是极具潜力的微生物光合底盘,也为光合生物制造技术开发高效的光驱固碳细胞工厂提供了重要平台。着眼于未来的规模化应用需求,蓝细菌光驱固碳细胞工厂需要在物质能量转化效率、工业过程中的生长和生产稳定性以及与工程过程的适配性这三方面进一步提升。现从光能的捕集和利用、碳源的固定和转化、逆境胁迫的适应以及工程过程的适配这四个角度,介绍了如何应用合成生物学工具和策略,人工设计、开发进而优化蓝细菌光驱固碳细胞工厂,以满足光合生物制造技术大规模应用的需要;最后,总结、介绍了本领域的最新研究进展,并对未来发展方向进行了展望。     

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。     

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。     

应用合成生物学策略优化光合蓝细菌底盘

光合蓝细菌具有一系列良好的特质,包括利用太阳能固定CO2、营养需求低、生长迅速以及遗传背景简单等.近年来,光合蓝细菌作为生产可再生燃料和精细化学制品的“自养型人工细胞工厂”引起了社会的广泛关注,促进了相关研究的升温.目前在应用合成生物学的技术和研究策略来优化光合蓝细菌作为底盘生物等方面已取得了一些令人鼓舞的进展.文中综述了近年来在光合蓝细菌底盘优化的方法、光合效率的提高以及各种耐受性蓝细菌底盘的构建方面的进展,并对光合蓝细菌底盘构建的工业应用价值进行了讨论.     

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。     

液相质谱联用的代谢组方法优化及其在蓝细菌分析中的应用

为了准确鉴定光合蓝细菌中的各种代谢物,需要对基于液相色谱–质谱联用仪(LC-MS)的代谢组学分析方法进行有针对性的优化。本研究选取了24种涉及中心碳代谢和能量代谢的代谢物作为LC-MS的检测目标,获得了每个代谢物的最适色谱分离条件和质谱参数;同时以光合蓝细菌Synechocystis sp.PCC6803为主要对象,针对性地优化了样品前处理条件,结果显示适当延长梯度洗脱顺序表的时间并将流速设为0.2 m L/min可以得到最佳的分离效果,同时选择80%(V/V)甲醇(-80?C)作为代谢物萃取剂。分析结果证明这一代谢组分析技术可以成功地应用到光合蓝细菌的研究中。     

吃进“废气”,吐出“宝贝”——记代谢工程改造光合细胞工厂生产异戊二烯

异戊二烯主要用于生产合成橡胶,还用于生产多种精细化工品及黏合剂和润滑剂。目前异戊二烯完全由石化原料生产。随着全球气候变暖和化石资源的日益短缺,构建以廉价生物质或CO2为原料的异戊二烯生物法合成线路已引起研究者的极大关注。中国科学院上海植物生理生态研究所杨琛课题组在蓝细菌中构建异戊二烯合成途径,利用代谢流量分析和代谢组学分析指导蓝细菌中异戊二烯合成途径的设计和改造,通过循环鉴定合成途径限速步骤和解除限速步骤,逐步提高异戊二烯合成途径的代谢通量,最终经过一系列改造后获得的工程菌可将光合作用所固定的碳的40%用于异戊二烯的合成,产量高达1.26 g/L。除了高效合成异戊二烯,该研究所构建的工程菌还可以作为平台,构建光合自养细胞工厂,合成各种萜类化合物。     

二氧化碳固定酶固碳效率及其对地衣芽孢杆菌代谢的影响

【背景】随着代谢工程与合成生物学的快速发展,通过对异养微生物进行代谢改造,利用生物法进行二氧化碳固定成为一个新的趋势。生物代谢途径中存在着大量固碳酶,这些酶尚待挖掘与应用,不同的酶固碳效率之间也缺少比较。【目的】在体外和体内对固碳功能和效率进行评价。【方法】选取3种固碳酶,即核酮糖1,5-二磷酸羧化加氧酶(ribose 1,5-diphosphate carboxylation oxygenase, RuBisCo)、磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase, PCK)和乙酰辅酶A羧化酶(acetyl coenzyme A carboxylase, ACC)在大肠杆菌中异源表达并纯化。测定纯酶的酶活,并建立无细胞催化实验-液质联用评价酶固碳能力的方法。在厌氧发酵条件下检测代谢指标,比较过表达固碳酶的地衣芽孢杆菌相较于原始菌的代谢差异。【结果】3种酶均实现可溶性表达,纯酶的比酶活分别为66.43、1.16和12.52 U/mg。通过体外无细胞催化实验,ACC在3种酶中表现出最高的固碳效率。分别过表达了PCK、ACC的重组地衣芽孢杆菌,厌氧发酵主产物乳酸的转化率从48.6%分别提升至58.1%和59.7%。【结论】可以通过体外、体内结合的方式对固碳酶的效率进行评价,该研究可为固碳酶在微生物遗传改造中理性、精准地应用提供参考。     

Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products

Metabolic engineering of cyanobacteria has the advantage that sunlight and CO₂ are the sole source of energy and carbon for these organisms. However, as photoautotrophs, cyanobacteria generally lack transporters to move hydrophilic primary metabolites across membranes. To address whether cyanobacteria could be engineered to produce and secrete organic primary metabolites, Synechococcus elongatus PCC7942 was engineered to express genes encoding an invertase and a glucose facilitator, which mediated secretion of glucose and fructose. Similarly, expression of lactate dehydrogenase- and lactate transporter-encoding genes allowed lactate accumulation in the extracellular medium. Expression of the relevant transporter was essential for secretion. Production of these molecules was further improved by expression of additional heterologous enzymes. Sugars secreted by the engineered cyanobacteria could be used to support Escherichia coli growth in the absence of additional nutrient sources. These results indicate that cyanobacteria can be engineered to produce and secrete high-value hydrophilic products.Metabolic engineering of photosynthetic microbes is attractive because of the efficient use of light energy by these organisms and the potential for CO₂ mitigation during production (21). Conventional terrestrial plants capture solar energy at low efficiencies (about 0.1 to 0.25% for corn and up to 1% for switchgrass), while fast-growing prokaryotic and eukaryotic microalgal species are about 1 order of magnitude more productive and their photosynthetic efficiencies can be >10% (12, 13). Genetic tools for engineering cyanobacterial species, including Synechococcus elongatus PCC7942 (Synechococcus), can be applied to metabolic engineering (7). For example, Deng and Coleman (8) expressed pyruvate decarboxylase and alcohol dehydrogenase in cyanobacteria to produce small amounts of ethanol, and Atsumi et al. recently described efficient synthesis of isobutanol using a four-step pathway established in Escherichia coli (2).Much attention has been focused on metabolic engineering to produce fuels. However, fuel molecules are generally toxic to microbes even at moderate concentrations. In addition, on a per-photon basis, the actual market value of fuels is at best comparable to, and generally lower than, the market value of other commodity organic compounds, such as sugars, lactic acid, and amino acids. Engineering cyanobacteria to produce and secrete hydrophilic or charged molecules would thus be economically desirable.Commonly used metabolic engineering organisms, such as E. coli and yeast (e.g., Saccharomyces cerevisiae), express a variety of transport systems for exporting waste products as well as importing nutrients. As photoautotrophs, cyanobacteria lack many of the transporters found in these organisms. In addition, while most microbes store energy by pumping protons across the plasma membrane, cyanobacteria store energy by transporting protons across the thylakoid membrane. In fact, cyanobacteria tend to alkalinize their growth medium in both laboratory and natural conditions (4), and thus how effective heterologous transporters can be in metabolic engineering of cyanobacteria is an open question. Here, we investigated whether heterologous transporters belonging to the major facilitator superfamily, in combination with relevant enzymes, could be introduced into cyanobacteria for production and secretion of useful products.     

肺炎克雷伯氏菌利用甘油生产1,3-PDO发酵优化的研究进展

发展可再生能源,尤其是生物能源,具有显著的能量收益和碳减排效益。随着石油等不可再生资源的减少,许多大宗传统石油化工产品正不断被使用可再生原料的生物制造产品替代。生物发酵法生产1,3-丙二醇（1,3-PDO）顺应了这一潮流,具有广阔的发展前景。提高微生物发酵竞争力,优化发酵法生产1,3-PDO水平,势必增加1,3-PDO的生产效益。对肺炎克雷伯氏菌（Klebsiella pneumoniae）发酵法进行1,3-PDO生产的代谢机理、菌株筛选和利用、发酵参数的选择和优化以及发酵工程策略的设计和监测等进行综述,为利用生物柴油副产物甘油生产有重要工业价值的1,3-PDO产品提供参考。     

Progress and perspective on cyanobacterial glycogen metabolism engineering

As important oxygenic photoautotrophs, cyanobacteria are also generally considered as one of the most promising microbial chassis for photosynthetic biomanufacturing. Diverse synthetic biology and metabolic engineering approaches have been developed to enable the efficient harnessing of carbon and energy flow toward the synthesis of desired metabolites in cyanobacterial cell factories. Glycogen metabolism works as the most important natural carbon sink mechanism and reserve carbon source, storing a large portion of carbon and energy from the Calvin-Benson-Bassham (CBB) cycle, and thus is traditionally recognized as a promising engineering target to optimize the efficacy of cyanobacterial cell factories. Multiple strategies and approaches have been designed and adopted to engineer glycogen metabolism in cyanobacteria, leading to the successful regulation of glycogen synthesis and storage contents in cyanobacteria cells. However, disturbed glycogen metabolism results in weakened cellular physiological functionalities, thereby diminishing the robustness of metabolism. In addition, the effects of glycogen removal as a metabolic engineering strategy to enhance photosynthetic biosynthesis are still controversial. This review focuses on the efforts and effects of glycogen metabolism engineering on the physiology and metabolism of cyanobacterial chassis strains and cell factories. The perspectives and prospects provided herein are expected to inspire novel strategies and tools to achieve ideal control over carbon and energy flow for biomanufacturing.     

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.     

Engineered cyanobacteria: teaching an old bug new tricks

Cyanobacteria have played an important role in the development of the Earth and have long been studied as model organisms for photosynthesis and the circadian rhythm. Recent developments have led to increased interest in the use of engineered cyanobacteria for the production of protein and chemical products. This review highlights the genetic tools and strategies for manipulation of cyanobacteria as well as previous accomplishments in the development of engineered cyanobacteria for applied use. Particular attention is given to the engineering of cyanobacteria for biofuel production, including both hydrocarbon and hydrogen fuels. Genetic engineering efforts to enhance cyanobacterial fitness are reviewed with an emphasis on physiological improvements for large-scale production. Lastly, a future outlook on engineered cyanobacteria is presented, highlighting the future areas of focus and technical challenges in this field. With the uncertainty of future energy security, it is an exciting time in applied cyanobacterial research, but we must take the time to learn from these past accomplishments before we can capitalize on the potential of these photosynthetic microorganisms.