, 许平 上海交通大学 生命科学技术学院，上海 200240
收稿日期：2020-10-07；接收日期：2021-01-19；网络出版时间：2021-01-25
基金项目：国家重点研发计划(No. 2018YFA0903600) 资助
作者简介：陶飞?? 上海交通大学研究员、博士生导师，国家重点研发计划青年首席，上海市青年科技启明星，上海市浦江人才。现任上海市生物工程学会合成生物学专业委员会委员和《合成生物学》期刊编委等。主要从事微生物合成生物学研究。在光驱动底盘开发和聚合物单体生产核心技术方面取得多项突破，以CO2为原料实现了多种高值化合物的生产。已发表SCI论文70余篇(含合作)。曾获教育部自然科学奖一等奖1项(排名第3).
摘要：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。
关键词：蓝细菌底盘合成生物学聚合物细胞工厂C1生物炼制
Advances of polymer-monomer production by cyanobacterial cell factory
Meiwen Qian, Chunlin Tan, Jun Ni, Fei Tao
, Ping Xu School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
Received: October 7, 2020; Accepted: January 19, 2021; Published: January 25, 2021
Supported by: National Key Research and Development Program of China (No. 2018YFA0903600)
Corresponding author: Fei Tao. Tel: +86-21-34206647; Fax: +86-21-34206723; E-mail: taofei@sjtu.edu.cn.
Abstract: Cyanobacteria is one of the promising microbial chassis in synthetic biology, which serves as a typical host for light-driven production. With the gradual depletion of fossil resources and intensification of global warming, the research on cyanobacterial cell factory using CO2 as carbon resource is ushering in a new wave. For a long time, research focus on cyanobacterial cell factory has mainly been the production of energy products, such as liquid fuels and hydrogen. One of the critical bottlenecks occurring in cyanobacterial cell factory is the poor economic performance, which is mainly caused by the inherent inefficiency of cyanobacteria. The problem is particularly prominent for these extremely cost-sensitive energy products. As an indispensable basis for modern industry, polymer monomers belong to the bulk chemicals with high added value. Therefore, increasing attention has been focused on polymer monomers which are superior in overcoming the economic barrier in commercialization of cyanobacterial cell factories. Here, we systematically review the progress on the production of polymer monomers using cyanobacteria, including the strategies for improving production, and the related technologies for the application of this important microbial cell factory. Finally, we summarize several issues in cyanobacterial synthetic biology and proposed future developing trends in this field.
Keywords: cyanobacterial chassissynthetic biologypolymercell factoryC1 biorefinery
蓝细菌，又称蓝藻、蓝绿藻等，是典型的光合单细胞原核微生物，能够直接利用CO2和阳光合成生物质，具有遗传可操作性强、光合效率高等特点，并且已经建立了基因编辑体系[1]。人们利用代谢工程、合成生物学技术，以蓝细菌为底盘细胞，通过合理的设计，导入外源代谢途径，已实现了多种化合物的合成，尤其是生物能源类化合物，如乙醇[2]、氢气[3]、脂肪酸[4]等。鉴于太阳能和CO2的可再生特性，建造蓝细菌细胞工厂进行物质的生产被认为是一种面向未来的化合物可持续生产方式[5]。
近十年蓝细菌细胞工厂的研究已经有了长足的进步，但是距离真正走向商业化还有很多重大挑战。比如，对连续照明的依赖性和缓慢的碳固定过程使得蓝细菌产物的滴度和效率远远低于工业可行性的要求[6]；微生物细胞工厂在克服自然进化所带来的障碍的同时会带来菌株的遗传不稳定性[7]的问题，必然导致生产力下降；而重分配代谢途径则是以牺牲生物量的积累来增加产品产量的[8]。这些问题最终会导致产品经济性的降低，削弱蓝细菌的细胞工厂的商业竞争力。
为了解决这些问题，一方面需要对光合效率的机制和瓶颈进行细致的研究，另一方面需要综合考虑产品本身的经济性问题，选择具有高光子经济性的产品进行细胞工厂的开发。已有不少研究尝试利用蓝细菌进行了多种植物天然产物的生产，比如白藜芦醇、姜黄素等[9]。这对于解决蓝细菌细胞工厂的经济性问题来说是一个不错的选择，为蓝细菌细胞工厂在当前技术阶段的商业化生产提供了新思路。
相比于上述香料物质、抗氧化剂、植物天然产物等高值化合物，聚合物的应用领域广，需求量大，与C1资源(CO、CO2、CH4等) 的特点及其开发利用的初衷具有非常好的契合度。同时，相比于能源物质，如乙醇、氢气等，聚合物具有更高的附加值，对于解决蓝细菌细胞工厂的经济性问题具有更大优势。除此之外，由于人们对环境问题的关注度日益提升，社会对于可降解生物塑料的需求日益高涨，生物基塑料取代传统塑料已成为大势所趋。这对基于蓝细菌细胞工厂的生物基聚合物生产而言是一次大的机遇。蓝细菌作为高效转化C1资源生产有机物的天然宿主，在聚合物单体合成领域已得到越来越多的关注[10]。根据蓝细菌生产的高附加值聚合物的合成前体，聚合物单体的种类可分为以下3大类：烯烃、醇类、羧酸(表 1)。本文将对这3类产品合成生物学产生方式(图 1) 及目前的研究进展进行综述，同时展望合成生物学发展所带来的契机及光驱动聚合物生产的前景。
表 1 蓝细菌聚合物单体的分类Table 1 The classification of polymer monomers
| Subject | Classification | Monomer |
| Polymer monomers | Alkene | Ethylene |
| Isoprene | ||
| Limonene | ||
| Phellandrene | ||
| Alcohol | Butanol | |
| 1, 2-propanediol | ||
| 1, 3-propanediol | ||
| 2, 3-butanediol | ||
| Carboxylic acid | Succinic acid | |
| Lactate | ||
| 3-hydroxypropionic acid | ||
| 3-hydroxybutanoic acid |
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| 图 1 蓝细菌聚合物单体的生物合成途径 Fig. 1 The biosynthesis pathways for plastic monomers in cyanobacteria. There are 12 different pathways belong to 3 types of monomers have been reported. The pathways mainly start from Calvin cycle, tricarboxylic acid cycle (TCA) cycle and the methylerythritol phosphate (MEP) pathway. The monomers are represented with their chemical structure. |
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1 聚合物单体在蓝细菌细胞工厂中的生产1.1 烯烃烯烃是当前石化工业中最为重要的单体化合物之一，也是合成塑料的主要单体，以乙烯和苯乙烯为代表的烯烃类单体广泛用于各种类型的塑料的合成，在食品、药品、化妆品领域占据重要地位(表 2)。利用光合自养微生物蓝细菌生产烯烃类化合物，有利于大宗材料化合物的合成，摆脱对化合物资源的依赖。
表 2 蓝细菌光合细胞工厂产烯烃类聚合物单体Table 2 Cyanobacterial cell factories for the production of alkene monomers
| Monomer | Polymer | Additional function | Chassis | Titer | Culture days | Enhancement Strategy | References |
| Ethylene | Polyethylene, polystyrene, PVC and polyester | Fuel, accelerate the ripening and chemical materials | 7 942 | 69.23 g/L | 7 d | Over-expression of efe | [11] |
| 6 803 | 87.67 g/(L·h) | 7 d | RBS screening and a single gene copy of efe, a glycogen-synthesis knockout mutant | [12] | |||
| 6 803 | 0.019 2 g/L | 1 d | Introduced efe gene, treated with the acetyl-CoA carboxylase inhibitor, cycloxydim | [13] | |||
| 6 803 | 278.54 g/L | 4 d | Partial deletion of ntcA, four-copy efe | [14] | |||
| 11 801 | 974.19 mmol/g | 12 d | A single copy of efe under the control of PcpcB | [25] | |||
| Isoprene | Rubber | Fuel, adhesives, lubricants, terpenoids precursors, generate vitamin A and steroid hormones | 6 803 | 1.26 g/L | 21 d | Engineering the methylerythritol phosphate pathway, plant-derived isoprene synthases, overexpression of IPP isomerase | [15] |
| 6 803 | 0.005 4 g/g | 4 d | Fusion constructs with the highly expressed cpcB gene | [16] | |||
| 6 803 | 0.012 3 g/g | 4 d | Increased the DMAPP reactant at the expense of IPP | [17] | |||
| 6 803 | 336 μg/g | 1 d | A plasmid-based strategy | [18] | |||
| 6 803 | 0.006 7 g/L | 7 d | Ribose 5-phosphate isomerase and ribulose 5-phosphate, 3-epimerase geranyl diphosphate synthase was expressed | [19] | |||
| Limonene | Polycarbonate, rubber | Pharmaceuticals, fragrance, solvents, medicines, materials and biofuels | 7 942 | 0.885 1 g/L | 5 d | Enhancing the downstream limonene carbon sink provide the substrate for the enzyme, a gas-stripping method | [20] |
| 6 803 | 1 mg/L | 150 h | Ipi, Dxs and IspD had the biggest impact on isoprene production | [21] | |||
| 6 803 | 0.002 8 g/g | 1 d | High amounts of heterologous terpene synthase, phycocyanin phellandrene synthase fusion | [26] | |||
| Phellandrene | Polyparallene and terpene resin | Spices, pharmaceuticals, cosmetics, cleaning products, biofuels | 6 803 | 0.003 2 g/L | 2 d | CpcB*PHLS was co-expressed with enzymes of | [22] |
| 6 803 | 0.010 g/L | 2 d | MVA pathway CpcB*PHLS | [23] | |||
| 6 803 | 0.005 9 g/L | 2 d | Overexpressed CpcB*PHLS fusion proteins | [24] |
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乙烯作为工业生产上最大规模的烯烃类化合物，以乙烯及其衍生物的聚合物形式存在于日常生活中，比如聚乙烯(Polyethylene，PE)、聚氯乙烯(Polyvinyl chloride，PVC) 等。人们以蓝细菌为底盘引入来自丁香假单胞菌Pseudomonas syringae中的乙烯生成酶基因(Ethylene-forming enzyme，efe) 成功实现了蓝细菌自养合成乙烯。Carbonell等[11]发现过表达关键酶基因efe能提高乙烯的生成效率；2017年，Veetil等[12]构建了糖原合成敲除突变体(ΔglgC)，整合了受Ptrc启动子控制的efe基因和作为核糖体结合位点的SD序列(5′-AGGAGG-3′) 使乙烯产量提高了两倍；Claudia等[13]为达到增产的目的，在蓝细菌中心碳代谢途径中，异源表达高活性的磷酸烯醇式丙酮酸羧化酶，增强了三羧酸循环的碳供应，提高了乙烯产量；此外，在蓝细菌代谢调控中，氮调控A (Nitrogen control A，NtcA) 是一级碳氮代谢过程中重要的全局转录调控因子，NtcA对控制efe表达的启动子PcpcB起负调控作用，NtcA部分缺失可使乙烯生成量增加23%，Huilin等[14]通过部分缺失NtcA及四拷贝efe获得重组菌株，促使乙烯生成速率增至(2 463±219) μL/(L·h·OD730)。
异戊二烯作为另一重要工业原料，可用于合成橡胶、粘合剂等，也可用于生产维生素A、类固醇等分子。2016年，Gao等[15]通过设计蓝细菌甲基赤藓糖醇磷酸途径(Methylerythritol phosphate，MEP) 以生产异戊二烯，在该途径中引入高活性的植物来源的异戊二烯合酶(Isoprene synthase，IspS)，过表达异戊烯焦磷酸(Isopentenyl pyrophosphate，IPP) 异构酶以增加异戊二烯前体二甲烯丙基焦磷酸(Dimethylallyl-diphosphate，DMAPP) 的产量，从而使异戊二烯产量大幅度提升；2017年，Chaves等[16]为了减轻IspS与藻蓝蛋白的高度表达的β亚基(cpcB蛋白) 融合后的表达障碍，制备了不同的cpcB*IspS融合构建体，高表达的cpcB为前导序列的融合体能使基因表达水平提高61–275倍，但前导cpcB融合蛋白易发生变构，IspS的比活性在融合转化子中会减弱，最终筛选出了最佳的产异戊二烯转化体，使得异戊二烯生产率比从0.2 mg/g细胞干重提高至5.4 mg/g细胞干重；为了进一步提高异戊二烯的生产活性，该研究团队在细胞水平上改进了反应物DMAPP和酶IspS，使产量达到了12.3 mg/g[17]；Pade等[18]发现IspS在标准条件下(0% NaCl) 与异戊二烯生产速率之间存在良好的相关性，补充NaCl可刺激IspS的mRNA水平提高，但受到前体水平不足的限制，异戊二烯产量降低，低浓度的NaCl反而导致异戊二烯产量提高。
与异戊二烯同属于萜类化合物的柠檬烯、水芹烯，常作为医药成分、燃料、香料使用，也具有很高的商业价值。将柠檬酸合酶在蓝细菌中异源表达，蓝细菌能够成功合成柠檬烯。磷酸戊糖途径是该合成过程的重要代谢途径。应用OptForce应变设计算法，过表达磷酸戊糖途径中的基因(核糖-5-磷酸异构酶和核酮糖-5-磷酸3-差向异构酶) 及来自植物大叶冷杉中的香叶基焦磷酸合酶能大幅度地优化柠檬烯的生物合成[19]；同时，过表达香叶基焦磷酸合成酶基因slr0739和异戊烯基焦磷酸异构酶基因sll1556、脱氧木酮糖-1-磷酸合成酶基因sll1945这3种MEP途径的酶，也能使柠檬烯的前体香叶基焦磷酸增加[20]。由于下游碳通量的增加造成柠檬烯的合成通量增加，会导致NADPH的积累[21]，微调ATP/NADPH可影响萜烯类生物的合成。
萜烯生物合成的靶标酶是异源萜烯合成酶，其中包括水芹烯合酶(β-phellandrene synthase，PHLS)，该酶催化活性很低，高水平的酶浓度可弥补这个缺陷，从而提高水芹烯的产率和产量。为了增强PHLS的表达，科学家把PHLS和藻蓝蛋白β亚基融合形成cpcB*IspS融合蛋白，该融合蛋白促进了水芹烯的合成[22]；将cpcB*IspS融合蛋白与香叶基焦磷酸合成酶在甲基戊酸途径异源共表达后，能增加萜类生物合成途径的碳通量，使代谢通量流向萜类前体异戊烯二磷酸和二甲烯丙基焦磷酸，促使在48 h内每克干细胞产生10 mg水芹烯[23]；Betterle等[24]利用融合结构作为蛋白质过表达载体，在蓝细菌中过表达卡那霉素先导序列nptI和氯霉素cmR耐性盒可以提高水芹烯的产生，nptI作为先导序列，促进了异源香叶基二磷酸合成酶(Geranyl-diphosphate synthase，GPPS)表达，cmR用于驱动异源生物合成基因的更高水平的表达，同时也避免了不良的内部重组。
1.2 醇类醇类化合物尤其是含有两个羟基的多元醇类化合物是合成聚酯的重要原料，这类化合物也可以在蓝细菌细胞工厂中合成(表 3)。另外，作为含能化合物的前体，其在生物能源方面的用途也引起了越来越多的重视。
表 3 蓝细菌光合细胞工厂产烯醇类聚合物单体Table 3 Cyanobacterial cell factories for production of alcohol monomers
| Monomer | Polymer | Additional function | Chassis | Titer | Cultural days | Enhancement strategy | References |
| Butanol | Poly butanol vinyl carbamate, poly-butanol terephthalate | Medicine, dewaxing agent, extractant biofuels and chemicals | 6 803 | 0.037 g/L | 8 d | Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions | [27] |
| 7 942 | 0.117 g/(L·d) | 4–5 d | Catalyzed by PduP, increase acetyl-CoA synthesis rate | [29] | |||
| 7 120 | 0.020 g/L | 10 d | Micro-oxic environment within heterocysts | [30] | |||
| 1, 2-propanediol | Polypropanediol | Antifreeze, heat transfer fluids, plasticizers, thermoset plastics, and cosmetics | 79 427 | 0.15 g/L | 10 d | NADPH pool | [31] |
| 6 803 | 1 g/L | 10 d | Insert mgsA, yqhD and adh | [32] | |||
| 1, 3-propanediol | Polyesters, polyethers, and polyurethanes, PTT | Paint, solvent, and antifreeze, food, medicine, cosmetics | 7 942 | 1.22 g/L | 20 d | A stronger promoter (Ptrc), optimize the induction timing | [35] |
| 7 120 | 0.046 mg/L | 20 d | Heterocysts | [36] | |||
| 7 942 | 0.040 g/L | 5 d | Dehydration of glycerol, co-cultivation | [41] | |||
| 7 942 | 0.288 g/L | 14 h | In a coenzyme B12-dependent manner | [42] | |||
| 2, 3-butanediol | Poly (butanediol succinate) | Solvent, biofuels | 7 942 | 3 g/L | 10 d | Supplementation with glucose or xylose | [37] |
| 7 942 | 1.1 g/L | 1 d | Increase carbon flux and redirect it towards carbon fixation | [38] | |||
| 7 942 | 0.761 g/L | 14 h | Utilize extracellular glycerol, l continuous light | [39] | |||
| 7 942 | 1.6 g/L | 16 d | The IPTG inducible promoter PLlacO1 | [43] |
表选项
正丁醇可用作生物燃料，也可作为聚合技术中生产各种化合物的中间体。Anfelt等[27]在充满氮气的条件下过表达磷酸转酮酶，使乙酰辅酶A水平增加了6倍，从而使正丁醇浓度从22 mg/L增加到37 mg/L，该研究证明了正丁醇生产过程中乙酰辅酶A遗传和营养调节的重要性；2016年，Shingo等[28]利用质粒pEL256转化菌株EL9构建菌株BUOH-SE，通过对酰基辅酶A的定量靶点分析和动力学分析，测定了细胞内乙酰辅酶A的浓度，发现丁酰辅酶A还原反应是正丁醇生产中的限速步骤，有效回收关键辅因子游离辅酶A起到促进丙酮酸转化为乙酰辅酶A的作用，最终，使得3 d内正丁醇的生产量达到86 mg/L；2018年，Artnice等[29]同样以BUOH-SE为背景菌株，构建了一株高丙醛脱氢酶活性的新菌株DC7，提高了游离辅酶A的再生率。为了利用DC7中乙酰辅酶A水平的提高来生产正丁醇，将解脂鸭跖草酶亚基的基因插入到aldA位点来实现DC7中乙酰辅酶A羧化酶的过表达，使生产效率达到117 mg/(L·d)。2019年，Higo等[30]为解决引入异源的氧敏感性酶而存在的生产力低的问题，构建了以蓝细菌异形胞光合驱动的厌氧生产为底盘的体系，利用异形胞生产正丁醇的产量比野生型蓝细菌高5倍。
1, 2-丙二醇(1, 2-propanediol, 1, 2-PDO)，一般应用于医药中间体、食品添加剂。在蓝细菌中引入甲基乙二醛合酶、甘油脱氢酶和醛还原酶基因实现1, 2-PDO的生产。然而，在产生1, 2-PDO的同时会产生还原性中间体羟基丙酮，为了最大化1, 2-PDO的积累，Li等[31]利用NADPH依赖的醇脱氢酶来实现目标，使1, 2-PDO的产量达到150 mg/L；2018年，Christian等[32]发现1, 2-PDO的产生与细胞内糖原的转化明显相关，降低糖原的碳通量可显著降低产品的滴度；为了进一步了解1, 2-PDO生产过程中与糖原转换的情况，尤其是与糖原之间碳的分配关系，在持续光照和黑暗条件分别检测了碳通量和生物量、储存化合物和产品之间的碳分配，发现1, 2-PDO中约1/4的碳来源于糖原，其余的碳来源于卡尔文-本森循环中的CO2[33]，即1, 2-PDO的合成依赖于光合有效辐射和糖原分解代谢，这对了解并提高1, 2-PDO的产量具有积极意义。
1, 3-丙二醇(1, 3-propanediol, 1, 3-PDO)是一种非石油基二元醇，将异源、高效的甘油生产和还原途径导入蓝细菌底盘，可氧化磷酸二羟基丙酮生成1, 3-PDO。然而，新构建的菌株在1, 3-PDO生产过程中积累了大量的中间代谢产物甘油。Hirokawa等[34]基于代谢流平衡分布模型计算机模拟分析了1, 3-PDO合成代谢途径，通过计算机模拟基因突变，发现破坏NADPH脱氢酶1 (NDH-1) 复合物能提高1, 3-PDO的产量，在培养20 d后，无ndhF1复合体的1, 3-PDO的滴度最高，达到0.338 g/L；为了增强合成代谢途径的后续反应，该研究团队采用强启动子Ptrc替换弱启动子PLlacO1控制表达策略，在适宜的基因表达诱导条件下，1, 3-PDO的效价达到了1.22 g/L[35]，与先前的研究相比提高了4倍多；2018年，为了解决氧敏感不相容的问题，Liu等[36]基于蓝细菌异形胞自然分化的空间隔离的策略，选用来源不同的1, 3-PDO生产基因与启动子串联排列，组装成1, 3-PDO光合生产模块，通过同源重组整合到鱼腥藻PCC7120菌株的染色体中，产量提高了1.7倍，达到46 mg/L。
2, 3-丁二醇(2, 3-butanediol, 2, 3-BD) 是橡胶、塑料等产品的重要化学原料，蓝细菌生产该产物会受到光照的严重限制。向专性光自养蓝细菌提供葡萄糖或木糖，处于黑暗条件下，10 d内2, 3-BD浓度可达3.0 g/L[37]，该研究证明了黑暗条件下的蓝细菌化学生产的可行性，也暗示蓝细菌细胞工厂的瓶颈主要在于光能的吸收和碳固定；为了形成更具工业应用性的生产方案，提高CO2和葡萄糖等碳源的利用率，Kanno等[38]改进了糖酵解途径和卡尔文-本森循环，增加了碳通量并重定向碳代谢，在连续光照条件下可生产12.6 g/L的2, 3-BD；而甘油作为蓝细菌光化学生产的额外碳源，异源的甘油呼吸途径使聚球藻PCC 7942能够利用胞外甘油，在连续光照条件下，该工程菌在48 h内生产了761 mg/L的2, 3-BD，比对照菌提高290%[39]；Nozzi等[40]选择高效生产2, 3-BD的聚球藻PCC 7002作为化学生产底盘，聚球藻PCC 7002对光、热和盐的耐受性更高，具有显著工程化应用和推广优势，生长速度更快，通过设计不同的遗传工具，比如茶碱核糖体开关异丙基-β-1-硫代半乳糖苷诱导的启动子，同时系统筛选不同的培养条件，比如操纵子排列和拷贝数、光强度、诱导剂浓度、诱导时的细胞密度和营养物浓度，在16 d生产试验后，该细胞工厂以100 mg/(L·d)的速率产生1.6 g/L的2, 3-BD。
1.3 羧酸羧酸类化合物，广泛应用于食品、药品、化妆品、高分子行业，尤其是羟基酸和二元羧酸类化合物可作为聚合物单体合成聚酯(表 4)，该类化合物对于各种材料的生产，具有重要的价值。
表 4 蓝细菌光合细胞工厂产羧酸类聚合物单体Table 4 Cyanobacterial cell factories for production of carboxylic monomers
| Monomer | Polymer | Additional function | Chassis | Titer | Cultural days | Enhancement strategy | References |
| Succinate | Polysuccinate | Food, fiber, medicine, pigment, detergent, surfactant | 11 801 | 0.93 g/L | 5 d | Knock out glgA, sdhB and overexpress SBPase, gltA, yjjPB | [45] |
| 7 942 | 0.43 g/L | 8 d | Expressed the genes encoding for phosphoenolpyruvate carboxylase and citrate synthase | [53] | |||
| 6 803 | 1 380 g/(L·h) | 3 d | Knock out ackA and overexpress sigE | [54] | |||
| Lactate | Polylactic acid | Biodegradable plastic, food, cosmetics, medicine | 7 002 | 1 g/L | 4 d | Delete the flv homologs or the Syn7002 pgr5 homolog, 25 ℃ and 0.04% CO2 | [46] |
| 7 942 | 1.23 g/L | 24 d | from DHAP in the Calvin cycle via methylglyoxal | [47] | |||
| 7 942 | 1.31 g/L | 10 d | Insert LdhD, introduce a lactic acid transporter and bubbling CO2-enriched air | [55] | |||
| 6 803 | 0.893 g/L | 14 d | Lactate dehydrogenase (LDH), co-expression of a heterologous pyruvate kinase | [56] | |||
| 3-hydroxypropionic acid | Poly(3-hydroxypropionic acid), poly (3-hydroxybutyric acid-co-3-hydroxypropionic acid) | Precursor of acrylic acid, crylamide, 1, 3-propanediol | 6 803 | 0.837 g/L | 6 d | Overexpress sll0385, sll1598 and sll0679 | [50] |
| 6 803 | 0.837 mg/L | 6 d | Reduce power supply, central carbon metabolism, the stress responses and enhance protein synthesis | [57] | |||
| 7 942 | 0.659 g/L | 2 d | Introduce malonyl-CoA and β-alanine dependent | [48] | |||
| UTEX 2973 | 0.068 mg/L | 7 d | Overexpress cscB, constructing a microbial consortium | [58] | |||
| 3-hydroxybutanoic acid | PHB PHA | Precursor of epilepsy, carbapenems and pheromones | 6 803 | 0.533 g/L | 21 d | Knock out slr1829 and slr1830, provide bicarbonate and atmospheric CO2 | [51] |
| 7 942 | 1.2 g/L | 21 d | Redesign ATP driving force | [52] |
表选项
琥珀酸是一种多功能化学品，可作为其他重要工业化学品(如1, 4-丁二醇、丁二烯) 的前体。科学家将编码α-酮戊二酸脱羧酶和琥珀酸半醛脱氢酶的基因表达到蓝细菌中，可得到产琥珀酸蓝细菌菌株。该菌株会由于α-酮戊二酸缺乏引起严重的生长迟缓问题，将异源磷酸烯醇式丙酮酸羧化酶和柠檬酸合成酶基因引入到该菌株中，菌株恢复正常生长并产生430 mg/L的琥珀酸[44]；Sengupta等[45]选择了具有较高光合作用和碳固定速率的蓝细菌S. elongatus PCC 11801作为宿主菌株，过表达景天庚酮糖-1, 7-二磷酸酶等并敲除糖原合酶A (glgA)基因和琥珀酸脱氢酶B亚基(sdhB)基因，最终使得该光养菌5 d内产生0.93 g/L琥珀酸，比已报道的最佳滴度高2.2倍，该工作表明了利用高光合速率底盘以形成更高产量的重用性。
乳酸作为形成重要绿色环保可降解材料聚乳酸(Polylactic acid，PLA) 的原材料，一般而言，在工程蓝细菌中的生产是在糖酵解过程中催化乳糖脱氢酶转化丙酮酸而实现的。对乳酸脱氢酶进行突变使得蓝细菌辅因子偏好性从NADH逆转为NADPH，乳酸的产量提高了3.6倍以上，为了进一步提高乳酸的产量，引入了乳酸转运体系并增加了CO2浓度，产物浓度最终达到80.7 mg/L[46]；蓝细菌细胞内Calvin循环的代谢通量高于糖酵解，利用Calvin循环代谢产物构建新的乳酸生成途径提高了蓝细菌的乳酸生产力，将来源于大肠杆菌的甲基乙二醛合酶基因、乳酸/H+转运体等基因导入聚球藻PCC 7942中，在培养24 d后，乳酸滴度达到1.23 g/L[47]。
3-羟基丙酸(3-hydroxypropionic acid，3-HP)是合成新型生物降解材料聚3-羟基丙酸(Poly(3-hydroxypropionate)，P3HP) 的重要的平台化学品，3-HP是通过甘油代谢产生的，而蓝细菌没有天然合成甘油或将甘油转化为3-HP的途径，为了克服该困难，Lan等[85]构建了两条替代途径：丙二酰辅酶A依赖途径和β-丙氨酸依赖途径，通过表达丙二酰辅酶A还原酶和丙二酸半醛还原酶基因，使得两途径合成3-HP的效价分别为665 mg/L、186 mg/L；而过表达NAD(P) 转氢酶基因来改善NADPH的供应、灭活PHA和乙酸酯生物合成的竞争途径等优化方法能将更多的碳通量引入3-HP生物合成中[49]；3-HP的产生与积累会影响细胞代谢，Wang等[50]通过综合分析蛋白质组和代谢组的数据发现，加强细胞代谢强度如增强ATP和NADPH的供应、中心碳代谢、应激反应和蛋白质合成过程，过表达3个转运蛋白基因：钴/镍、锰和磷酸转运基因(即sll0385、sll1598和sll0679) 能确保蓝细菌的正常生长及3-HP产量的高效积累。
3-羟基丁酸(3-hydroxybutanoic acid，3-HB)是合成可生物降解塑料聚羟脂肪酸酯(Polyhydroxyalkanoates，PHA) 和许多精细化学品(如抗生素) 的重要成分。从集胞藻PCC 6803基因组中删除基因slr1829和slr1830 (编码PHB聚合酶)，失活竞争途径可促进3-HB的产生，在人工培养21 d后，产量高达533.4 mg/L[51]，该研究首次证明了直接利用光能和CO2生产3-HB的可行性；在3-HB的生产中，由于异源宿主的内环境不同于天然宿主且人工合成的非天然代谢途径难以控制等问题，Ku等[52]在聚球藻PCC 7942中引入了以ATP水解为基础的驱动模块，并为过量碳通量提供了可逆出口，累积的3-HB效价为1.2 g/L，该研究除了增加了3-HB产量外，还突出强调了路径可逆性用来平衡驱动力和中间体积累的重要性。
2 合成生物学的应用和前景在蓝细菌的光合驱动生产过程中，越来越多的研究人员应用合成生物学手段在蓝细菌底盘细胞中合成目标产物，并致力于将CO2直接转化为一系列有价值的化学产品，从而进一步分支成许多其他珍贵和有用的天然产物，应用于医药、燃料、化妆品等行业(图 2)。合成生物学的各个环节比如底盘选择和改造、元件设计与优化、高效遗传操作和基因编辑技术等，都对蓝细菌细胞工厂的创建具有重要的影响。
 |
| 图 2 基于蓝细菌细胞工厂的聚合物生产和应用前景 Fig. 2 The prospect of polymer production based on cyanobacterial cell factories. Photon energy drives water splitting to producing ATP, NADPH, and O2. Carbon dioxide is assimilated and captured in the Calvin cycle with the cost of NADPH and ATP. The fixed carbon is then directed to plastic monomers or directly to valuable polymers using synthetic gene clusters. |
| 图选项 |
蓝细菌底盘细胞的选择及改造对蓝细菌生物合成具有关键性影响。在培养过程中，蓝细菌中生物量的积累会受到来自环境的各种压力及其合成的最终产物的抑制，而提高蓝细菌抗压能力可潜在地增强蓝细菌底盘的坚固性和目标产物的最终产量。Xie等[59]探索了蓝细菌对环境扰动和产品毒性作出反应的机制，发现设计转运蛋白编码基因是增强细胞对生物和非生物胁迫抗性的重要举措；Luan等[60]通过重塑蓝细菌的代谢和生理结构，使蓝细菌能够更有效地吸收和利用太阳能和CO2；Ni等[9]系统地研究了各种策略，包括引入抗反馈抑制酶，创建功能性融合蛋白以及增加丙二酰辅酶A的供应等，这些合成生物学方法为提高蓝细菌底盘细胞工厂的生产率提供了新的方向。
功能单元的构建是蓝细菌合成生物学必不可少的环节。2018年，Ni等[61]将2-苯乙醇途径和一个人工反馈抑制抗性模块引入到蓝细菌低通量的莽草酸途径中来创建强大的代谢库，使得超过30%的碳通量被重定向到莽草酸途径，用于碳固定、芳香族合成及增加氧气释放，这项研究为提高光合作用效率提供了设计借鉴。为了解决工程化蓝细菌环境释放的风险问题，Zhou等[62]整合铁离子诱导型启动子PisiAB和毒素/抗毒素模块，诱导菌株程序性自杀，这项工作为工程化蓝细菌的生物控制提供了有用工具。
CRISPR-Cas体系是一种新近开发的RNA介导的基因组编辑体系，利用高效、精准的CRISPR-Cas体系对蓝细菌进行基因组工程设计能有效地触发染色体上的双链断裂(Double strand break，DSB) 并引起细胞死亡。通过带有基因盒和侧翼同源臂的模板质粒的共转化，利用CRISPR- Cas9介导的DSB可实现精确的基因整合，最终加快了获得均一且稳定的重组菌株的进程[63]；Yao等[64]利用CRISPRi干扰系统(dCas12a-mediated CRISPR interference system，CRISPRi-dCas12a) 介导的基因抑制有效地阻止转录起始从而促进蓝细菌细胞工厂的建设，更高效地生产高附加值化学品。
高通量计算和实验方法的开发为优质蓝细菌数据、元件和理性设计奠定了基础。Nakajima等[65]通过应用同位素非稳态代谢通量估算了野生型和突变体ΔnrtABCD代谢通量分布，最终揭示了低氮环境下代谢适应的机制；2019年，Hing等[66]将同位素非平稳代谢通量分析与蛋白质组学结合在一起共同对光合作用中主要的碳固定途径卡尔文-本森循环进行调控，提高了该途径的性能和效率，这项研究对未来的代谢工程工作做了重要的理论指导。蓝细菌代谢通路、细胞工厂等合成生物学体系的高通量工程构建和优化必将加速蓝细菌细胞产物的产业化进程。
总之，合成生物学作为极具发展潜力的技术，可赋予蓝细菌细胞全新的生物学功能特性，为蓝细菌产物规模化推广应用做出了巨大贡献。
3 总结与展望蓝细菌作为光合生物研究领域重要的模式生物，对于以突破光合作用自然进化极限为目标的研究而言，具有重要的科学价值。以聚合物生产为导向的蓝细菌细胞工厂研究能把C1资源化与环境友好的生物基塑料结合起来，有利于解决当前蓝细菌细胞工厂商业化所面临的效率低和经济性差等问题。可以预期，聚合物单体的光驱动合成将会成为未来蓝细菌细胞工厂研究的主战场。
当前蓝细菌细胞工厂生产的聚合物单体的种类、机制及技术的研究已经初具规模，对于后续开发新的聚合物单体合成途径，全面深入研究蓝细菌细胞工厂生产聚合物提供了必要的基础信息。代谢工程和合成生物学的迅速发展，也将助力于挖掘蓝细菌细胞工厂在高附加值聚合物合成前体方面的潜力，可为工业、医药、新型聚合物等领域带来广阔的前景。
蓝细菌细胞工厂的合成生物学技术领域仍面临着许多挑战，如基因组编辑工具尚未完全成熟、代谢模型的适配性不高等都是该领域亟待解决的问题。可以预期的是，随着合成生物学以及代谢科学的发展，蓝细菌代谢规律将被逐步阐明，蓝细菌细胞工厂的开发将越来越有章可循，越来越趋于理性、精准和高效。
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