微藻叶绿体细胞器工厂研究进展

doi:10.12211/2096-8280.2022-044

合成生物学 ›› 2022, Vol. 3 ›› Issue (6): 1218-1234.DOI: 10.12211/2096-8280.2022-044

微藻叶绿体细胞器工厂研究进展

朱振¹^,², 田晶², 江静¹^,³, 王旺银¹, 曹旭鹏¹

^1.中国科学院大连化学物理研究所，催化国家重点实验室，洁净能源国家实验室（筹），辽宁大连 116023
^2.大连工业大学，生物工程学院，辽宁大连 116034
^3.中国科学技术大学化学与材料科学学院，安徽合肥 230026

收稿日期:2022-08-08 修回日期:2022-09-28 出版日期:2022-12-31 发布日期:2023-01-17
作者简介:朱振（1991—），女，博士研究生。研究方向为合成生物学和微藻可控培养研究。E-mail：zhuzhen@dicp.ac.cn
曹旭鹏（1978—），男，研究员，博士生导师。致力于太阳能生物转化研究。。E-mail：c_x_p@dicp.ac.cn
基金资助:
国家自然科学基金(21878285);国家自然科学基金委“人工光合成”基础科学中心项目(22088102)

Progress in microalgae chloroplast organelle factory development

Zhen ZHU¹^,², Jing TIAN², Jing JIANG¹^,³, Wangyin WANG¹, Xupeng CAO¹

^1.China State Key Laboratory of Catalysis，Dalian Institute of Chemical Physics，Chinese Academy of Sciences，Dalian National Laboratory for Clean Energy，Dalian 116023，Liaoning，China
^2.School of Bioengineering，Dalian Polytechnic University，Dalian 116034，Liaoning，China
^3.School of Chemical and Materials Science，University of Science and Technology of China，Hefei 230026，Anhui，China

Received:2022-08-08 Revised:2022-09-28 Online:2022-12-31 Published:2023-01-17

摘要/Abstract

摘要：

微藻是重要的太阳能驱动CO₂生物转化的生物，建立微藻叶绿体细胞器工厂是通过合成生物学手段实现“碳中和”的潜在途径之一。这是因为微藻叶绿体是碳同化以及后续碳水化合物、脂肪酸、天然色素、氨基酸等重要合成器官，与高等植物细胞内具备多个相对较小的叶绿体不同，大部分微藻仅拥有一个占细胞体积50%以上的大叶绿体，更有利于获得纯净的株系，有望在食品、水产、医药、化学品、生物燃料等领域占据重要地位。微藻改造及微藻叶绿体细胞器工厂研究尚处于起步阶段，本文系统通过对现有转化、表达技术进展进行汇总和简要分析，对比了叶绿体直接转化和基于叶绿体转运肽的核转化叶绿体表达不同策略的优缺点，为后续发展提供借鉴。其中，靶向叶绿体基因组的直接转化策略应用较广泛，主要集中在莱茵衣藻中，已成功表达了100多种不同的蛋白质，但是叶绿体基因组可插入位点有限和调控手段相对缺乏；通过利用叶绿体转运肽，叶绿体中90%以上的蛋白都是核编码并被可控递送到叶绿体内，因此近年来基于叶绿体转运肽的核转化叶绿体定位表达技术的关注度得到了提升，并且已经在固碳、油脂生产调节方面展示出了一定优势。

关键词: 碳中和, 莱茵衣藻, 叶绿体转运肽, 叶绿体细胞器工厂, 合成生物学

Abstract:

Microalgae are important solar-driven CO₂ biotransformation organisms. The chloroplasts of microalgae are important organelles for carbon assimilation and subsequent synthesis of carbohydrates, fatty acids, natural pigments, and amino acids. Unlike higher plant cells, which have multiple relatively small chloroplasts, most microalgae only have one large chloroplast that accounts for more than 50% of the cell volume. This makes it more conducive for us to obtain pure strains and it is expected to be used in food, aquatic industry, medicine, chemical products, biofuels and other fields by the establishment of microalgal chloroplast organelle factories. The construction of microalgae chloroplast organelle factories is one of the potential ways to achieve “carbon neutrality” by means of synthetic biology. The researches on microalgae transformation and microalgae chloroplast organelle factory are still in their infancy. There are still a lot of scientific and technical questions to be answered before microalgae chloroplast organelle factory can be applied at a large scale. In this mini review, the current progress of chloroplast transformation and expression technology in microalgae has been systematically summarized and briefly analyzed, and different approaches are compared, especially regarding the transformation strategies, i.e., direct chloroplast transformation and the transformation of nuclear-encoded chloroplast-targeted genes based on chimeric chloroplast transit peptides. The direct transformation strategy targeting the chloroplast genome is widely used. In Chlamydomonasreinhardtii, the most studied species, more than 100 different proteins have been successfully expressed. However, the chloroplast genome has limited insertion sites and the available regulation machineries on the exogenous genes' expression are rare. By using chloroplast signal peptides, more than 90% of native chloroplast proteins are nuclear-encoded and controllably delivered to the chloroplast. In recent years, the strategy of nuclear transformation and chloroplast-targeting expression based on chimeric chloroplast signal peptide has gained attention, having shown advantages in carbon fixation and oil production regulation. Some perspectives were also discussed. In the global effort for carbon neutralization, the microalgae chloroplast organelle factories will be good carriers to convert CO₂ to complex biomass by artificial and natural hybrid photosynthesis as a solution to food, energy, and environmental problems.

Key words: carbon neutrality, Chlamydomonasreinhardtii, chloroplast transit peptide, chloroplast organelle factory, synthesis biology

中图分类号:

Q812

朱振, 田晶, 江静, 王旺银, 曹旭鹏. 微藻叶绿体细胞器工厂研究进展[J]. 合成生物学, 2022, 3(6): 1218-1234.

Zhen ZHU, Jing TIAN, Jing JIANG, Wangyin WANG, Xupeng CAO. Progress in microalgae chloroplast organelle factory development[J]. Synthetic Biology Journal, 2022, 3(6): 1218-1234.

图/表 4

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藻株	表达系统	表达水平	描述	文献
C.reinhardtii	pACTBVP1vector (atpX-CTBVP1),atpA promoter, rbcL terminator	3 % TSP	CTB-VP1融合基因保留了其蛋白的神经节苷脂GM1亲和力和抗原性，并证明绿藻叶绿体作为生产黏膜疫苗的潜力	[55]
C.reinhardtii	psbA promoter and 5′ UTR, psbA 3′ UTR	ND	利用微藻生产具有可溶性和酶活性的抗毒素，并延长了植入人类B细胞肿瘤小鼠的生存时间	[56]
C.reinhardtii	pSRSapI and pASapI vector,psaA-1 promoter, rbcL 3′ UTR	约1.3 mg/g DB	利用莱茵衣藻叶绿体生产抗人类病原体肺炎链球菌特有的内溶素Cpl-1和Pal并具有抗菌活性	[57]
C.reinhardtii	psbA promoter and 5′ UTR	>5 % TSP	利用莱茵衣藻叶绿体稳定表达具有生物活性且可溶性的哺乳类蛋白（MSAA）	[58]
C.reinhardtii	atpA or rbcL promoters and 5′ UTR	0.5 % TSP	首次实现利用莱茵衣藻叶绿体积累可溶性的，正确折叠的lsc抗体	[59]
C.reinhardtii	pASapI vector, psbH, aadA	6.77 mg/L 22 mg/L DA	首次实现中试规模下的利用莱茵衣藻缺壁株的叶绿体表达细胞色素P450和二萜合酶TPS4	[60]
C.reinhardtii	psbA, atpApsbD promoter	21% TCP	VP28基因的密码子优化会影响蛋白质的积累	[45]
H.pluvialis	16S-trnI/trnA-23S region,psbA and rbcL promoter, terminator	ND	首次实现利用雨生红球藻叶绿体成功表达抗菌肽	[61]
C.vulgaris	16S-trnI/trnA-23S region, rbcL promoter, psbA terminator, aadA	ND	首次在小球藻叶绿体中成功实现2个密码子优化后的抗菌肽共表达	[62]

藻株	表达系统	表达水平	描述	文献
C.reinhardtii	pACTBVP1vector (atpX-CTBVP1),atpA promoter, rbcL terminator	3 % TSP	CTB-VP1融合基因保留了其蛋白的神经节苷脂GM1亲和力和抗原性，并证明绿藻叶绿体作为生产黏膜疫苗的潜力	[55]
C.reinhardtii	psbA promoter and 5′ UTR, psbA 3′ UTR	ND	利用微藻生产具有可溶性和酶活性的抗毒素，并延长了植入人类B细胞肿瘤小鼠的生存时间	[56]
C.reinhardtii	pSRSapI and pASapI vector,psaA-1 promoter, rbcL 3′ UTR	约1.3 mg/g DB	利用莱茵衣藻叶绿体生产抗人类病原体肺炎链球菌特有的内溶素Cpl-1和Pal并具有抗菌活性	[57]
C.reinhardtii	psbA promoter and 5′ UTR	>5 % TSP	利用莱茵衣藻叶绿体稳定表达具有生物活性且可溶性的哺乳类蛋白（MSAA）	[58]
C.reinhardtii	atpA or rbcL promoters and 5′ UTR	0.5 % TSP	首次实现利用莱茵衣藻叶绿体积累可溶性的，正确折叠的lsc抗体	[59]
C.reinhardtii	pASapI vector, psbH, aadA	6.77 mg/L 22 mg/L DA	首次实现中试规模下的利用莱茵衣藻缺壁株的叶绿体表达细胞色素P450和二萜合酶TPS4	[60]
C.reinhardtii	psbA, atpApsbD promoter	21% TCP	VP28基因的密码子优化会影响蛋白质的积累	[45]
H.pluvialis	16S-trnI/trnA-23S region,psbA and rbcL promoter, terminator	ND	首次实现利用雨生红球藻叶绿体成功表达抗菌肽	[61]
C.vulgaris	16S-trnI/trnA-23S region, rbcL promoter, psbA terminator, aadA	ND	首次在小球藻叶绿体中成功实现2个密码子优化后的抗菌肽共表达	[62]

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微藻叶绿体细胞器工厂研究进展

Progress in microalgae chloroplast organelle factory development

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