时间： 2022年6月17日（周五）9:00-11:00
会议号：ZOOM 565 530 4673 密码123456
主办：www.bob.com、湖北洪山实验室
承办：生命科学技术学院
报告人1：陈太钰9:00-10:00
题目：利用Rubisco 的基因工程改造来优化植物光合作用
Improvement of plant Photosynthesis by Rubisco genetic Engineering
摘要：1，5-2-磷酸核酮糖羧化酶/加氧酶(Rubisco, Ribulose-1,5-bisphosphate carboxylase/oxygenase) 介导着植物光合作用过程中CO2的初始固定。植物Rubisco的催化速率极低且不能有效区别CO2和O2，当其以O2为底物时将导致光合产物的严重流失(光呼吸)。为了克服这些缺陷，光合生物进化出各种二氧化碳浓缩机制（CO2-concentrating mechanisms，CCMs）来提升Rubisco活性位点周围的CO2浓度。蓝细菌和部分变形菌进化出了以羧酶体（Carboxysome, CB）为核心的CCM。羧酶体是由壳蛋白和内核组成的类似于病毒的蛋白只复合物，其中Rubisco和碳酸酐酶（carbonic anhydrases，CA）被壳蛋白包裹在CB中。羧酶体壳蛋白对碳酸氢根具有选择性通透性，而对CO2和O2具有天然的屏蔽作用。碳酸氢根在进入羧酶体后在碳酸酐酶的催化下生成CO2，后者进而被Rubisco固定。所以羧酶体在其内部给Rubisco营造出一个高浓度CO2和低浓度O2的环境，抑制其氧化活性；同时，结合高速率Rubisco和高效的无机碳转运系统，光合细菌具有高效的CO2固定效率，每年贡献了约全球25%的碳固定。但是大部分作物为C3植物，并没有类似的CCM。在作物中引入高效Rubisco及其CCM，优化作物Rubisco的碳固定效率，抑制其氧化活性将从源头上增加作物产量。而Rubisco的表达与组装有众多伴侣蛋白的参与，涉及复杂的装配过程，且羧酶体复合物自身也包含多达8个不同蛋白(复合体)组成，由9 个基因编码。虽然已有多个在植物叶绿体中表达外源Rubisco的报道，但是外源Rubisco在转基因植物叶绿体的组装效率极低，Rubisco的产量只有野生型植物的10%左右,同时转基因植株在高CO2下比野生型植株生长更慢。如何突破高速率Rubisco在植物中的表达与组装，成功组装具有完整功能的羧酶体是利用细菌羧酶体提升作物光合作用的关键。在过去的三年时间里，我们通过叶绿体转化成功在植物叶绿体中高效表达与组装出Halothiobacillus neapolitanus来源高速率Rubisco。转基因植株的Rubisco含量达到野生型的40%且在1% CO2浓度下表现出和野生型植株一样的生长速率。基于已成功表达的Rubisco系统，我们进一步在烟草叶绿体中转化羧酶体表达操纵子（包含9个基因）并组装出完整功能羧酶体复合物。研究成果在利用以羧酶体为核心CCM来提升作物光合作用能力上获得突破性进展，为后续优化CCM在作物中利用打下良好基础。
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) directs the primary carbon fixation in photosynthesis. Rubisco has inherently the poor capability in discriminating between CO2 and O2 and slow catalytic rate and photorespiration will cause a large amount of carbon fixation loss when Rubisco uses O2 as the substrate. To overcome these limitations, photosynthetic organisms have evolved various CO2-concentrating mechanisms (CCM) to increase CO2 concentration in the vicinity of Rubisco. Cyanobacteria and many proteobacteria have evolved the carboxysomes (CBs) as the central CO2-fixing organelles in CCM. CBs is an icosahedral virus-like proteinaceous complex, in which Rubisco and carbonic anhydrases (CAs) are encapsulated. The proteinaceous shell has a selective permeability to HCO3- and a natural barrier to CO2 and O2. HCO3- will be dehydrated to CO2 by CA and then fixed by Rubisco in the lumen of CBs. Thus, a high CO2/low O2 concentration environment will be created in the lumen of CBs for Rubisco and hence inhibited its oxygenase activity. Based on fast Rubisco and efficient Ci uptake system, photosynthetic bacterium obtain high photosynthetic capacity and annually contribute 25% of carbon fixation on a global scale. However, an overwhelming majority of crops, namely C3 plants, lack any form of CCMs. Therefore, introducing fast Rubisco and CCMs into crops represents a promising strategy to optimize Rubisco carbon fixation ability, inhibiting its oxygenase activity and hence increasing crop yield. However, the biogenesis of Rubisco involves many cognate chaperones and complex folding and assembling procedures, and CB is composed of up to 8 proteins (complex) coding by 9 genes. Several attempts have been carried out to express different exogenous Rubiscos in high plant chloroplasts; however, the expression efficiency of exogenous Rubiscos is extremely low in transgenic plants resulting in a low yield of Rubisco (10% of that in wild-type plants) and worse plant growth performance under 1% CO2 condition. Therefore, increasing the expression efficiency of faster Rubisco and assembling a fully functional carboxysome in high plant chloroplasts is the bottleneck of the application of faster Rubisco and CCM to enhance crop carbon fixation capacity. In the past three years, we successfully expressed a faster Rubisco from Halothiobacillus neapolitanus with a high yield of Rubisco (40% of that in wild-type plants), and the transgenic plant displayed a comparable growth rate with wild-type plants. Based on the high efficient Rubisco express system, a CB expressing operon including up to 9 target genes was transformed into tobacco chloroplast and a fully functional CB complex was assembled in tobacco chloroplast. Our results have made breakthrough progress in the application of CCM with CB as the central CO2-fixing organelles to enhance crop photosynthesis capacity and indicated promising anticipation in the optimization of CCM in crops.
报告人2：吴晟 10:00-11:00
题目：微生物合成植物激素独脚金内脂及其生物合成途径研究
Microbial production of plant hormone strigoactone and studies on its biosynthetic pathways
摘要：独脚金内酯（Strigolactone）是一类重要的植物激素，它在植物生长和发育过程中起着重要的作用。然而独脚金内酯难以获取，这成为了制约其研究和应用的重要因素。本研究整合运用生物信息学，合成生物学及代谢工程等手段，建立了利用大肠杆菌-酿酒酵母混菌体系来生产多种天然独脚金内酯的微生物合成平台：以木糖为原料合成了包括独脚金内酯前体物质carlactone，carlactonoic acid及三种重要独脚金内酯产物5-deoxystrigol (5DS; 6.65 ± 1.71 微克/升)， 4-deoxyorobanchol (3.46 ± 0.28 微克/升) 和orobanchol (OB; 19.36 ± 5.20 微克/升)。此平台不仅能用来快速鉴定参与独脚金内酯生物合成途径的基因 (2周之内)，还能定量比较相关酶的体内催化活性。通过代谢工程克服限速步骤以及发酵过程优化，一种典型的独角金内酯5DS的产量提高到了47.3 微克/升。这项工作为研究独脚金内酯的生物合成和进化研究提供了一个独特的平台，并为进一步开发微生物合成独脚金内酯奠定了基础。
Strigolactones (SLs) are a class of phytohormones playing diverse roles in plant growth and development, yet the limited access to SLs is largely impeding SL-based foundational investigations and applications. Here, we developed Escherichia coli–Saccharomyces cerevisiae consortia to establish a microbial biosynthetic platform for the synthesis of various SLs, including carlactone, carlactonoic acid, 5-deoxystrigol (5DS; 6.65 ± 1.71 mg/liter), 4-deoxyorobanchol (3.46 ± 0.28 mg/liter), and orobanchol (OB; 19.36 ± 5.20 mg/liter). The SL-producing platform enabled us to conduct functional identification of CYP722Cs from various plants as either OB or 5DS synthase. It also allowed us to quantitatively compare known variants of plant SL biosynthetic enzymes in the microbial system. The titer of 5DS was further enhanced through pathway engineering to 47.3 mg/liter. This work provides a unique platform for investigating SL biosynthesis and evolution and lays the foundation for developing SL microbial production process.