SLC家族碳酸氢根转运体在微拟球藻固碳通路中的功能机制研究

The understanding and engineering of carbon dioxide concentration mechanism (CCM) is of particular importance to molecular breeding of industrial microalgae for large-scale biological carbon fixation. In the model industrial oleaginous microalgae Nannochloropsis oceanica (Nanno), it is very likely bicarbonate transporters (BCTs) play a pivotal role in their ‘compressed yet efficient’ CCM, however the underlying mechanisms are not well understood. To tackle this challenge, I propose a hypothesis explain how the CO2 concentration near carbon fixation site is elevated in Nanno: the core of Nanno CCM is composed of two SLC-family BCTs and one functional carbonic anhydrase (CA); the two BCTs, via their distinct localization and additive function, collaboratively take up bicarbonate from extracellular environment and transport it to the CA located at the endoplasmic reticulum lumen, and then this CA converts the bicarbonate to CO2. To test this hypothesis, firstly, I will verify the importance and essentiality of this BCT-based bicarbonate transportation system in the CCM. Secondly, I will probe and validate their in vivo localizations and activities in CCM, via a combination of targeted genome engineering tools (gene knock down, gene editing and overexpression), so as to understand the labor division and physiological significance of the two BCTs. Finally, based on these findings, I will rationally engineer the endogenous BCTs, and possibly transplant and functionalize exogenous BCTs in Nanno, in order to elevate the cellular carbon fixation rate. These endeavors will establish the first inorganic carbon transmembrane transportation model in industrial oleaginous microalgae, and may lead to a new strategy for strain development for superior carbon fixation.

二氧化碳浓缩机制(CCM)的理解和改造对于工业固碳微藻的分子育种具有重要意义。在模式工业产油微藻海洋微拟球藻（Nanno）中，碳酸氢根转运体BCTs很可能是其“简洁而高效”CCM的关键一环，但其功能机制未知。基于前期工作，申请人提出以下假说：Nanno精简的CCM由两个SLC家族BCTs和一个功能碳酸酐酶CA构成，其中，两个BCTs通过空间分工和功能叠加将碳酸氢根从胞外运输到位于内质网基质的CA，并由此CA转化成CO2以提高固碳位点CO2浓度。为验证此假说：首先，将确认转运环节对CCM的重要性和必要性；其次，基于基因敲低、基因编辑和过表达，对两个BCTs的空间分工和体内活性进行剖析，理解其功能分工及生理意义；进而，通过内源和外源BCTs的理性设计、改造与移植实现细胞固碳速率的提高。这些努力将建立首个工业油藻无机碳跨膜转运机制模型，为工业固碳微藻的分子育种提供新思路。

二氧化碳浓缩机制（CCM）的理解和改造对于工业微藻的分子育种具有重要意义。在模式工业产油微藻海洋微拟球藻（Nanno）中，碳酸氢根转运体BCTs可能是其“简洁而高效”CCM的关键，但其功能机制未知。基于前期工作，申请人提出假设：Nanno精简的CCM由两个SLC4家族BCTs和一个碳酸酐酶NoCA5构成，其中，两个BCTs通过空间分工和功能叠加将碳酸氢根运输到位于内质网基质的NoCA5，并由此CA转化成CO2以提高固碳位点CO2浓度。为验证此假设，首先将确认转运环节对CCM的重要性；其次，基于基因编辑和过表达，对两个BCTs的空间分工和体内活性进行剖析，理解其功能分工及生理意义；进而通过内源和外源BCTs的理性设计和改造实现细胞固碳速率的提高。在研究中，BCT抑制剂实验证实了BCT的转运功能对于Nanno吸收碳酸氢根具有重要作用；对NoBCT1和NoBCT2分别敲除，发现在低CO2下与野生型相比，敲除NoBCT1损害了藻株的生长（生长速率降低14.0%和21.7%，生物量降低约21.1%和13.4%），无机碳亲和性（降低0.99倍和1.48倍）, 碳酸氢根利用率（溶液剩余碳酸氢根高50.9%和41.8%）；敲低NoBCT2的藻株表型与野生型相差不大；结合NoBCT1和NoBCT2同时敲除株的表型（生长速率降低23.6% 和24.9%，无机碳亲和性降低1.03倍和1.97倍，溶液剩余碳酸氢根高85.3%和145%），说明位于叶绿体的NoBCT1是Nanno CCM的关键组分。为提高固碳效率，同时过表达NoBCT1、NoCA5和外源硅藻的BCT，发现突变株在低CO2培养下的生长和碳酸氢根利用率相比野生型显著提高（生长速率提高约36.9%和15.4%，溶液剩余碳酸氢根低56.2%和43.7%）。本项目验证了BCTs在Nanno CCM中的重要功能，为工业固碳微藻的分子育种提供新思路。