聚球藻UTEX 2973 高光适应相关基因筛选及其糖类化合物合成潜力研究

蓝细菌能够进行光合作用，是研究植物中光合及抗逆等生理过程机理的重要模式微生物，具有重要的理论研究意义。生物能源为可再生能源，是化石类能源的替代品之一，然而如何高效率、低成本地获取糖原料，是限制生物能源发展和应用的瓶颈问题之一。蓝细菌可以进行光合作用，并且具有生长速度快、遗传操作简便等优势，是生物能源生产中理想的细胞工厂。 聚球藻UTEX 2973 是目前报道的生长速度最快的蓝细菌，具有高温、高光耐受能力，而聚球藻PCC 7942 的高光、高温耐受性稍差。考虑到聚球藻UTEX 2973 与聚球藻PCC 7942 的基因组仅存在0.2%的差异，通过研究聚球藻UTEX 2973 与聚球藻PCC7942 的差异基因，我们尝试对高光耐受相关的基因进行鉴定；另外，对聚球藻UTEX 2973 合成糖类化合物的能力进行初步的探索。 本研究的主要内容及结果如下： （1）通过将聚球藻UTEX 2973 与聚球藻PCC 7942 存在于基因中的55 个 SNPs 位点互补到聚球藻PCC 7942 中进行高光筛选，鉴定了聚球藻UTEX 2973中与高光适应相关的基因ATP synthase FoF1 subunit alpha，将聚球藻PCC 7942中该基因突变为聚球藻UTEX 2973 中的序列后，得到的聚球藻PCC 7942 突变株也具有了一定的高光耐受能力，证明ATP synthase FoF1 确实是与高光耐受相关的重要基因； （2）通过对聚球藻UTEX 2973 糖原积累的检测，发现在正常BG11 培养下糖原积累量达到其细胞干重的51%，并且积累糖原的同时其生物质也迅速积累； （3）聚球藻UTEX 2973 与聚球藻PCC 7942 有相似的耐盐能力，在不同盐浓度下的蔗糖生产能力也较为类似；将蔗糖转运蛋白CscB 在聚球藻UTEX 2973中过表达，成功实现了胞内蔗糖的外运并且得到的最高蔗糖产量3.34 g L-1； （4）相同浓度的KCl 与NaCl 在胁迫聚球藻UTEX 2973 生产蔗糖上具有相 似的能力，但是KCl 对藻细胞产生的损伤较小，使得使用KCl 作为胁迫物质时可以更好的生产蔗糖。使用含有KCl 的培养基以半连续的方式培养聚球藻UTEX2973，在培养21 天时共积累了8.7 g L-1 的蔗糖，同时蓝细菌胞内糖原维持在干重的40%左右。Cyanobacteria are a group of prokaryotes that perform oxygenic photosynthesis, and were seen as research models in studying mechanisms in plants, such as photosynthesis and stress resistance. Biofuel was one of the promising substitutes for fossil fuel, however the lack of efficiency and economic accesses to sugar feedstock is one of the bottleneck problems impeding the development and commercialization of biofuels. Besides the ability of photosynthesis, cyanobacteira have faster growth speed and easier genetic operation systems, and thus draw much attention recently both in the field of academic and industry. Synechococcus elongatus UTEX 2973 is the fastest growing cyanobacterium reported up to now, whose genome sequence is 99.8% identical to that of Synechococcus elongatus PCC 7942. Synechococcus UTEX 2973 has better tolerance to high light and high temperature than Synechococcus PCC 7942. Thus in this work, we tried to identify the high light tolerance related genes in Synechococcus UTEX 2973 based on the differences between two Synechococcus genomes, and explored the potential of Synechococcus UTEX 2973 for sugar feedstock production. The main research contents and results are summarized as follows: Firstly, the atpA gene encoding ATP synthase FoF1 subunit alpha was found to be responsible for high light acclimation in Synechococcus UTEX 2973 FoF1 through gene complementary experiments. The resulting Synechococcus PCC 7942 mutant harboring the same single nucleotide polymorphism site with Synechococcus UTEX 2973 possesses tolerance to high light condition, which proved that ATP synthase FoF1 was involved in the high-light acclimation of Synechococcus UTEX 2973. Secondly, Synechococcus UTEX 2973 accumulated glycogen of up to 51% of dry cell weight (DCW) during its fast accumulation of biomass under nitrogen replete condition. Thirdly, Synechococcus UTEX 2973 possesses similar ability of salt tolerance, and has similar sucrose accumulation level under salt stress. Synechococcus UTEX 2973 can secrete sucrose into medium after overexpression of a sucrose transporter CscB. And a highest extracellular sucrose accumulation of 3.34 g L-1 was achieved in the CscB-expressing mutant of Synechococcus UTEX 2973. Finally, we found that KCl displays the same ability to induce sucrose production in Synechococcus UTEX 2973 as NaCl. However, KCl causes much lower damage to cells comparing with NaCl, which makes KCl a better choice for sucrose production. Semi-continuous cultivation using medium containing KCl was performed, 8.7 g L-1 of sucrose was accumulated after 21 days of cultivation, and glycogen was maintained at 40% of DCW