微生物代谢工程研究组知识库

碳源在异养微生物发酵生产生物能源与生物基化学品过程中占据着关键地位并且是主要的成本来源，传统的碳源主要来源于甘蔗、玉米等粮食作物及木质纤维素生物质，但在应用过程中存在各自的核心障碍，限制了其进一步发展的空间。蓝藻作为一种光能自养原核微生物，能够利用光能和二氧化碳合成有机物，遗传背景和遗传操作相对简单。由于一些蓝藻在盐胁迫条件下可以产生蔗糖，近年来引起了广泛的研究和重视。本研究一方面通过基因工程手段，对聚球藻PCC 7942蔗糖代谢相关的关键基因和蔗糖转运基因进行了研究；另一方面在产蔗糖基因工程藻株的基础上对于蓝藻蔗糖的下游应用进行了初步的探索。 本论文的主要研究内容和结果如下： 第一，glgC基因的表达与蔗糖合成的关系研究。利用茶碱控制的核糖体开关ENYC4控制glgC的表达，用不同的茶碱浓度来控制glgC的表达水平。结果发现在正常培养期间glgC的表达水平越高，糖原的积累越多。在盐胁迫后糖原很快分解，蔗糖迅速合成，而且盐胁迫前糖原越多，蔗糖的产量也越高。 第二，蔗糖转运蛋白基因及蔗糖合成关键基因筛选。分别构建了在中性位点3的位置过表达来源于拟南芥的atsuc4，玉米的zmsut1，甘蔗的shsut1和小肠结肠炎耶尔森菌的scry的聚球藻PCC 7942基因工程藻株，随后将得到的突变藻株进行培养及盐胁迫并分析了胞外蔗糖的产量，结果显示在聚球藻PCC 7942中过表达这些蔗糖转运基因并未起到蔗糖转运的作用。 从不同蓝藻中选取了10个蔗糖磷酸合成酶基因（sps）和3个蔗糖合成酶基因（ss），将选取的基因的N端加上his-tag并在大肠杆菌BL21（DE3）中进行表达和纯化，结果发现大部分蛋白不可溶。 第三，产蔗糖蓝藻突变株全生物质作为营养物质用于微生物发酵研究。首先，我们对聚球藻突变株及野生型藻株的细胞生物质成分进行了测定，发现细胞生物质中超过80% 的成分是蛋白质、糖类及脂类。随后，确定了用盐酸分别处理细胞生物质及含有蔗糖的蓝藻培养液的预处理方法，利用该方法蓝藻培养液中的蔗糖可以完全被水解成葡萄糖和果糖。最后，我们用不同的水解液去培养大肠杆菌及酵母菌，发现大肠杆菌及酵母菌均在混合水解液中可以正常的生长及发酵产乙醇，说明蓝藻生产的蔗糖和细胞生物质经过简单处理后不仅可以为微生物的生长和发酵提供营养物质而且体系中不含有任何的抑制性物质。

Starting materials for microbial heterotrophic fermentation represent one of the most important factors and take up a majority of the cost for production of biofuels and biochemicals. Traditionally, carbohydrate feedstocks were typically derived from cellulosic biomass and agriculture crops such as sugarcanes and corns. Both have their core obstacles which refrain the further development. Cyanobacteria, photoautotrophic prokaryotic microorganisms, can synthesize organic compounds with solar and CO2. Besides, their genetic background is relatively simple and genetic tools for metabolic engineering are established. Recently, more and more attentions have been drawn into that some cyanobacteria can synthsize sucrose under salt stress. In this study, on one hand, the key genes in sucrose synthesis and sucrose transportation were studied in Synechococcus sp. PCC 7942. On the other hand, the processes of applications of sucrose produced by cyanobacteria were tentatively explored. First, the relationship of glgC expression level and sucrose production was studied. The riboswich ENYC4 controlled by adding different concentrations of theophylline was applied to control the expression level of glgC, and the result showed that the higher glgC expression level was applied, the more glycogen was accumulated. Glycogen was decomposed and sucrose was accumulated rapidly under salt shock. Second, sucrose transporter genes and key genes for sucrose synthesis were screened. atsuc4 gene from Arabidopsis, zmsut1 gene from Zea mays, shsut1 gene from sugarcane and scry from Yersinia enterocolitica were cloned and introduced into neutral site 3 of Synechococcus sp. PCC 7942, and the production of sucrose in cultured was analyzed. The result showed that the selected transporter genes had no efficiency for sucrose transportation. Ten different sps genes and three sucrose synthetase genes (ss) from different cyanobacteria species were cloned and introduced into E. coli BL21(DE3) with his-tag at N terminal，and the proteins encoded by the selected genes were extracted, but most proteins were insoluble. Third, cyanobacterial nutrients with sucrose production can be used for microbial fermentation. We analyzed the components of the sucrose production mutant and the total content of protein, lipid and carbonhydrate was more than 80%. Then, the pretreatment method was determined that the cellular biomass and sucrose in cell-free culture was hydrolyzed by HCl separately, and the sucrose in cell-free culture was completely hydrolyzed into glucose and fructose. Cultivated with the neutralized hydrolysates, Escherichia coli and Saccharomyces cerevisiae, the mostly used industrial hosts, showed a desirable growth and ethanol fermentation without any inhibition.