Improving heterologous protein expression in Synechocystis sp. PCC 6803 for alpha-bisabolene production
Improving heterologous protein expression in Synechocystis sp. PCC 6803 for alpha-bisabolene production
复制标题
DOI：
10.1016/j.mec.2019.e00117
复制
发表时间：
2020-06-01
影响因子：
5.2
通讯作者：
Peebles, Christie A. M.
中科院分区：
文献类型：
--
作者：
Sebesta, Jacob;Peebles, Christie A. M.
蓝藻生物燃料具有降低生物燃料生产的成本和气候影响的潜力，因为初级碳固定和燃料转化是在蓝藻培养过程中同时完成的。因此，蓝藻生物燃料不依赖于异养生物所需的昂贵的有机碳原料，这减少了对耕地和淡水等农业资源的竞争。然而，使用蓝藻细菌获得的大多数感兴趣分子的已发表的产品滴度落后于使用酵母和大肠杆菌（E. coli）培养物获得的产品滴度。在集胞藻属中。 PCC 6803 (S. 6803) 中，我们尝试提高倍半萜、红没药烯的产品滴度，将其转化为红没药烷（一种可能的柴油替代品）。我们使用来自大冷杉 (Abies grandis) 的红没药烯合酶的五种不同密码子使用序列测试了 19 株转基因 S.6803。每个密码子使用序列至少测试了三个核糖体结合位点（大多数是使用 RBS 计算器设计的）。我们还测试了带有和不带有来自大肠杆菌的法呢基焦磷酸合酶基因的菌株。在连续光照下生长五天后，红没药烯滴度从未检测到到 7.8 mg/L。测量了红没药烯合酶丰度，发现其与滴度密切相关。还对选定的菌株进行了 12:12 光照：黑暗循环的测试，在相同的光照时间后达到了相似的滴度。还在暴露于最大光强度为 1600 mu mol 光子 m(-2) s(-1) 的模拟室外光模式的光生物反应器中测试了一种工程菌株。在此，生长 36 天后红没药烯滴度达到 22.2 mg/L。为了将这些滴度提高到报道的工程大肠杆菌的水平，我们需要显着提高控制蓝细菌（如 S. 6803）基因表达的能力，并共同利用其他代谢工程方法。
Cyanobacterial biofuels have the potential to reduce the cost and climate impacts of biofuel production because primary carbon fixation and conversion to fuel are completed together in the cultivation of the cyanobacteria. Cyanobacterial biofuels, therefore, do not rely on costly organic carbon feedstocks that heterotrophs require, which reduces competition for agricultural resources such as arable land and freshwater. However, the published product titer achieved for most molecules of interest using cyanobacteria lag behind what has been achieved using yeast and Escherichia coli (E. coli) cultures. In Synechocystis sp. PCC 6803 (S. 6803), we attempted to increase the product titer of the sesquiterpene, bisabolene, which may be converted to bisabolane, a possible diesel replacement. We tested 19 strains of genetically modified S. 6803 with five different codon usage sequences of the bisabolene synthase from the grand fir tree (Abies grandis). At least three ribosome binding sites (most designed using the RBS Calculator) were tested for each codon usage sequence. We also tested strains with and without the farnesyl pyrophosphate synthase gene from E. coli. Bisabolene titers after five days of growth in continuous light ranged from un-detected to 7.8 mg/L. Bisabolene synthase abundance was measured and found to be well correlated with titer. Select strains were also tested in 12:12 light:dark cycles, where similar titers were reached after the same amount of light exposure time. One engineered strain was also tested in photobioreactors exposed to a simulated outdoor light pattern with maximum light intensity of 1600 mu mol photons m(-2) s(-1). Here, the bisabolene titer reached 22.2 mg/L after 36 days of growth. Dramatic improvements in our ability to control gene expression in cyanobacteria such as S. 6803, and the co-utilization of additional metabolic engineering methods, are needed in order for these titers to improve to the levels reported for engineered E. coli.