College of Life Science and Technology, Beijing University of Chemical Technology, 15, Beisanhuan East Road, Chaoyang District, Beijing 100029, China; Cyanoworks, LLC, 1771 Haskell Rd., Olean, NY 14760, USA.
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 31-007 Kraków, Poland.
Environ Pollut. 2018 Jun;237:926-935. doi: 10.1016/j.envpol.2018.01.071. Epub 2018 Feb 15.
In this report, we establish proof-of-principle demonstrating for the first time genetic engineering of a photoautotrophic microorganism for bioremediation of naturally occurring cyanotoxins. In model cyanobacterium Synechocystis sp. PCC 6803 we have heterologously expressed Sphingopyxis sp. USTB-05 microcystinase (MlrA) bearing a 23 amino acid N-terminus secretion peptide from native Synechocystis sp. PCC 6803 PilA (sll1694). The resultant whole cell biocatalyst displayed about 3 times higher activity against microcystin-LR compared to a native MlrA host (Sphingomonas sp. ACM 3962), normalized for optical density. In addition, MlrA activity was found to be almost entirely located in the cyanobacterial cytosolic fraction, despite the presence of the secretion tag, with crude cellular extracts showing MlrA activity comparable to extracts from MlrA expressing E. coli. Furthermore, despite approximately 9.4-fold higher initial MlrA activity of a whole cell E. coli biocatalyst, utilization of a photoautotrophic chassis resulted in prolonged stability of MlrA activity when cultured under semi-natural conditions (using lake water), with the heterologous MlrA biocatalytic activity of the E. coli culture disappearing after 4 days, while the cyanobacterial host displayed activity (3% of initial activity) after 9 days. In addition, the cyanobacterial cell density was maintained over the duration of this experiment while the cell density of the E. coli culture rapidly declined. Lastly, failure to establish a stable cyanobacterial isolate expressing native MlrA (without the N-terminus tag) via the strong cpcB560 promoter draws attention to the use of peptide tags to positively modulate expression of potentially toxic proteins.
在本报告中，我们首次建立了一个证明，证明了对光自养微生物进行基因工程以生物修复天然存在的蓝藻毒素的原理。在模型蓝藻集胞藻 PCC 6803 中，我们异源表达了 Sphingopyxis sp. USTB-05 微囊藻酶 (MlrA)，其带有来自天然集胞藻 PCC 6803 PilA (sll1694) 的 23 个氨基酸 N 端分泌肽。与天然 MlrA 宿主（鞘氨醇单胞菌 ACM 3962）相比，所得的全细胞生物催化剂对微囊藻-LR 的活性提高了约 3 倍，以光密度为归一化。此外，尽管存在分泌标签，但 MlrA 活性几乎完全位于蓝藻的细胞质部分，粗细胞提取物显示出与表达 MlrA 的大肠杆菌提取物相当的 MlrA 活性。此外，尽管全细胞大肠杆菌生物催化剂的初始 MlrA 活性高约 9.4 倍，但在半自然条件（使用湖水）下培养时，光自养底盘的利用导致 MlrA 活性的稳定性延长，异源 MlrA 生物催化大肠杆菌培养物的活性在 4 天后消失，而蓝藻宿主在 9 天后显示出活性（初始活性的 3%）。此外，在实验过程中维持了蓝藻细胞密度，而大肠杆菌培养物的细胞密度迅速下降。最后，未能通过强 cpcB560 启动子建立稳定表达天然 MlrA（无 N 端标签）的蓝藻分离株，引起了人们对使用肽标签来积极调节潜在毒性蛋白表达的关注。
