Liu Ying-Xing, Zhuo Xiao-Zhen, Li Si-Yu
Department of Chemical Engineering, National Chung Hsing University, Taichung, Taiwan.
Innovation and Development Center of Sustainable Agriculture, National Chung Hsing University, Taichung, Taiwan.
Synth Biol (Oxf). 2022 Aug 17;7(1):ysac014. doi: 10.1093/synbio/ysac014. eCollection 2022.
The strong transcriptional activity of the virulent gene in has been proven to be anthrax toxin activator (AtxA)-regulated. However, the obscure transcription mechanism hinders practical applications of this strong promoter. In this study, a 509-bp DNA fragment [termed 509sequence, (-508)-(+1) relative to the P2 transcription start site] was cloned upstream of rbs-GFPuv as pTOL02B to elucidate the AtxA-regulated transcription. The 509sequence was dissected into the -10 sequence, -35 sequence, AT tract, SLI/SLII and upstream site. In conjunction with the heterologous co-expression of AtxA (under the control of the T7 promoter), the -10 sequence (TATACT) was sufficient for the AtxA-regulated transcription. Integration of pTOL02F + pTOLAtxA as pTOL03F showed that the AtxA-regulated transcription exhibited a strong specific fluorescence intensity/common analytical chemistry term (OD) of 40 597 ± 446 and an induction/repression ratio of 122. An improved induction/repression ratio of 276 was achieved by cultivating /pTOL03F in M9 minimal medium. The newly developed promoter system termed P consists of AtxA, the -10 sequence and RNA polymerase. These three elements synergistically and cooperatively formed a previously undiscovered transcription system, which exhibited a tight-control, high-level, modulable and stationary-phase-specific transcription. The P was used for expression for the stationary-phase polyhydroxybutyrate production, and the results showed that a PHB yield, content and titer of 0.20 ± 0.27 g/g-glucose, 68 ± 11% and 1.5 ± 0.4 g/l can be obtained. The positive inducible P, in contrast to negative inducible, should be a useful tool to diversify the gene information flow in synthetic biology. Graphical Abstract.
已证明炭疽杆菌中毒力基因的强转录活性受炭疽毒素激活剂(AtxA)调控。然而,其模糊的转录机制阻碍了这种强启动子的实际应用。在本研究中,将一个509 bp的DNA片段[称为509序列,相对于P2转录起始位点为(-508)-(+1)]克隆到rbs-GFPuv上游,构建成pTOL02B,以阐明AtxA调控的转录。509序列被分解为-10序列、-35序列、AT序列、SLI/SLII和上游位点。与AtxA的异源共表达(在T7启动子控制下)相结合,-10序列(TATACT)足以实现AtxA调控的转录。将pTOL02F + pTOLAtxA整合为pTOL03F表明,AtxA调控的转录表现出40597±446的强特异性荧光强度/共同分析化学术语(OD)和122的诱导/抑制比。通过在M9基本培养基中培养/pTOL03F,诱导/抑制比提高到了276。新开发的称为P的启动子系统由AtxA、-10序列和RNA聚合酶组成。这三个元件协同形成了一个以前未发现的转录系统,该系统表现出严格控制、高水平、可调节和稳定期特异性转录。P被用于稳定期聚羟基丁酸酯生产的表达,结果表明,聚羟基丁酸酯的产量、含量和滴度分别为0.20±0.27 g/g-葡萄糖、68±11%和1.5±0.4 g/l。与负诱导型相比,正诱导型P应该是在合成生物学中使基因信息流多样化的有用工具。图形摘要。