四溴双酚A胁迫响应分子元件的功能

Functional characteristics of the gene cluster in response to tetrabromobisphenol A stress

摘要:

目的 为了解析四溴双酚A (tetrabromobisphenol A, TBBPA)胁迫响应显著上调的chr1_2605-chr1_2604基因簇的分子功能及应用潜力，分别研究了chr1_2605及chr1_2604分子元件在TBBPA特异性识别及降解中的作用。方法 利用合成生物学方法构建传感细胞食异源物鞘氨醇菌(Sphingobium xenophagum) C1 (pBBR-2605-HiBiT)和降解细胞大肠埃希氏菌(Escherichia coli) BL21(DE3, pET30b-2604)。通过荧光素酶活性检测方法分析底盘细胞中chr1_2605元件对不同污染物的响应特征，并采用高效液相色谱法测定降解细胞中chr1_2604元件对TBBPA的降解效率。结果 异生物质响应转录因子Chr1_2605仅对TBBPA表现出高度特异性的响应功能，基于其构建的传感细胞S. xenophagum C1 (pBBR-2605-HiBiT)对TBBPA具有良好的线性响应范围与灵敏度，最低检测限为0.010-0.050 μmol/L；α-酮戊二酸/Fe依赖性双加氧酶Chr1_2604对TBBPA具有高效的降解功能，基于其构建的降解细胞E. coli BL21(DE3, pET30b-2604)在3 d内对2.0 mg/L TBBPA的降解率可达44.415% [0.296 mg/(L·d)]，显著高于目前报道的大部分天然菌株在非共代谢条件下对TBBPA的降解率。结论 本研究发现并证实了chr1_2605-chr1_2604分子元件可以特异性识别并高效降解TBBPA，其中异生物质响应转录因子Chr1_2605能够精准识别TBBPA，α-酮戊二酸/Fe依赖性双加氧酶Chr1_2604可以高效降解TBBPA。

Abstract:

Objective To understand the molecular functions and potential applications of the significantly up-regulated gene cluster chr1_2605-chr1_2604 in response to tetrabromobisphenol A (TBBPA) stress, we investigated the roles of chr1_2605 and chr1_2604 in the specific recognition and efficient degradation of TBBPA.Methods Synthetic biology methods were employed to construct Sphingobium xenophagum C1 (pBBR-2605-HiBiT) and Escherichia coli BL21(DE3, pET30b-2604) as chassis cells for biosensing and degrading, respectively. The response characteristics of Chr1_2605 in the chassis cells to different pollutants were analyzed by the luciferase activity assay. Additionally, the degradation activity of TBBPA by Chr1_2604 in the chassis cells was determined by high-performance liquid chromatography.Results The xenobiotic-responsive element Chr1_2605 exhibited a highly specific response to TBBPA. The Chr1_2605-based chassis cell of S. xenophagum C1 (pBBR-2605-HiBiT) demonstrated high responsivity and sensitivity to TBBPA, with a limit of detection ranging from 0.010 to 0.050 μmol/L. The 2-oxoglutarate/Fe-dependent dioxygenase Chr1_2604 in the chassis cell of E. coli BL21(DE3, pET30b-2604) displayed the degradation rate of 44.415% for 2.0 mg/L TBBPA within 3 d [0.296 mg/(L·d)], which was significantly higher than those of most reported microbial strains under non-co-metabolic conditions.Conclusion The chr1_2605-chr1_2604 gene cluster can accurately recognize and degrade TBBPA. Specifically, the xenobiotic-responsive element Chr1_2605 specifically recognizes TBBPA, whereas the 2-oxoglutarate/Fe-dependent dioxygenase Chr1_2604 efficiently degrades TBBPA.