Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan 46241, Republic of Korea.
College of Pharmacy and Research Institute for Drug Development, Pusan National University, Busan 46241, Republic of Korea.
ACS Synth Biol. 2024 Aug 16;13(8):2587-2599. doi: 10.1021/acssynbio.4c00423. Epub 2024 Aug 7.
Genetic code expansion (GCE) is a powerful strategy that expands the genetic code of an organism for incorporating noncanonical amino acids into proteins using engineered tRNAs and aminoacyl-tRNA synthetases (aaRSs). While GCE has opened up new possibilities for synthetic biology, little is known about the potential side effects of exogenous aaRS/tRNA pairs. In this study, we investigated the impact of exogenous aaRS and amber suppressor tRNA on gene expression in . We discovered that in DH10β Δ, transformed with the F1RP/F2P two-hybrid system, the high consumption rate of cellular adenosine triphosphate by exogenous aaRS/tRNA at elevated temperatures induces temperature sensitivity in the expression of genes regulated by the cyclic AMP receptor protein (CRP). We harnessed this temperature sensitivity to create a novel biological AND gate in , responsive to both -benzoylphenylalanine (BzF) and low temperature, using a BzF-dependent variant of chorismate mutase and split subunits of adenylate cyclase. Our study provides new insights into the unexpected effects of exogenous aaRS/tRNA pairs and offers a new approach for constructing a biological logic gate.
遗传密码扩展 (GCE) 是一种强大的策略，它利用工程化的 tRNA 和氨酰-tRNA 合成酶 (aaRS) 将非规范氨基酸掺入蛋白质中，从而扩展生物体的遗传密码。虽然 GCE 为合成生物学开辟了新的可能性，但对于外源 aaRS/tRNA 对的潜在副作用知之甚少。在这项研究中，我们研究了外源 aaRS 和琥珀终止 tRNA 对. 基因表达的影响。我们发现，在 DH10β Δ 中，转化为 F1RP/F2P 双杂交系统后，高温下外源 aaRS/tRNA 的高细胞三磷酸腺苷消耗率会导致环 AMP 受体蛋白 (CRP) 调控基因的表达产生温度敏感性。我们利用这种温度敏感性在. 中创建了一个新的生物与门，该与门对 -苯甲酰苯丙氨酸 (BzF) 和低温都有响应，使用依赖 BzF 的分支酸变位酶变体和分裂的 腺苷酸环化酶亚基。我们的研究提供了对外源 aaRS/tRNA 对意外影响的新见解，并为构建生物逻辑门提供了一种新方法。
