Development of inducible promoters for regulating gene expression in Clostridium tyrobutyricum for biobutanol production,Biotechnology and Bioengineering

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Development of inducible promoters for regulating gene expression in Clostridium tyrobutyricum for biobutanol production
Biotechnology and Bioengineering ( IF 3.5 ) Pub Date : 2024-03-28 , DOI: 10.1002/bit.28701
Jun Feng _{1,

2} , Qingke Wang ₂ , Zhen Qin ₂ , Xiaolong Guo ₁ , Hongxin Fu ₁ , Shang-Tian Yang ₂ , Jufang Wang _{1,

3}

Affiliation

Clostridium tyrobutyricum is an anaerobe known for its ability to produce short-chain fatty acids, alcohols, and esters. We aimed to develop inducible promoters for fine-tuning gene expression in C. tyrobutyricum. Synthetic inducible promoters were created by employing an Escherichia coli lac operator to regulate the thiolase promoter (PCathl) from Clostridium acetobutylicum, with the best one (LacI-Pto4s) showing a 5.86-fold dynamic range with isopropyl β- d-thiogalactoside (IPTG) induction. A LT-Pt7 system with a dynamic range of 11.6-fold was then created by combining LacI-Pto4s with a T7 expression system composing of RNA polymerase (T7RNAP) and Pt7lac promoter. Furthermore, two inducible expression systems BgaR-PbgaLA and BgaR-PbgaLB with a dynamic range of ~40-fold were developed by optimizing a lactose-inducible expression system from Clostridium perfringens with modified 5′ untranslated region (5′ UTR) and ribosome-binding site (RBS). BgaR-PbgaLB was then used to regulate the expressions of a bifunctional aldehyde/alcohol dehydrogenase encoded by adhE2 and butyryl-CoA/acetate Co-A transferase encoded by cat1 in C. tyrobutyricum wild type and Δcat1::adhE2, respectively, demonstrating its efficient inducible gene regulation. The regulated cat1 expression also confirmed that the Cat1-catalyzed reaction was responsible for acetate assimilation in C. tyrobutyricum. The inducible promoters offer new tools for tuning gene expression in C. tyrobutyricum for industrial applications.

中文翻译：

开发用于调节酪丁酸梭菌基因表达的诱导型启动子以生产生物丁醇

酪丁酸梭菌是一种厌氧菌，以其产生短链脂肪酸、醇和酯的能力而闻名。我们的目的是开发诱导型启动子，用于微调酪丁酸梭菌中的基因表达。通过使用大肠杆菌 lac操纵子来调节来自丙酮丁醇梭菌的硫解酶启动子 (PCa thl )，创建了合成的诱导型启动子，其中最好的启动子 (LacI-Pto4s) 显示出与异丙基 β- d-硫代半乳糖苷 (IPTG) 5.86 倍的动态范围）就职。然后，通过将 LacI-Pto4s 与由 RNA 聚合酶（T7RNAP）和 Pt7 lac启动子组成的 T7 表达系统相结合，创建了动态范围为 11.6 倍的 LT-Pt7 系统。此外，通过优化来自产气荚膜梭菌的乳糖诱导表达系统（具有修饰的 5' 非翻译区 (5' UTR) 和核糖体结合），开发了两个动态范围约为 40 倍的诱导表达系统 BgaR-PbgaLA 和 BgaR-PbgaLB网站（苏格兰皇家银行）。然后使用BgaR-PbgaLB调节由adh E2编码的双功能醛/醇脱氢酶和由cat 1编码的丁酰-CoA/乙酸Co-A转移酶在C.tyrobutyricum野生型和Δcat 1:: adh E2中的表达，分别证明了其有效的诱导基因调控。受调节的cat 1 表达也证实了 Cat1 催化的反应是酪丁酸梭菌中乙酸同化的原因。诱导型启动子为工业应用中调节酪丁酸梭菌基因表达提供了新工具。

更新日期：2024-03-28

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