| Autor: |
Qiao Q; School of Biological Engineering, Tianjin University of Science and Technology, Tianjin 300457, China.; Key Laboratory of Engineering Biology for Low-carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.; National Center of Technology Innovation for Synthetic Biology, Tianjin 300308, China., Ning S; School of Biological Engineering, Tianjin University of Science and Technology, Tianjin 300457, China.; Key Laboratory of Engineering Biology for Low-carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.; National Center of Technology Innovation for Synthetic Biology, Tianjin 300308, China., Wang R; Shenzhen Kexing Tianhe Biotechnology Co., Ltd., Shenzhen 518000, Guangdong, China., Zheng Y; School of Biological Engineering, Tianjin University of Science and Technology, Tianjin 300457, China., Lu F; School of Biological Engineering, Tianjin University of Science and Technology, Tianjin 300457, China., Chen J; Key Laboratory of Engineering Biology for Low-carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.; National Center of Technology Innovation for Synthetic Biology, Tianjin 300308, China., Liu J; Key Laboratory of Engineering Biology for Low-carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.; National Center of Technology Innovation for Synthetic Biology, Tianjin 300308, China., Zheng P; School of Biological Engineering, Tianjin University of Science and Technology, Tianjin 300457, China.; Key Laboratory of Engineering Biology for Low-carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.; National Center of Technology Innovation for Synthetic Biology, Tianjin 300308, China. |
| Abstrakt: |
Corynebacterium glutamicum is a major workhorse in the industrial production of branched-chain amino acids (BCAAs). The acetohydroxyacid synthase (AHAS) encoded by ilvBN is a key enzyme in the biosynthesis of BCAAs. Enhancing AHAS expression is essential for engineering BCAA producers. However, at present, the available studies only used limited promoters to regulate AHAS expression, which is insufficient for achieving efficient regulation. Herein, we first employed a previously developed reporter system to screen out a strong constitutive promoter P gpmA * from six candidate promoters for expressing ilvBN . P gpmA * showcased the expression strength 23.3-fold that of the native promoter P ilvBN . Moreover, three synthetic RBS libraries based on the promoter P gpmA * were constructed and evaluated by plate fluorescence imaging. The results revealed that "R (9) N (6) " was the best mutant library. A total of 36 RBS mutants with enhanced strength were further screened by evaluation in 96-deep-well plates, and the highest strength reached up to 62.3-fold that of P ilvBN . Finally, the promoter P gpmA * was combined with three RBS mutants (WT, RBS18, and RBS36) to fine-tune the expression of ilvBN S155F for L-valine biosynthesis, respectively. Increased expression strength led to enhanced L-valine production, with titers of 1.17, 1.38, and 2.29 g/L, respectively. The combination of RBS18 strain with the further overexpression of ilvC produced 7.57 g/L L-valine. The regulatory elements obtained in this study can be utilized to modulate AHAS expression for BCAA production in C . glutamicum . Additionally, this strategy can guide the efficient expression regulation of other key enzymes. |
