Utilizing 5′ UTR Engineering Enables Fine-Tuning of Multiple Genes within Operons to Balance Metabolic Flux in <i>Bacillus subtilis</i>
Jiajia You,
Yifan Wang,
Kang Wang,
Yuxuan Du,
Xiaoling Zhang,
Xian Zhang,
Taowei Yang,
Xuewei Pan,
Zhiming Rao
Affiliations
Jiajia You
Key Laboratory of Industrial Biotechnology of the Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi 214122, China
Yifan Wang
Department of Food Science and Technology, Texas A & M University, College Station, TX 77843, USA
Kang Wang
Key Laboratory of Industrial Biotechnology of the Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi 214122, China
Yuxuan Du
Key Laboratory of Industrial Biotechnology of the Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi 214122, China
Xiaoling Zhang
Key Laboratory of Industrial Biotechnology of the Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi 214122, China
Xian Zhang
Key Laboratory of Industrial Biotechnology of the Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi 214122, China
Taowei Yang
Key Laboratory of Industrial Biotechnology of the Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi 214122, China
Xuewei Pan
Key Laboratory of Industrial Biotechnology of the Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi 214122, China
Zhiming Rao
Key Laboratory of Industrial Biotechnology of the Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi 214122, China
The application of synthetic biology tools to modulate gene expression to increase yield has been thoroughly demonstrated as an effective and convenient approach in industrial production. In this study, we employed a high-throughput screening strategy to identify a 5′ UTR sequence from the genome of B. subtilis 168. This sequence resulted in a 5.8-fold increase in the expression level of EGFP. By utilizing the 5′ UTR sequence to overexpress individual genes within the rib operon, it was determined that the genes ribD and ribAB serve as rate-limiting enzymes in the riboflavin synthesis pathway. Constructing a 5′ UTR library to regulate EGFP expression resulted in a variation range in gene expression levels exceeding 100-fold. Employing the same 5′ UTR library to regulate the expression of EGFP and mCherry within the operon led to a change in the expression ratio of these two genes by over 10,000-fold. So, employing a 5′ UTR library to modulate the expression of the rib operon gene and construct a synthetic rib operon resulted in a 2.09-fold increase in riboflavin production. These results indicate that the 5′ UTR sequence identified and characterized in this study can serve as a versatile synthetic biology toolkit for achieving complex metabolic network reconstruction. This toolkit can facilitate the fine-tuning of gene expression to produce target products.
