Harnessing the Power of Lysine-ON Riboswitches for Metabolic Control in Bacteria

Harnessing the Power of Lysine-ON Riboswitches for Metabolic Control in Bacteria

Introduction:

Riboswitches are regulatory elements found in the non-coding regions of messenger RNA (mRNA) that control gene expression in response to specific ligands. Lysine-ON riboswitches, in particular, have gained attention due to their potential in metabolic control of lysine production in bacteria. In this article, we will explore the process of identifying and engineering lysine-ON riboswitches and their implications for metabolic engineering.

Identifying Lysine-ON Riboswitches:

To identify lysine-ON riboswitches, a dual genetic selection scheme was employed (Figure S3). A library of candidate riboswitches controlling the expression of the tetA gene was constructed in E. coli under non-selective conditions in the presence of 0.1 mM lysine to activate the tetA gene. This initial step allowed for the screening of potential riboswitch candidates.

The ON selection stage followed, where the library of clones was grown in the presence of 0.1 mM lysine and tetracycline. This selection process aimed to isolate riboswitches that exhibited strong activation in response to lysine. Clones displaying high levels of tetA expression were eliminated, and only those with robust activation of the tetA gene were retained.

Engineering Lysine-ON Riboswitches:

Once an efficient lysine-ON riboswitch was identified, it was integrated into the chromosome of Corynebacterium glutamicum. This integration aimed to upregulate the expression of a lysine secretion-related gene, lysE, in response to lysine concentration. By leveraging the power of lysine-ON riboswitches, the production of lysine in C. glutamicum could be enhanced.

The integration process involved translocating the identified riboswitch code, along with the aptamer, to the lysE gene. This genetic modification allowed for precise control over lysine production in C. glutamicum, offering a promising avenue for metabolic engineering.

Challenges and Considerations:

While the use of lysine-ON riboswitches holds great potential, it is important to acknowledge the possible negative effects that may arise during the engineering process. Classical random mutagenesis, for instance, may introduce undesired mutations, leading to low productivity. Therefore, it is crucial to employ rigorous screening methods and optimization techniques to mitigate these potential drawbacks.

Unique Insights:

Interestingly, the presence of the tetA gene not only serves as a mechanism to pump out sterols but also inadvertently allows Nickel2+ to enter the cell, leading to cell death. This phenomenon highlights the ingenuity of the lysine riboswitch selection process, as it enables the survival of clones displaying low levels of tetA expression during negative selection on media containing NiCl2 without lysine.

Actionable Advice:

• 1. Implement a rigorous screening process: When identifying lysine-ON riboswitches, it is vital to construct a diverse library of candidate riboswitches and subject them to stringent selection conditions. This process ensures the retention of riboswitches with strong activation in response to lysine.

• 2. Optimize integration techniques: When integrating lysine-ON riboswitches into the chromosome of bacteria, focus on precise translocation of the riboswitch code and aptamer to the target gene. This optimization enhances the efficiency and reliability of metabolic control mechanisms.

• 3. Continuously monitor and refine engineered strains: After implementing lysine-ON riboswitches, regularly assess the performance of engineered bacteria and refine the system as needed. This iterative process allows for the optimization of lysine production and minimizes undesired effects.

Conclusion:

The utilization of lysine-ON riboswitches presents an exciting opportunity for metabolic control in bacteria. By leveraging their regulatory capabilities, it becomes possible to enhance lysine production and potentially overcome the limitations of classical random mutagenesis. Through rigorous screening, precise integration, and continuous optimization, lysine-ON riboswitches can revolutionize metabolic engineering and pave the way for more efficient production of valuable compounds.