Engineering a Lysine-ON Riboswitch for Enhanced Lysine Production in Corynebacterium glutamicum

Engineering a Lysine-ON Riboswitch for Enhanced Lysine Production in Corynebacterium glutamicum

Introduction:

In the pursuit of improving lysine production in Corynebacterium glutamicum, researchers have explored the use of lysine-ON riboswitches. These riboswitches can upregulate the expression of lysine secretion-related genes in response to lysine concentration, thereby enhancing lysine production. However, the process of engineering these riboswitches comes with potential challenges and negative effects. This article delves into the techniques and strategies used to engineer a lysine-ON riboswitch, highlighting the benefits and drawbacks associated with this approach.

The Dual Genetic Selection Scheme:

To identify efficient lysine-ON riboswitches, a dual genetic selection scheme was employed. This scheme involved the construction of a library of candidate riboswitches controlling tetA gene expression in E. coli. The selection process consisted of multiple steps:

1. ON Selection:

The library of candidate riboswitches was grown in the presence of 0.1 mM lysine to activate the tetA gene. This allowed the selection of riboswitches that were responsive to lysine concentration. Only the riboswitches that exhibited activation of the tetA gene were retained for further evaluation.

2. Negative Selection:

Following tetA activation, the surviving clones were grown in the presence of 0.1 mM lysine and tetracycline. This step aimed to select only the riboswitches that could maintain lysine activation of the tetA gene while being resistant to the inhibitory effects of tetracycline. By subjecting the clones to this negative selection, researchers ensured that only the most robust riboswitches were chosen.

3. Expression Readjustment:

The surviving clones were then grown under non-selective conditions in the absence of lysine. This step allowed the expression of the tetA gene to readjust, ensuring that the selected riboswitches were stable and functional even in the absence of lysine.

4. Final Negative Selection:

To further narrow down the selection, clones were grown on media containing NiCl2 but without lysine. The presence of tetA allowed Nickel2+ to enter the cell, resulting in cell death. Only the clones with low levels of tetA expression could survive this negative selection step, indicating that these riboswitches were efficient in avoiding cell death caused by Nickel2+.

Integration into C. glutamicum Chromosome:

After successful screening of an efficient lysine-ON riboswitch, it was integrated into the C. glutamicum chromosome. This integration aimed to upregulate the expression of the lysine secretion-related gene, lysE, in response to lysine concentration. By incorporating the riboswitch into the bacterial chromosome, researchers achieved a more controlled and stable system for enhanced lysine production.

Unique Insight:

Interestingly, the presence of the tetA gene not only facilitated the selection of the lysine-ON riboswitch but also inadvertently allowed the entry of Nickel2+ into the cell, leading to negative selection. This dual purpose of tetA highlights the ingenious nature of the selection scheme employed.

Actionable Advice:

• 1. Optimize Screening Parameters: When employing a dual genetic selection scheme, it is crucial to carefully optimize the screening parameters, such as lysine concentration and the presence of inhibitory substances. This will ensure the selection of robust riboswitches that can withstand varying conditions.

• 2. Consider Additional Gene Integration: While the integration of the lysine-ON riboswitch into the chromosome of C. glutamicum proved effective, it may be worthwhile to explore the integration of other genes involved in lysine production. This approach could potentially further enhance lysine production and metabolic control.

• 3. Utilize High-Throughput Techniques: To expedite the engineering process and increase the chances of identifying efficient riboswitches, researchers should consider employing high-throughput techniques, such as next-generation sequencing and robotic screening. These techniques can help analyze a larger pool of candidates and accelerate the discovery of optimal lysine-ON riboswitches.

Conclusion:

The engineering of a lysine-ON riboswitch for metabolic control of lysine production in C. glutamicum holds great promise for the biotechnology industry. Through a dual genetic selection scheme and integration into the bacterial chromosome, researchers have made significant progress in enhancing lysine production. By optimizing screening parameters, considering additional gene integration, and utilizing high-throughput techniques, further advancements can be achieved in the field of metabolic control and engineering riboswitches.