DESIGN AND CONSTRUCTION OF THE MVAA GENE WITH MODIFIED RIBOSOME BINDING SITE FOR THE OPTIMIZATION OF THE HETEROLOGOUS MEVALONATE PATHWAY IN BACILLUS SUBTILIS

• Main Author: Mullesa, Jeremy
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/82563
• Institution: Institut Teknologi Bandung

To enhance and accelerate terpenoid production, a semi-synthetic approach involving genetic engineering in microbes has become a sustainable alternative for production. Previous research constructed and expressed the mevalonate pathway using the pDR111 vector in Bacillus subtilis host cells. However, the upper mevalonate pathway expressed yielded low levels of mevalonate. This study aims to optimize the upper mevalonate pathway, which consists of three key genes (atoB, mvaS, and mvaA), on the plasmid pDR111_atoB_mvaS_mvaA for expression in B. subtilis. The mvaA gene plays a crucial role as it converts the intermediate product HMG-CoA into mevalonate. To enhance the expression strength of the mvaA gene, we designed and constructed a strong Ribosome Binding Site (RBS) for mvaA. The RBS design was created and analyzed using in silico De Novo DNA methods, RNA folding, and literature review. The designed RBS was constructed using PCR and confirmed through sequencing analysis. Subsequently, the reconstructed plasmid was transformed into B. subtilis host cells. The results showed that the RBS design can be used to regulate the expression of the mvaA gene, leading to optimal expression of the mevalonate pathway. This was evidenced by an increased translation rate of the new RBS and improved RNA secondary structure ComPared to the native RBS on the plasmid pDR111_atoB_mvaS_mvaA.