Rib Leader Modification for Riboflavin Production
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for enhancing riboflavin production in microorganisms, such as Bacillus subtilis, face limitations in achieving saturating mRNA levels and stability, leading to suboptimal yields due to feedback inhibition and instability of expression cassettes with multiple promoter copies.
Innovation Solution
Modification of the rib leader sequence by deleting terminator sequences and introducing ribO mutations to reduce regulatory repression, thereby increasing full-length mRNA stability and transcription rates, and using strong constitutive promoters to enhance riboflavin production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If terminator sequences and regulatory elements are present in the rib leader, then transcription is regulated and mRNA stability is maintained, but riboflavin production is limited due to feedback inhibition and suboptimal mRNA levels
Solution Approach 1:
The patent removes terminator sequences and regulatory elements from the rib leader region to eliminate feedback inhibition and increase mRNA stability. Specifically, deletions are made at positions that correspond to terminator structures, allowing full-length mRNA transcripts to accumulate without premature termination, thereby resolving the contradiction between regulated transcription and high productivity
Solution Approach 2:
Instead of relying on natural regulatory mechanisms to control transcription, the patent inverts the approach by deleting the regulatory elements themselves. This allows the system to operate in a deregulated state where mRNA is continuously produced at high levels without the normal feedback control, achieving maximum productivity
2Productivity
If multiple promoter copies are used to enhance riboflavin production, then transcription rates increase, but expression cassette stability decreases leading to suboptimal yields
Solution Approach 1:
The patent removes the problematic multiple promoter copy structure and replaces it with a single modified rib leader containing deleted terminator sequences. This extraction of the unstable multi-promoter system eliminates the instability issue while maintaining high transcription rates through the deregulated rib leader
Solution Approach 2:
The patent changes the structural parameters of the leader sequence by deleting specific nucleotide regions corresponding to terminators. This parameter change in the DNA sequence structure transforms the transcriptional properties, allowing stable, high-level expression without requiring multiple promoter copies
3Productivity
If rib leader regulatory elements are intact, then transcription control is maintained, but full-length mRNA stability and transcription rates are suboptimal
Solution Approach 1:
The patent extracts and removes the terminator sequences from the rib leader region, eliminating the molecular structures that cause premature transcription termination. This removal allows RNA polymerase to continue transcription through the full length of the operon, dramatically increasing the rate of full-length mRNA production
Solution Approach 2:
The patent performs preliminary deletion of terminator sequences in the rib leader before transcription occurs. By pre-modifying the DNA template to remove termination signals, the system is prepared in advance to allow uninterrupted transcription, achieving high transcription rates without requiring ongoing regulatory adjustments
Data Source
AI summary
The present invention provides an improved biotechnological production of riboflavin (also referred herein as vitamin B2) through modification in the operon containing the riboflavin biosynthetic genes (rib operon), in particular modifications of/in the leader sequences (rib leader) upstream of the corresponding riboflavin biosynthetic genes (rib operon). Furthermore, the present invention relates to genetically engineered microorganisms carrying said modified sequences, processes to generate said modified sequences/microorganisms and the use thereof for production of riboflavin.