Chloroplast Transit Peptide Selection for DMO Processing
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Solution Overview
Problem
Current methods for targeting dicamba monooxygenase (DMO) to chloroplasts in plants are inefficient, leading to incomplete processing and reduced herbicide tolerance, which complicates regulatory approval and increases production costs due to additional proteins and regulatory hurdles.
Innovation Solution
A recombinant DNA molecule comprising a DNA sequence encoding a chloroplast transit peptide (CTP) operably linked to a DNA sequence encoding dicamba monooxygenase, using specific CTP sequences such as those from Arabidopsis thaliana or Pisum sativum, to ensure correct processing and localization of DMO in chloroplasts, thereby enhancing herbicide tolerance and simplifying product characterization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a pea Rubisco small subunit CTP (27 aa sequence) is used to target DMO to chloroplasts, then DMO can be localized to chloroplasts, but incomplete processing occurs generating additional protein bands (∼41 kDa) that adversely impact DMO activity and create regulatory hurdles
Solution Approach 1:
The patent changes the amino acid sequence parameters of the CTP by selecting alternative sequences (Arabidopsis thaliana CTP or modified pea CTP with reduced sequence) to achieve complete and accurate processing of DMO protein, eliminating the incomplete processing issue with the original pea RbcS CTP
Solution Approach 2:
The patent extracts only the essential CTP sequence without the problematic 27 aa coding region of the pea Rubisco enzyme, using a reduced or modified CTP sequence that maintains targeting function while enabling complete processing
2Reliability
If alternative CTP sequences (maize cab-m7 or pea glutathione reductase) are used to target DMO to plastids, then DMO can be targeted to plastids, but processing efficiency is reduced and no correct processing data is obtained
Solution Approach 1:
The patent changes the CTP sequence parameters to match sequences known to work efficiently in chloroplasts (Arabidopsis thaliana or optimized pea sequences), improving both targeting capability and processing efficiency simultaneously
3Reliability
If additional amino acids from CTP coding regions are retained in the final protein, then the protein can be localized to chloroplasts, but the extra amino acids adversely impact DMO activity and create regulatory characterization efforts
Solution Approach 1:
The patent removes the problematic additional amino acids from the CTP coding region while retaining the essential targeting sequence, achieving complete processing that eliminates extra residues and simplifies product characterization
Solution Approach 2:
The patent modifies the CTP sequence parameters to ensure complete cleavage and removal of the CTP portion, leaving only the mature DMO protein without additional amino acids that would complicate characterization
Data Source
Figure 1
AI summary
The invention provides for identification and use of certain chloroplast transit peptides for efficient processing and localization of dicamba monooxygenase (DMO) enzyme in transgenic plants. Methods for producing dicamba tolerant plants, methods for controlling weed growth, and methods for producing food, feed, and other products are also provided, as well as seed that confers tolerance to dicamba when it is applied pre- or post-emergence.