RiPP Production via Sulfide and Mevalonate Addition
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Solution Overview
Problem
Current methods for producing ribosomally synthesized and posttranslationally modified peptides (RiPPs) face challenges with low and variable yields, particularly in cyanobactin RiPP production, despite optimizations in transcription, translation, and folding.
Innovation Solution
The use of recombinant cells expressing tru or pat proteins, cultured in the presence of an exogenous sulfide source and mevalonate, to enhance the production of RiPPs, achieving high yields by modulating substrate preference and enzymatic activity through the addition of cysteine and mevalonate.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional methods for producing RiPPs are used with optimizations in transcription, translation, and folding, then the production process is established, but the yield remains low and variable
Solution Approach 1:
The patent changes the chemical parameters of the culture medium by adding exogenous sulfide sources (such as sodium sulfide or cysteine) and mevalonate. This modifies the substrate availability and enzymatic activity parameters, transforming the production system from low-yield conventional methods to high-yield consistent production achieving up to 27 mg/L RiPP with improved reliability
Solution Approach 2:
The patent introduces intermediary substances (sulfide sources and mevalonate) into the culture system that mediate between the precursor peptide substrate and the final RiPP product. These intermediaries facilitate the enzymatic modification steps, particularly for prenylated cyanobactins, enabling consistent high-yield production by bridging the gap between substrate availability and product formation
2Productivity
If exogenous sulfide source and mevalonate are added to culture medium, then RiPP yield increases significantly, but the process complexity increases
Solution Approach 1:
The patent modifies the culture medium composition by adding specific compounds (sulfide sources at 0.1-10 mM and mevalonate at 0.1-10 mM). These parameter changes are simple to implement and maintain, requiring only standard laboratory additions to the culture medium without complex equipment or process changes, thus achieving high productivity without significant complexity increase
Data Source
AI summary
The disclosure provides recombinant cells and methods for producing a ribosomally synthesized and posttranslationally modified peptide (RiPP), as well as RiPP libraries and methods for producing RiPP libraries.