Auto-replicative Nucleic Acid for Antibiotic-Free Fermentation
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Solution Overview
Problem
The use of antibiotics as selection agents in microbial cell production leads to environmental contamination and an energetic burden on cells, particularly when large resistance-conferring genes are expressed constitutively, negatively impacting product yield.
Innovation Solution
A method involving a microbial host with an auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence that confers an advantage, with controlled genetic activity, optionally accompanied by a second sequence involved in product production, allowing for selective survival and reduced energetic burden.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If antibiotics are used as selection agents with resistance-conferring genes, then microbial host survival is improved, but environmental contamination and energetic burden increase
Solution Approach 1:
The patent extracts the harmful antibiotic selection system and replaces it with a bacteriocin-based system. The resistance gene is replaced by an immunity gene that confers resistance to bacteriocin without requiring antibiotic presence, thereby eliminating environmental contamination while maintaining selective pressure for host survival.
Solution Approach 2:
The patent changes the selection mechanism from antibiotic-dependent to bacteriocin-dependent. By using bacteriocin as the selection agent and immunity gene as the resistance mechanism, the system achieves the same selective function without the harmful environmental effects of antibiotics.
2Reliability
If large resistance-conferring genes are expressed constitutively, then microbial host survival is improved, but product yield decreases due to energetic burden
Solution Approach 1:
The patent implements inducible expression of the immunity gene rather than constitutive expression. The immunity gene is expressed only when bacteriocin is present, allowing the cell to conserve energy during periods when resistance is not needed, thereby improving product yield while maintaining survival capability when required.
Solution Approach 2:
The patent transitions from static constitutive expression to dynamic inducible expression of the immunity gene. This allows the expression level to adjust according to environmental conditions (presence of bacteriocin), optimizing the balance between survival and energy conservation for product production.
Data Source
AI summary
Some embodiments relate to a method for producing a product of interest with a microbial host using an auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence whose genetic activity confers an advantage to the host, optionally wherein the genetic activity of said first nucleic acid molecule is controlled.