Engineering Dynamic Tuning of Gene Copy Number Via Plasmid Replication Control
- Type: Conference Presentation
- Conference Type: AIChE Annual Meeting
- Presentation Date: November 11, 2021
- Duration: 18 minutes
- Skill Level: Intermediate
- PDHs: 0.50
Dynamic metabolic pathway regulation has been proved efficient in lowering accumulation of toxic intermediates and reducing the stress caused by competition between cell growth and production in metabolic engineering. Various regulation toolsets have been developed to dynamically control gene expression at different levels. With the increased pathway complexities and growing demand for multiplex regulations, how to efficiently regulate complex pathways containing multi-targets becomes increasingly important. Here, we developed a strategy to dynamically control the plasmid replication and achieve multiplex adjustment of gene copy numbers. We first demonstrated the feasibility for inducible control of plasmid replication in medium-copy plasmids. To enable the control of high-copy plasmid, CRISPRi-mediated inhibition was integrated into the system and achieved an over 40% reduction in copy number of high-copy plasmid. On validating the control of both high-copy and medium-copy plasmids, two genetic circuits with different dynamic behaviors were constructed using the p-coumaric acid responsive regulator PadR. The dynamic gene copy number control strategy was then applied in regulating the p-coumaric acid biosynthesis. The dynamic regulation enabled better cell growth and enhanced the p-coumaric acid titer, demonstrating the great potential of applying dynamic gene copy number regulation for controlling complex biosynthetic pathways in future metabolic engineering.
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|AIChE Member Credits||0.5|
|AIChE Graduate Student Members||Free|
|AIChE Undergraduate Student Members||Free|