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Random Chromosomal Integration and Screening Yields E. coli K-12 Derivatives Capable of Efficient Sucrose Utilization
David N Carruthers1, Tatyana E Saleski1, Scott A Scholz2
1Chemical Engineering Department, University of Michigan, Ann Arbor, Michigan 48109, United States.
ACS Synthetic Biology
|November 25, 2020
Summary
Randomly integrating genes into the bacterial chromosome using Tn5 transposase enables efficient sucrose metabolism and improves isobutanol production. This method optimizes heterologous gene expression for enhanced growth and bioproduction.
Area of Science:
- Microbiology
- Synthetic Biology
- Metabolic Engineering
Background:
- Chromosomal gene expression offers advantages over episomal methods but is hard to optimize.
- Variability in chromosomal gene expression is influenced by genomic context.
Purpose of the Study:
- To optimize heterologous gene expression via random chromosomal integration.
- To assess the efficiency of sucrose catabolism and its impact on cellular growth and biochemical production.
Main Methods:
- Utilized Tn5 transposase for random integration of the sucrose catabolism (csc) operon into the E. coli K-12 chromosome.
- Screened transposon library for isolates with varying growth rates on sucrose.
- Transduced the csc locus into evolved strains and an isobutanol production strain.
Main Results:
- Achieved growth rates on sucrose comparable to E. coli's growth on glucose (μmax = 0.70 ± 0.03 h⁻¹).
- Demonstrated efficient csc expression and that sucrose catabolism is not growth-limiting.
- Produced high titers of isobutanol (7.56 ± 0.25 g/L) using sucrose as the sole carbon source.
Conclusions:
- Random integration is an effective strategy for optimizing heterologous gene expression.
- This approach enhances both cellular growth and biochemical production phenotypes.
- Facilitates metabolic engineering for improved bioprocesses.
