Sequential flux optimization to improve GABA synthesis from acetate in Escherichia coli
- Title
- Sequential flux optimization to improve GABA synthesis from acetate in Escherichia coli
- Authors
- 백선우
- Date Issued
- 2024
- Publisher
- 포항공과대학교
- Abstract
- Strategic allocation of metabolic flux is essential for achieving higher
production performance in genetically engineered organisms. Flux optimization
between cell growth and chemical production has led to the establishment of
cost-effective chemical production methods in microbial cell factories. This
effect is amplified when utilizing a low-cost carbon source. γ-aminobutyric acid
(GABA) is an important component in pharmaceutical industry and for the
production of biodegradable polymers. Acetate is an attractive and cost-effective
substrate for the biosynthetic production of value-added chemicals, including
GABA. However, a balanced distribution of acetate-derived flux is essential for optimizing production performance without compromising growth. In this study,
we demonstrated GABA production from acetate by focusing on optimizing the
metabolic flux at critical nodes.
To construct a E. coli W strain capable of producing and accumulating
GABA by utilizing acetate as a sole carbon source, three genes were deleted (iclR,
transcriptional regulator of aceBAK genes which is essential for acetate
metabolism, gabT and puuE genes, 4-aminobutyrate aminotransferase, which
turns GABA into succinate-semialdehyde) and a mutated gadB gene (gadB
E89Q Δ452-466) that shows activity at around pH 7 was expressed by cloning it
into a pETDuet-1 vector. The resulting strain was named WGM strain, After the
initial production of GABA from acetate in the WGM strain, flux rewiring of the
sequential metabolite nodes led to a considerable improvement in GABA
production. Initial upregulation of the aceA gene in the glyoxylate shunt at the
isocitrate node and subsequent downregulation of the sucA gene in the
tricarboxylic acid cycle at the α-ketoglutarate node amplified metabolic flux
directed to GABA. The resulting strain produced 2.54 g/L GABA from 5 g/L
acetate in 24 h, with 0.34 g/g yield.
Through stepwise flux optimization at key metabolic nodes, engineered
E. coli W produced GABA from acetate with high efficiency. These findings
suggest that delicate flux balancing using an economical substrate can contribute
to the cost-effective production of GABA.
- URI
- http://postech.dcollection.net/common/orgView/200000808659
https://oasis.postech.ac.kr/handle/2014.oak/124017
- Article Type
- Thesis
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