Engineering Escherichia coli for selective 1-decanol production using the reverse β-oxidation (rBOX) pathway

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Biotechnology

Background:

• 1-Decanol is a valuable chemical with applications in pharmaceuticals, fragrances, and the broader chemical industry.
• Efficient and selective microbial production of 1-decanol is desirable for industrial sustainability.
• Existing methods for 1-decanol synthesis often face challenges with yield, titer, and specificity.

Purpose of the Study:

• To engineer Escherichia coli for the selective biosynthesis of 1-decanol.
• To optimize the reverse beta-oxidation (rBOX) pathway and termination modules for efficient 1-decanol production.
• To enhance 1-decanol production through metabolic engineering strategies and fermentation optimization.

Main Methods:

• Engineered E. coli utilizing the core reverse beta-oxidation (rBOX) pathway and specific termination enzymes.
• Screening of acyl-CoA reductase termination enzymes and regulation of rBOX pathway expression.
• Metabolic engineering including pyruvate dissimilation optimization (pyruvate dehydrogenase) and thioesterase knockout (YigI).
• Bi-phasic fermentation with dodecane overlay to mitigate toxicity and enhance transport.
• Optimization of pathway expression, cell growth conditions (oxygen availability), and media composition (rich vs. minimal).

Main Results:

• Achieved an initial 1-decanol titer of 1.4 g/L, improved to 1.9 g/L through further engineering.
• Bi-phasic fermentation yielded 4.4 g/L of 1-decanol (0.21 g/g yield) in 36 hours.
• Optimized conditions led to production of 6.1 g/L (0.26 g/g yield) in shake flasks and 10.05 g/L (0.2 g/g yield) in a bioreactor using rich medium.
• Minimal medium fermentation achieved 2.8 g/L (0.14 g/g yield) with 100% specificity.
• The achieved titers, yields, and purity are significantly higher (at least 10-fold) than previously reported.

Conclusions:

• The engineered E. coli strain demonstrates significant potential for the industrial-scale production of 1-decanol.
• Combining the rBOX pathway with specific termination enzymes and chassis engineering is an effective strategy for selective alcohol production.
• The developed metabolic engineering and fermentation approaches provide a robust platform for producing valuable long-chain alcohols.