Utilization of fadA knockout mutant Pseudomonas putida for overproduction of medium chain-length-polyhydroxyalkanoate

Area of Science:

• Microbial biotechnology
• Metabolic engineering
• Polymer science

Background:

• The fatty acid beta-oxidation pathway is crucial for cellular energy.
• Polyhydroxyalkanoates (PHAs) are biodegradable polymers with diverse applications.
• Engineering microbial hosts for enhanced PHA production is an active research area.

Purpose of the Study:

• To engineer P. putida KCTC1639 for increased production of medium chain-length polyhydroxyalkanoate (mcl-PHA).
• To investigate the effect of blocking the fadBA operon on metabolic flux towards mcl-PHA.
• To optimize cultivation conditions for maximizing mcl-PHA yield.

Main Methods:

• Genetic modification of P. putida KCTC1639 to create a fadA knockout mutant.
• Cultivation of the mutant strain under controlled fed-batch conditions.
• Supplementation with succinate to enhance growth and polymer production.

Main Results:

• Blocking the fadBA operon successfully redirected metabolic intermediates to mcl-PHA biosynthesis.
• Succinate addition (150 mg l(-1)) significantly stimulated both cell growth and mcl-PHA production.
• Fed-batch cultivation achieved a high mcl-PHA concentration of 8 g l(-1) with a cell dry weight of 10.3 g l(-1) within 60 hours.

Conclusions:

• Metabolic engineering by fadBA operon knockout is an effective strategy for enhancing mcl-PHA production in P. putida.
• Succinate serves as a beneficial supplement for improving microbial cell factory performance in mcl-PHA synthesis.
• The developed fed-batch process demonstrates high-yield mcl-PHA accumulation, paving the way for industrial applications.