Advanced pathway engineering for phototrophic putrescine production

Area of Science:

• Synthetic Biology
• Metabolic Engineering
• Algal Biotechnology
• Green Chemistry

Background:

• Putrescine (1,4-diaminobutane), a vital polyamine, is crucial for cellular functions and a key precursor for polyamide synthesis in industry.
• The green microalga Chlamydomonas reinhardtii naturally accumulates putrescine, presenting an opportunity for sustainable bio-production of this valuable chemical.
• Advances in genetic engineering offer tools to harness C. reinhardtii as a 'green cell factory' for sustainable base chemical production.

Purpose of the Study:

• To systematically investigate and engineer the native putrescine metabolism in Chlamydomonas reinhardtii.
• To establish the first CO2-based bio-production of putrescine using synthetic biology and metabolic engineering.
• To maximize putrescine titres for industrial applications.

Main Methods:

• CRISPR/Cas9 gene editing was employed to knockout key enzymes in the polyamine biosynthesis pathway.
• Engineered overexpression of ornithine decarboxylases (ODCs) and recombinant arginases were utilized.
• Functional knockout of putrescine degradation pathways (e.g., AMX2) and high cell density cultivation were performed.

Main Results:

• Ornithine decarboxylase 1 (ODC1) was identified as a critical enzyme for putrescine accumulation, while amine oxidase 2 (AMX2) is the primary degradation enzyme.
• Engineered overexpression of ODCs increased cellular putrescine levels by 4.5-fold, with some bacterial ODCs showing substrate promiscuity (co-producing cadaverine and 4-aminobutanol).
• Final pathway engineering, including arginase overexpression and AMX2 knockout, resulted in a 10-fold increase in putrescine titres, achieving 200 mg/L within 10 days.

Conclusions:

• The study successfully established a CO2-based bio-production platform for putrescine in Chlamydomonas reinhardtii.
• Metabolic engineering strategies, including enzyme knockout and overexpression, significantly enhanced putrescine accumulation.
• The engineered C. reinhardtii system offers a sustainable and efficient route for industrial putrescine production.