Title: Engineering Pseudomonas putida KT2440 for efficient 3-HP production and its evaluation using different carbon sources
Abstract:
The industrial production of 3-hydroxypropionic acid (3-HP) from glycerol is limited by the high cost of the essential cofactor coenzyme B12. This study engineered Pseudomonas putida KT2440, a native coenzyme B12-producing strain, as a potential host for 3-HP biosynthesis. Two synthetic modules were introduced in strain KT2440: the glycerol synthesis module, consisting of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase, and a 3-HP synthesis module, including glycerol dehydratase, glycerol dehydratase reactivase, and aldehyde dehydrogenase. The recombinant strain produced 35 mM 3-HP in 24 hr using glucose as a carbon source. To enhance production, genes involved in 3-HP degradation (3hpdH, 3hibdHIV, 3hibdHII, and 3hibdHI) and the glycerol kinase (glpK) gene were sequentially deleted, redirecting glycerol flux toward 3-HP biosynthesis. The resulting mutant strain achieved a titer of 403 mM 3-HP in 42 hr, with a yield of 0.91 mol/mol glycerol. Strain KT2440 showed a significantly low requirement for coenzyme B12, supporting a more economically viable production platform. Substituting glucose with gluconate as a carbon source significantly improved the 3-HP titer to 630 mM in 42 hr with a yield of 0.97 mol/mol glycerol. This increase in 3-HP production could be attributed to alleviating the generation of glyceraldehyde-3-phosphate, which directly inhibits the activity of glycerol dehydratase. Overall, this work demonstrates the potential of P. putida KT2440 as an efficient and economical microbial chassis for 3-HP production from renewable carbon sources. The findings highlight both the metabolic flexibility and industrial applicability of this engineered strain for biotechnological production of valuable platform chemicals.
