Engineering Pseudomonas putida for isoprenoid production by manipulating endogenous and shunt pathways supplying...

The soil bacterium Pseudomonas putida is a promising platform for the production of industrially valuable natural compounds. In the case of isoprenoids, the availability of biosynthetic precursors is a major limiting factor. In P. putida and most other bacteria, these precursors are produced from pyruvate and glyceraldehyde 3-phosphate by the methylerythritol 4-phosphate (MEP) pathway, whereas other bacteria synthesize the same precursors from acetyl-CoA using the unrelated mevalonate (MVA) pathway.

Here we explored different strategies to increase the supply of isoprenoid precursors in P. putida cells using lycopene as a read-out. Because we were not aiming at producing high isoprenoid titers but were primarily interested in finding ways to enhance the metabolic flux to isoprenoids, we engineered the well-characterized P. putida strain KT2440 to produce low but detectable levels of lycopene under conditions in which MEP pathway steps were not saturated. Then, we compared lycopene production in cells expressing the Myxococcus xanthus MVA pathway genes or endogenous MEP pathway genes (dxs, dxr, idi) under the control of IPTG-induced and stress-regulated promoters. We also tested a shunt pathway producing isoprenoid precursors from ribulose 5-phosphate using a mutant version of the Escherichia coli ribB gene.

The most successful combination led to a 50-fold increase in lycopene levels, indicating that P. putida can be successfully engineered to substantially increase the supply of metabolic substrates for the production of industrially valuable isoprenoids.