Abstract
BackgroundThe 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.ResultsHere 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.ConclusionsThe 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.
Highlights
The soil bacterium Pseudomonas putida is a promising platform for the production of industrially valu‐ able natural compounds
Some P. putida strains can tolerate much higher level of isoprenoids than other microorganisms
Pseudomonas putida KT2440 can be engineered for lycopene production To use lycopene as a read-out of isoprenoid precursor availability in P. putida, a lycopene-producing operon (LYC) containing the crtE, crtB and crtI genes from Pantoea ananatis [16], was expressed in the strain KT2440. crtE encodes the enzyme geranylgeranyl diphosphate (GGPP) synthase, which catalyzes the addition of three molecules of isopentenyl diphosphate (IPP) to one DMAPP unit to yield GGPP, the immediate precursor for carotenoids
Summary
The soil bacterium Pseudomonas putida is a promising platform for the production of industrially valu‐ able natural compounds. Some P. putida strains can tolerate much higher level of isoprenoids than other microorganisms (e.g. its growth is not affected by concentrations of the monoterpene geranic acid that are toxic to E. coli or S. cerevisiae [4]). Several industrial processes based on this bacterium are being exploited for the production of fine chemicals (e.g. 2-quinoxalinecarboxylic acid, 5-methylpirazine-2-carboxylic acid, chiral amines and 4-(hydroxypyridin-3-yl)-4-oxobutyrate), and the production of many different compounds, including isoprenoids, is under study [6]. Despite the low number of available studies, the achievement of substantial yields of isoprenoids such as geranic acid and zeaxanthin and the high tolerance of P. putida to some of these compounds, usually toxic for other microbial models, illustrate the high potential of this bacterium to become a platform of choice for the industrial production of valuable isoprenoids. An important conclusion of these studies is that enhancing the supply of isoprenoid precursors is crucial for success as the bacterium only produces minor amounts of such precursors [7]
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