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

Abstract

Background 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. Results 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. Conclusions 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.

Engineering isoprenoids production in metabolically versatile microbial host Pseudomonas putida

“…2 , 7 ). These results are consistent with a previous report [ 26 ] and the reason might be the result of the distinct central metabolism in P. putida and its different flux distribution with acetyl-CoA (Additional file 1 : Figure S1). For isoprenol, we also engineered the IPP-bypass MVA pathway, and it showed advantages compared with using the MEP and the original MVA pathway.…”

“…For example, there have been a few literatures for isoprenoids production in P. putida , and mostly, the endogenous MEP pathway was engineered for the isoprenoids production and frequently focused on the oxidation of terpenes using P450 enzymes as P. putida is known to be tolerant to oxidative stress [ 25 ]. The heterologous MVA pathway was also expressed in P. putida , but the performance was not as good as what was shown in E. coli when a similar engineering strategy was attempted, and only a low productivity and titers were achieved [ 26 ].…”

Diversifying Isoprenoid Platforms via Atypical Carbon Substrates and Non-model Microorganisms

“…Expression of the nDXP shunt enabled a 4-fold increase in MEP derived bisabolene production ( Kirby et al, 2015 ). This approach was further demonstrated in P. putida by expression of the mutant ribB gene, but with low efficiency ( Hernandez-Arranz et al, 2019 ). In an analogous work, promiscuous activity of fructose-6-phosphate aldolase in E. coli was used to generate D-ribulose from the glycolaldehyde and hydroxyacetone.…”

Narrative of a versatile and adept species Pseudomonas putida

“…For example, among the compounds produced by P. putida are polyhydroxyalkanoate (PHA) biopolymers that offer an alternative to petroleumbased plastics [126]. P. putida is also a promising alternative platform for production of rhamnolipids, biodegradable bacterial biosurfactants, after introducing two genes of the rhamnolipid synthesis pathway from P. aeruginosa [127,128], and for the production of isoprenoids (terpenoids) with a diverse range of applications in areas such as the pharmaceutical industry [129]. In addition, lignocellulosic biomass from catalytic pyrolysis containing aromatic compounds could be converted by P. putida capable of utilizing such substrates into added-value products such as cis-cis muconate [125,130,131].…”