Pseudomonas 2.0: genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression

Abstract

Background: Because of its adaptability to sites polluted with toxic chemicals, the model soil bacterium Pseudomonas putida is naturally endowed with a number of metabolic and stress-endurance qualities which have considerable value for hosting energy-demanding and redox reactions thereof. The growing body of knowledge on P. putida strain KT2440 has been exploited for the rational design of a derivative strain in which the genome has been heavily edited in order to construct a robust microbial cell factory. Results: Eleven non-adjacent genomic deletions, which span 300 genes (i.e., 4.3% of the entire P. putida KT2440 genome), were eliminated; thereby enhancing desirable traits and eliminating attributes which are detrimental in an expression host. Since ATP and NAD(P)H availability -as well as genetic instability, are generally considered to be major bottlenecks for the performance of platform strains, a suite of functions that drain high-energy phosphate from the cells and/or consume NAD(P)H were targeted in particular, the whole flagellar machinery. Four prophages, two transposons, and three components of DNA restriction-modification systems were eliminated as well. The resulting strain (P. putida EM383) displayed growth properties (i.e., lag times, biomass yield, and specific growth rates) clearly superior to the precursor wild-type strain KT2440. Furthermore, it tolerated endogenous oxidative stress, acquired and replicated exogenous DNA, and survived better in stationary phase. The performance of a bi-cistronic GFP-LuxCDABE reporter system as a proxy of combined metabolic vitality, revealed that the deletions in P. putida strain EM383 brought about an increase of >50% in the overall physiological vigour.

Improved Thermotolerance of Genome‐Reduced Pseudomonas putida EM42 Enables Effective Functioning of the PL/cI857 System

“…The resulting non‐motile strain turned out to be way superior to the wild‐type counterpart in enduring different types of physicochemical stresses. And – most important for the sake of the present work – genome‐reduced strains increased their intracellular levels of ATP and NAD(P)H by >20% . Additional benefits of the genome‐reduced strain include a better genomic stability upon deletion of prophages and other mobile elements …”

“…E. coli CC118 strain was used for cloning purposes. P. putida strain KT2440 and its genome‐reduced derivative EM42 have been described before . Rich LB liquid and solid media were routinely used for bacterial growth and supplemented when necessary with 50 μg mL −1 kanamycin (Km) or 150 μg mL −1 ampicillin (Ap) or 30 μg mL −1 .…”

“…putida EM42 is a genome-edited variant of P. putida KT2440, itself a plasmid-less derivative of isolate P. putida mt-2. [9,10] In order to increase its predictability and make it more suited for industrial applications, P. putida EM42 was deleted of all prophages and other instability determinants that account for 4.3% of the genome and approximately 300 genes. This included elimination of the entire flagellar machinery.…”

“…Andmost important for the sake of the present workgenome-reduced strains increased their intracellular levels of ATP and NAD(P)H by >20%. [9] Additional benefits of the genome-reduced strain include a better genomic stability upon deletion of prophages and other mobile elements. [9,10] In this work, we have investigated whether the ATP surplus and superior stress endurance of P. putida EM42 could be exploited for adapting to this bacterium the well-known heatinducible system based on regulatory parts recruited from the E. coli lambda phage.…”

“…[9] Additional benefits of the genome-reduced strain include a better genomic stability upon deletion of prophages and other mobile elements. [9,10] In this work, we have investigated whether the ATP surplus and superior stress endurance of P. putida EM42 could be exploited for adapting to this bacterium the well-known heatinducible system based on regulatory parts recruited from the E. coli lambda phage. Typically, the expression device includes either (or both) of the strong P L /P R promoters and the cI857 gene, that encodes a thermo-labile variant of the cognate repressor cI.…”