Abstract
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. 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. The rationally modified P. putida strain allowed for the better functional expression of implanted genes by directly improving the metabolic currency that sustains the gene expression flow, instead of resorting to the classical genetic approaches (e.g., increasing the promoter strength in the DNA constructs of interest).
Highlights
Since the onset of the recombinant DNA era, heterologous gene expression has been one of the pillars of contemporary Metabolic Engineering [1]
The rationally modified P. putida strain allowed for the better functional expression of implanted genes by directly improving the metabolic currency that sustains the gene expression flow, instead of resorting to the classical genetic approaches
These sequences are genuinely parasitic, and they make cells more sensitive to DNA damage and, when induced, they cause stochastic lysis in the bacterial population. It comes the 54 insertion sequences (IS) and other mobile DNA elements borne by P. putida, which account for ~1% of the genome of P. putida KT2440 [37,38], and which are poised to counterselect knocked-in constructs that may burden the host [39,40]
Summary
Since the onset of the recombinant DNA era, heterologous gene expression has been one of the pillars of contemporary Metabolic Engineering [1]. The implicit assumption is that DNA acts as a sort of software which, if entered in a reading machine already in place (the host), will result in the expression of the genes at stake at the user’s will [2,3] This somewhat naïve concept has proven, very successful, and the number of genes and pathways that have chaperoning ability of the host cells [8,9], [ii] the stress caused by the encoded biological activities (e.g., enzymes and their metabolic products) on the endogenous biochemical network [10,11], and [iii] the drain of metabolic currency that is diverted into production of the implanted gene(s) and/or pathway(s), a phenomenon termed metabolic burden [12,13]. 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
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