Abstract
BackgroundThe implementation of novel platform organisms to be used as microbial cell factories in industrial applications is currently the subject of intense research. Ongoing efforts include the adoption of Pseudomonas putida KT2440 variants with a reduced genome as the functional chassis for biotechnological purposes. In these strains, dispensable functions removed include flagellar motility (1.1% of the genome) and a number of open reading frames expected to improve genotypic and phenotypic stability of the cells upon deletion (3.2% of the genome).ResultsIn this study, two previously constructed multiple-deletion P. putida strains were systematically evaluated as microbial cell factories for heterologous protein production and compared to the parental bacterium (strain KT2440) with regards to several industrially-relevant physiological traits. Energetic parameters were quantified at different controlled growth rates in continuous cultivations and both strains had a higher adenosine triphosphate content, increased adenylate energy charges, and diminished maintenance demands than the wild-type strain. Under all the conditions tested the mutants also grew faster, had enhanced biomass yields and showed higher viability, and displayed increased plasmid stability than the parental strain. In addition to small-scale shaken-flask cultivations, the performance of the genome-streamlined strains was evaluated in larger scale bioreactor batch cultivations taking a step towards industrial growth conditions. When the production of the green fluorescent protein (used as a model heterologous protein) was assessed in these cultures, the mutants reached a recombinant protein yield with respect to biomass up to 40% higher than that of P. putida KT2440.ConclusionsThe two streamlined-genome derivatives of P. putida KT2440 outcompeted the parental strain in every industrially-relevant trait assessed, particularly under the working conditions of a bioreactor. Our results demonstrate that these genome-streamlined bacteria are not only robust microbial cell factories on their own, but also a promising foundation for further biotechnological applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0207-7) contains supplementary material, which is available to authorized users.
Highlights
The implementation of novel platform organisms to be used as microbial cell factories in industrial applications is currently the subject of intense research
Building on the concepts outlined above, we advocate the choice of Pseudomonas putida strains as microbial platforms pre-endowed with metabolic and stressendurance traits that are optimal for biotechnological needs [12]
Sequencing of the 6,181,863-bp long genome of P. putida KT2440 brought forth a significant advance in the potential applications of this bacterium [17,18]
Summary
The implementation of novel platform organisms to be used as microbial cell factories in industrial applications is currently the subject of intense research. The physiological effects of freeing the bacterium of all the viral DNA encoded in its extant chromosome (represented by not less than four prophages) were likewise explored [24] While such genetic manipulations conferred interesting biotechnological properties to the bacterial chassis, the industrial worth of a reduced genome P. putida strain has not been systematically explored hitherto. As a matter of fact, the rational engineering of cell factories tailored for optimized protein synthesis and process performance has traditionally focused on protein related issues (such as optimized codon usage, expression systems, folding characteristics, etc.), together with biochemical engineering aspects (i.e., bioreactor setup and control), while the basic properties of the biocatalyst proper are generally left to its default physiological values Expanding this scope by focusing on platform engineering opens the door to optimized strains offering low energy demands as a prerequisite for further improvements in production yield
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