Engineering redox metabolism of Pseudomonas putida KT2440

  • Engineering des Redox Metabolismus von Pseudomonas putida KT2440

Zobel, Sebastian; Blank, Lars Mathias (Thesis advisor); Büchs, Jochen (Thesis advisor)

1. Auflage. - Aachen : Apprimus Verlag (2016, 2017)
Dissertation / PhD Thesis

In: Applied microbiology 4
Page(s)/Article-Nr.: 1 Online-Ressource (XII, 99 Seiten) : Diagramme

Dissertation, RWTH Aachen University, 2016

Abstract

Pseudomonas putida is a remarkable soil microorganism and emerges as biotechnological platform host, which despite its remarkable natural abilities needs to be optimized to meet the challenges associated with industrial processes, e.g., homogenic behavior of a producing culture and an efficient supply of reducing equivalents. The overall goal of this thesis was twofold, i) the development of a genetic tool, which minimizes expression variability leading to a consistent phenotype of P. putida and ii) increasing the NADH regeneration rate leading to hyper producing strain suited for redox biocatalysis. The heterologous expression device was designed in a way that it integrates into a single site of the chromosome circumventing copy number variations of classical plasmid based devices. It is composed of a synthetic promoter, a translational coupler and a gene of interest. The synthetic promoter is hence the only sequence determining the strength of gene expression. The reliability of this device was demonstrated by fluorescence measurements with genomically integrated constructs exhibiting a stable expression during the main stage of growth. By this means a calibrated synthetic promoter library was developed exhibiting a 30-fold range of activities. The modular structure of the heterologous expression device allows simple exchange of the gene of interest and hence a community tool is presented.The aim of increasing the NADH regeneration rate was pursued by two different approaches. Since the central carbon metabolism is the main source of NADH, the first approach aimed at increasing the glucose uptake rate, which in E. coli was shown to be controlled by the demand for ATP. However, this strategy adapted to P. putida KT2440 did not lead to an increased glucose uptake rate, since it is likely equipped with mechanisms sensing and thereby preventing wastage of ATP. With the second approach we took advantage of P. putida KT2440 being equipped with an NAD+-dependent formate dehydrogenase converting formate to CO2 and NADH. Co-feed experiments of glucose and formate indicated that formate can be used as an electron source for NADH as an increased biomass yield on glucose was observed. Thus, a co-feed of formate constitutes a promising approach of increasing the NADH regeneration rate likely supporting redox biocatalysis with P. putida KT2440 under process relevant conditions. In addition, based on these results we developed an easy and fast whole cell assay for the estimation of NADH regeneration rates and the screening of redox enhancing substrates.All together this thesis contributes to the progress of implementing P. putida KT2440 as versatile and industrially relevant workhorse with the successful development of a reliable expression tool and the simple formate co-feeding strategy of increasing the regeneration rate of the main redox cofactor NADH.