Engineering Pseudomonas for the production of reduced compounds

Nies, Salome Clarissa; Blank, Lars M. (Thesis advisor); Wierckx, Nick (Thesis advisor)

1. Auflage. - Aachen : Apprimus Verlag (2022)
Book, Dissertation / PhD Thesis

In: Applied microbiology 27
Page(s)/Article-Nr.: 1 Online-Ressource : Illustrationen, Diagramme

Dissertation, RWTH Aachen University, 2021

Abstract

Reduced, valuable compounds, such as biofuels, can be produced using metabolically engineered microbes. The production of such molecules often relies on redox-cofactor-dependent biocatalysis in microbial production hosts. The prerequisite for outstanding performance requires a basic understanding of the redox metabolism of the production host, a simple redox chassis, and a target molecule for which a metabolic pathway exist. In this thesis, these prerequisites are addressed using the obligate aerobe Pseudomonas taiwanensis VLB120 as chassis and methyl ketones as products of interest. The essentiality of the three NADH dehydrogenases of the respiratory chain of P. taiwanensis VLB120 was investigated. A mutant lacking all three NADH dehydrogenase seemed inviable, whereas the mutant deficient in both type-2 dehydrogenases (∆∆ndh) showed a clear phenotype with biphasic growth behavior and a strongly reduced growth rate in the second growth phase. In-depth analyses of the metabolism of the generated mutants revealed a redirection of metabolic fluxes through increased ROS formation in ∆∆ndh with concomitant growth reduction. This metabolic flexibility maintained a stable, viable phenotype. For redox biocatalysis in an obligate aerobe bacterium, the availability of NADH is key. Hence, we established a redox chassis that potentially has more electrons for synthesis, by reducing energy/electron consumption of the chassis. The deletion of the ATP-dependent ABC transporter and the polyhydroxyalkanoate synthesis operon were combined with the NADH dehydrogenase deletion mutants for the redox chassis. The deletions had only weak effect on phenotype, so an NADH-consuming reaction, of which the rate can be easily monitored is indispensable to study the impact of each gene deletion. Methyl ketones, which are industrially produced from petroleum-based hydrocarbons, were chosen as representatives of highly reduced platform chemicals. The implementation of published strategies resulted in 2.1 g Laq-1 methyl ketones in fed-batch fermentation. Further metabolic optimization motivated by metabolic modeling increased production by eliminating competing reactions to 9.8 g of Laq-1 methyl ketones (corresponding to 69.3 g of Lorg-1 in the in situ extraction phase), which was 53% of the maximum theoretical yield. This achievement represents a 4-fold improvement in product titer compared to the initial production strain and the highest titer of recombinantly produced methyl ketones reported to date. The best methyl ketone production strategy was evaluated in the potential Pseudomonas redox chassis but produced 1.5-fold less than the best-published strain. In summary, the in-depth analysis and understanding of redox metabolism in P. taiwanensis allowed superior production of highly reduced chemicals, thereby contributing to the envisaged bioeconomy.