Metabolic engineering of Pseudomonas taiwanensis VLB120 for sustainable production of 4-Hydroxybenzoate

Lenzen, Christoph; Blank, Lars M. (Thesis advisor); Wierckx, Nick (Thesis advisor)

1. Auflage. - Aachen : Apprimus (2020)
Book, Dissertation / PhD Thesis

In: Applied microbiology 17
Page(s)/Article-Nr.: 1 Online-Ressource (XVIII, 153 Seiten) : Illustrationen, Diagramme

Dissertation, RWTH Aachen University, 2019

Abstract

The aromatic compound 4-hydroxybenzoate and its derivatives, the parabens, find applications in everyday life. Current production routes for aromatics such as 4-hydroxybenzoate are mainly based on chemical catalysis and depend on the intensive use of energy and fossil resources. Due to the decreasing availability of the latter, the rising demand for aromatics and the non-ecofriendly way of formation, there is an urgent need for finding more efficient and sustainable syntheses. The present work focused on the development of a Pseudomonas-based whole-cell biocatalyst for the bioconversion of 4-hydroxybenzoate from renewable substrates such as glucose or glycerol. Besides the remarkable and versatile metabolism of this genus and its native high tolerance towards toxic compounds such as solvents, the species Pseudomonas taiwanensis VLB120 was chosen as a production host, since it accepts five carbon sugars such as xylose as sole carbon source and it is not regarded as a pathogenic organism. Former attempts to biotechnologically produce 4-hydroxybenzoate using different Pseudomonads and other species were indeed successful, but several studies resulted in only minor yields or required the supplementation of additional metabolites due to auxotrophies. Therefore, the objective was to enable high-yield 4-hydroxybenzoate biosynthesis solely from one specific carbon source. In order to exploit the full potential of metabolic engineering, rational as well as non-rational techniques were applied for host development. By introducing a heterologous production pathway based on tyrosine, eliminating and downregulating competing pathways and overexpressing key genes, strain P. taiwanensis VLB120 CL4.3 produced 4-hydroxybenzoate with a C-mol yield of 19.0% on glucose, whereas strain P. taiwanensis VLB120 CL3.3 reached 29.6% when grown on glycerol in batch mode, and a titer of 9.9 g l-1 during pulsed fed-batch fermentations. A non-rational approach for improved 4-hydroxybenzoate production was applied by random chemical mutagenesis and subsequent high-throughput screening via flow cytometry using a developed fluorescence-based biosensor. The best identified strain, P. taiwanensis VLB120 CL1gfp2 P2H08, produced 31% more 4-hydroxybenzoate than the non-mutated strain. Although further rational engineering of this strain did not result in better production performance, the introduced mutations can still give valuable insights into future engineering targets. Further improvement of production performance was achieved by exploiting the metabolic demand concept. In doing so, the main metabolic routes from glucose to acetyl-CoA were disrupted while leaving the acetyl-CoA generating 4-hydroxybenzoate production pathway intact in order to establish a growth-coupled production. On glucose as sole carbon source, stain P. taiwanensis VLB120 CL5.4 produced 4-hydroxybenzoate with a C-mol yield of 21.0%, supporting the assumption that the metabolic demand concept can be used for more efficient aromatics production. The results gained in this work underline the huge potential of P. taiwanensis VLB120 as a host for sustainable industrial bioconversion of aromatics and may be the basis for further engineering in order to promote the biotechnological formation of valuable compounds as a promising alternative to current chemical production routes.