Engineering Acinetobacter baylyi ADP1 : New tools and strategies for enhanced production of chemicals using non-conventional substrates
Luo, Jin (2022)
Tampere University
2022
Tekniikan ja luonnontieteiden tohtoriohjelma - Doctoral Programme in Engineering and Natural Sciences
Tekniikan ja luonnontieteiden tiedekunta - Faculty of Engineering and Natural Sciences
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Väitöspäivä
2022-10-28
Julkaisun pysyvä osoite on
https://urn.fi/URN:ISBN:978-952-03-2608-1
https://urn.fi/URN:ISBN:978-952-03-2608-1
Tiivistelmä
An intriguing feature of microbial cell factories is the possibility to use abundant industrial side streams as raw materials for chemical production. Lignin-rich streams represent underutilized side-products from lignocellulose-based industries. The aromatic-catabolizing capability of certain microorganisms opens up the possibility for biological lignin valorization. Despite that, the toxicity of lignin-derived aromatics poses a challenge to the process. Another challenge is optimizing the efficiency of the cell factories concerning the production metrics, such as titer and yield.
Acinetobacter baylyi ADP1 is an emerging model organism and has been recognized as a potential candidate for biological lignin valorization. In this thesis, adaptive laboratory evolution was employed to improve the tolerance of A. baylyi ADP1 towards common lignin-derived aromatics. The evolved strain was further exploited to produce a non-native product, 1-undecene, from ferulate. The beneficial mutations derived from the evolution were investigated by whole-genome sequencing and a novel reverse-engineering methodology. It was concluded that the mutations in the aromatic-specific transport system play important roles in conferring aromatic tolerance by reducing the entry of aromatics into periplasm or cytosol.
Metabolic engineering efforts were taken to enhance the production of the native product, wax esters, by rewiring the metabolic pathways. A significant improvement in wax ester production was achieved by enhanced production of acetyl-CoA and driving force from the downstream pathway by aceA deletion and acr1 overexpression. Given that cell size can affect product accumulation capability and downstream processes, the impact of cell morphology on wax ester production was studied. The established inducible clustered regularly interspaced short palindromic repeats interference (CRISPRi) system was employed to increase the cell size by repressing the essential cell division gene ftsZ. The large-sized cells sustained efficient wax ester production in combination with the previously established engineering strategy.
To sum up, the current work demonstrated Acinetobacter baylyi ADP1 as a suitable bacterial chassis for the utilization of lignin-derived aromatics and producing oleochemicals. Metabolic engineering strategies were implemented, and engineering tools were developed to improve the cell properties concerning substrate tolerance and production efficiency.
Acinetobacter baylyi ADP1 is an emerging model organism and has been recognized as a potential candidate for biological lignin valorization. In this thesis, adaptive laboratory evolution was employed to improve the tolerance of A. baylyi ADP1 towards common lignin-derived aromatics. The evolved strain was further exploited to produce a non-native product, 1-undecene, from ferulate. The beneficial mutations derived from the evolution were investigated by whole-genome sequencing and a novel reverse-engineering methodology. It was concluded that the mutations in the aromatic-specific transport system play important roles in conferring aromatic tolerance by reducing the entry of aromatics into periplasm or cytosol.
Metabolic engineering efforts were taken to enhance the production of the native product, wax esters, by rewiring the metabolic pathways. A significant improvement in wax ester production was achieved by enhanced production of acetyl-CoA and driving force from the downstream pathway by aceA deletion and acr1 overexpression. Given that cell size can affect product accumulation capability and downstream processes, the impact of cell morphology on wax ester production was studied. The established inducible clustered regularly interspaced short palindromic repeats interference (CRISPRi) system was employed to increase the cell size by repressing the essential cell division gene ftsZ. The large-sized cells sustained efficient wax ester production in combination with the previously established engineering strategy.
To sum up, the current work demonstrated Acinetobacter baylyi ADP1 as a suitable bacterial chassis for the utilization of lignin-derived aromatics and producing oleochemicals. Metabolic engineering strategies were implemented, and engineering tools were developed to improve the cell properties concerning substrate tolerance and production efficiency.
Kokoelmat
- Väitöskirjat [4847]