Developing synthetic biology tools for acetogens

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Redfern, Elizabeth (2022) Developing synthetic biology tools for acetogens. PhD thesis, University of Nottingham.

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Abstract

Acetogenic bacteria are of industrial interest due to their capacity for autotrophic growth on single carbon (C1) feedstocks such as CO or CO2 and H2. Both Eubacterium limosum and Clostridium carboxidivorans can autotrophically produce four carbon (C4) products, with C. carboxidivorans also able to produce six carbon (C6) products, such as hexanol and hexanoate. E. limosum is also capable of autotrophic growth on methanol. This study outlines efforts to further the industrial potential of these strains through the development of DNA transfer methods, synthetic biology toolkits and a large transposon mutant library elucidating gene essentiality under industrially relevant conditions.

To establish DNA transfer in C. carboxidivorans, preliminary genotypic characterisation of the ten RM systems was performed. This substantial barrier was then overcome by the application of multiple circumvention strategies including disruption of key recognition motifs in the shuttle vector sequence, heterologous expression of native methyltransferases in E. coli conjugal donors, and isolation of hyper-receptive recipient strains with modifications in the fabK gene. Through complementation of these strategies a reliable conjugative DNA transfer method was established. Utilising this method, an inducible exogenous CRISPR-Cas9 system was introduced and used to knockout CcaP7ORF17830P, one of the Type II RM system genes. In implementing this genetic modification system multiple auxiliary genetic tools were demonstrated, including a catP reporter gene and theophylline inducible riboswitch.

Also reported herein is the generation of a saturated E. limosum NG-6849 transposon insertional mutant pool with 179,161 unique insertional mutants. High throughput sequencing of this mutant pool, grown either exclusively on methanol or on glucose, was used to evaluate the respective gene essentiality. This revealed 103 disparate genes required solely for growth on methanol, and not glucose. Due to mutant overgrowth within the methanol conditions, this process also revealed key candidate genes whose disruption may correlate with improved methanol growth kinetics.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Minton, Nigel
Zhang, Ying
Humphreys, Christopher
Keywords: Acetogenic, Bacteria, Synthetic biology tools
Subjects: Q Science > QR Microbiology > QR 75 Bacteria. Cyanobacteria
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 69764
Depositing User: Redfern, Elizabeth
Date Deposited: 31 Dec 2022 04:40
Last Modified: 31 Dec 2022 04:40
URI: https://eprints.nottingham.ac.uk/id/eprint/69764

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