Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.719280
Title: Kinetics and structure-guided characterisation and engineering of aldehyde deformylating oxygenase (ADO) for a renewable microbial biofuel platform
Author: Menon, Navya
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2015
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Abstract:
The increased demand for an alternative form of fuel has raised a great interest towards exploring various metabolic pathways and enzymes in several microbial species for hydrocarbon production. In recent years, cyanobacteria have emerged as an attractive microbial host and cyanobacterial metabolic pathways were targeted for engineering to produce "drop in" fuels such as propane and butane. Whilst appealing, practicalities for producing biofuels in cyanobacteria remain challenging, requiring the identification and engineering of natural biocatalysts and their integration into metabolic processes. Cyanobacterial hydrocarbon biosynthesis arises from fatty acid metabolism involving a potential enzyme, aldehyde deformylating oxygenase (ADO), which catalyses the decarbonylation of long-chain fatty aldehydes to alkanes, mainly in the conversion of octadecanal (C17H35CHO) to heptadecane (C17H36) and formate. The substrate specificity and preferences for long-chain aldehyde by ADO necessitates a detailed kinetic and structural characterisation in order to optimise/engineer this enzyme for future biotechnological applications. Thus, the main objective was to identify a potential ADO enzyme that can be optimised for shorter chain alkane production. By studying the substrate specificity and reaction kinetics of different ADO enzymes, it was found that ADO from Prochlorococcus marinus MIT 9313 (PmADO) is a potential target for short chain alkane production. The crystal structural of PmADO was solved and further GC-MS analysis was carried out to identify the chemical origin of a mixture of long-chain fatty acid in the active site, originated from E. coli cells during recombinant over-expression and purification. It was suggested that the structure-guided protein engineering for short-chain alkane production should be carried out along with the removal of this adventitious ligand from the active site in order to increase the alkane production. Four important residues present at the entrance of the ligand-binding cavity were targeted and saturated mutagenesis was performed on PmADO to identify variants that excluded the long fatty acid ligands from the active site but have specificity and higher conversion rates for shorter chain aldehydes. This identified two variants, V41Y and A134F, with the A134F variant that not only exhibiting an improved activity and turnover value of PmADO by four-fold but also improved binding affinity for butyraldehyde by 2 times. Finally the improved variants were incorporated in a host organism (E. coli) and the possibilities for the development of a microbial platform for renewable propane synthesis based on a fermentative clostridial butanol pathway were explored. Four pathways were designed namely atoB-adhE2, atoB-TPC7, nphT7-adhE2 and nphT7-TPC7 routes, which utilise CoA intermediates selected to incorporate ADO as the terminal enzyme. When PmADO was co-expressed with these pathways, the engineered E. coli host produced propane. The atoB-TPC7-ADO pathway was the most effective in producing propane (220 ± 3 μg/L). By (i) deleting competing pathways, (ii) including a previously designed A134F variant ofPmADO with an enhanced specificity towards short-chain substrates, and (iii) including a ferredoxin-based electron supply system, the propane titre was increased up to 3.40 ± 0.19 mg/L. It was also shown that the best propane producing pathways are scalable in a 250 mL flask and in a large-scale (up to 30 L) fermentor setup. This thesis focuses on the detailed kinetics and structure-guided characterisation and engineering studies on the ADO enzyme for the development of a renewable microbial biofuel platform.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.719280  DOI: Not available
Keywords: Clostridial butanol pathway ; Propane ; Direct-fuel ; Protein engineering ; Biofuels ; Aldehyde deformylating oxygenase (ADO) ; Enzymes
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