Directed Evolution and Synthetic Biology for the Production of Next Generation Biofuels

  • Lucy Green

Student thesis: Phd


With the increasing demand for sustainable, renewable fuels, the microbial production of hydrocarbons, such as liquid petroleum gasses (LPGs), has become an attractive solution. The enzyme aldehyde deformylating oxygenase (ADO) is responsible for catalysis of aldehydes to alkanes in cyanobacteria. Although ADO has been successfully employed as the terminal enzyme in many hydrocarbon producing pathways, it has a notoriously low catalytic activity. Attempts to increase this activity have mostly comprised of rational design approaches, all with modest improvements. However, an appropriate turnover for industrial scale fermentation of LPGs still remains elusive. Directed evolution is a process which introduces mutation to an enzyme’s sequence, and then screens or selects variants with a desired fitness criteria (such as activity, solubility or substrate specificity). As amino acids across the whole protein can attribute to its activity, and mutating a single residue can disrupt epistatic interactions, the entire gene needs to be exposed to mutation if an improved variant is to be recovered. The following thesis describes a directed evolution workflow which was employed to improve the activity of ADO through the assembly of large, comprehensive DNA libraries and ultra-highthroughput screening via fluorescence activated cell sorting. The previously reported A134F variant was used to amplify libraries through both the targeted GeneORator method and error-prone PCR, and an alkane biosensor was used to monitor propane production in vivo. Through the optimisation of this process, an ADO variant with a 1000% increase in activity was discovered. Pathways were then assembled with the variant, yielding a significant increase in LPG production. The halotolarent organism Halomonas bluephagenesis was also explored as a chassis for low-cost fermentation of LPGs. This work demonstrates how directed evolution can be a powerful tool for increasing the activity of flux controlling enzymes within synthetic biology pathways.
Date of Award1 Aug 2021
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorDouglas Kell (Supervisor) & Nigel Scrutton (Supervisor)


  • Synthetic Biology
  • Metabolic Engineering
  • DNA library assembly
  • Directed Evolution
  • Biofuels

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