Development of genetic engineering tools for the cyanobacterium Synechocystis PCC 6803 for advanced biofuel production

• Main Author: Al-Haj, L. A.
• Published: University College London (University of London) 2014
• Subjects: 570
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.626614

Cyanobacteria hold significant potential as platforms for the production of a wide variety of high-value products and biofuel molecules such as biohydrogen, isoprenoids and alkanes. Currently, the genome sequences of over 120 cyanobacterial species are publicly available, and techniques for the genetic manipulation of a few species are well established. However, more advanced metabolic engineering technologies are required for high-throughput production/evaluation of modified strains with improved biofuel characteristics. The most popular species for genetic studies is the freshwater species, Synechocystis sp. PCC 6803 – not least because it is naturally transformable and is a facultative phototroph. We demonstrated in chapter 3, that a deletion of an exonuclease gene in Synechocystis greatly increases transformation rates, probably through the promotion of non-HR events. However the mutant generated did not display random insertional mutagenesis but rather foreign DNA was being targeted to specific locations in the genome. On the other hand, targeted gene knock-outs and knock-ins are easily achieved in Synechocystis via homologous recombination (HR), but the creation of each transforming plasmid is rather laborious and involves several PCR and cloning steps. Chapter 4 of this thesis describes detailed study conducted to determine the minimum length of homologous DNA sequence flanking the foreign DNA that is necessary for efficient integration into the cyanobacterial genome. Our findings suggest that as little as 50 bases is sufficient for HR and open up the possibility of a quick, single step PCR strategy using long-tail primers to achieve any desirable knock-out or knock-in. However, the efficiency of the PCR needs to be optimised in order to make the technique more efficient. Chapter 5 describes genetic modification of the isoprenoid pathway for production of the novel high-value product geraniol (C10 monoterpene) and the fuel molecule farnesene (C15 sesquiterpene). Finally, chapter 6 exploits the possibility of expressing a biological plug-in for facilitated subtract delivery and product removal of hydrocarbons in Synechocystis.