TRANSFORMATION AND EXPRESSION OF RECOMBINANT PROTEINS IN MICROALGAE

Abstract

Microalgae are attractive platforms for the production of high-value products such as recombinant proteins due to the low-cost and the short timeframe of the production process. Moreover, microalgal culture is scalable to relatively high volumes and densities under controlled conditions facilitating biosafety and their expression systems enable the expression of complex recombinant proteins. However, the lack of reproducible genetic transformation systems for most of the microalgae with potential in biotechnology and the low accumulation levels of recombinant proteins are still major limitations for the progress of this technology. This thesis reports the development and test of new molecular tools for the genetic engineering of commercially important microalgae. For this aim, the chloroplast genome of the haptophyte alga Tisochrysis lutea was sequenced, assembled and characterised in chapter 3. In chapter 4, contrary to previous reports, the stable and uniform expression of the recombinant protein Viral Protein 28 from White Spot Syndrome Virus was achieved in several lines of a plastid engineered Chlamydomonas reinhardtii strain. The accumulation level was estimated at 1% of total cellular protein under the regulation of psbD regulatory regions. Reproducible transformation of marine microalgae is the first step for their use as hosts for high-value products. In this regard, the assembly of transformation constructs suitable for Dunaliella tertiolecta and Tisochrysis lutea was accomplished in chapter 5 and 6 of this research. The limited number of efficient selectable markers available for marine algae has driven the development of herbicide based selection markers. The use of algal herbicide resistant genes is desirable for biosafety and commercialisation regulations since the use of antibiotics is avoided. In section 5 of this thesis, a mutated Dunaliella salina gene encoding phytoene desaturase carrying a F131V amino acid substitution was tested in Dunaliella tertiolecta as selectable marker for norflurazon based selection of chloroplast transformants, whilst a mutated Dunaliella tertiolecta gene encoding phytoene desaturase with a R265C amino acid substitution was explored in nuclear transformation of Dunaliella tertiolecta. Finally, an engineered Tisochrysis lutea phytoene desaturase containing a R268T amino acid substitution allowed the recovery of norflurazon resistant lines carrying the selectable marker. The adaptation of available transformation technologies enabled the nuclear transformation of both marine algae using the bacterial ble selectable marker gene under the control of the Dunaliella tertiolecta rbcS and the Tisochrysis lutea actin regulatory regions and zeocin selection.

Date of Award	31 Dec 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Robin Curtis (Supervisor) & Anil Day (Supervisor)