Szaub, JB; (2013) Genetic engineering of green microalgae for the production of biofules and high value products. Doctoral thesis , UCL (University College London).

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Abstract

A major consideration in the exploitation of microalgae as biotechnology platforms is choosing robust, fast-growing strains that are amenable to genetic manipulation. The freshwater green alga Chlorella sorokiniana has been reported as one of the fastest growing and thermotolerant species, and studies in this thesis have confirmed strain UTEX1230 as the most productive strain of C. sorokiniana with doubling time under optimal growth conditions of less than three hours. Furthermore, the strain showed robust growth at elevated temperatures and salinities. In order to enhance the productivity of this strain, mutants with reduced biochemical and functional PSII antenna size were isolated. TAM4 was confirmed to have a truncated antenna and able to achieve higher cell density than WT, particularly in cultures under decreased irradiation. The possibility of genetic engineering this strain has been explored by developing molecular tools for both chloroplast and nuclear transformation. For chloroplast transformation, various regions of the organelle’s genome have been cloned and sequenced, and used in the construction of transformation vectors. However, no stable chloroplast transformant lines were obtained following microparticle bombardment. For nuclear transformation, cycloheximide-resistant mutants have been isolated and shown to possess specific missense mutations within the RPL41 gene. Such a mutant allele should prove useful as a dominant marker. Genetic engineering of the chloroplast genome has been well established for another microalga Chlamydomonas reinhardtii. This system was exploited in three biotechnological applications: 1) generation of alkane producing strains by introducing genes encoding for acyl reductase and aldehyde decarbonylase. 2) expression of a vaccine candidate major capsid protein L1 of the human papillomavirus. 3) expression of a potent HIV-inactivating protein cyanovirin-N. In all cases, stable transformant lines were obtained and molecular analysis confirmed the successful integration of the transgenes into the genome. The detailed biochemical analysis of the lines is presented in the thesis.

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