Many species of microalgae have attracted attention from industrial biotechnology with the goal of harnessing specific physiological traits in applications including fuel, food, feed and high value medicinal products. However, to realise their full potential certain species will require optimisation through engineering. This study focussed on molecular tool development to address bottlenecks hindering the engineering of two microalgal species: the green alga Chlorella vulgaris and the marine diatom Phaeodactylum tricornutum, both exhibit rapid growth rates and high density cultures making them prominent candidates for use in numerous applications. Work in this thesis was sponsored by the algal biotechnology company Algenuity, Stewartby, Bedfordshire.

Progress in engineering of Chlorella has been slowed by the lack of reliable transformation protocols. In this study transformation protocols based on electroporation and biolistics were investigated with the goal of reliably transforming Algenuity’s proprietary C. vulgaris strain. The literature surrounding these transformation methods was systematically reviewed and informed the protocol development process. RNAseq analysis of the closely related species Chlorella variabilis informed the design and assembly of a Golden Gate (GG) based Chlorella MoClo toolkit including promoters, terminators and antibiotic resistance genes. Regulatory elements from a Chlorella virus and Chlamydomonas reinhardtii were also included in the toolkit. From these parts antibiotic resistance cassettes were assembled and used in trials to develop an electroporation or biolistic transformation protocol for C. vulgaris. Attempts to transform the proprietary strain by these means were unsuccessful but a transformation technique based on Escherichia coli conjugation was successfully employed enabling the characterisation of the parts assembled. Results of conjugative transformation suggest that use of the C. reinhardtii RbcS2 intron1 in antibiotic resistance CDS reduces transformation efficiency and that use of the C. variabilis EF1 promoter drives better expression than the HSP70-RbcS2 fusion promoter from C. reinhardtii.

The molecular toolbox of the marine diatom P. tricornutum is more advanced than for C. vulgaris but lacks strong, inducible promoters. As such the AP1 promoter (P_AP1), upregulated under conditions of phosphate stress, and the METE promoter (P_METE), upregulated in the absence of vitamin B₁₂ (B₁₂), were investigated accordingly to bring the engineering potential of P. tricornutum inline with other synthetic biology (SynBio) chassis. Reporter constructs were assembled with the promoters of interest cloned upstream of a Venus fluorescent protein and used to transform P. tricornutum. Initial characterisations of P_AP1::Venus lines suggested that it suffered from leaky expression. Incorporation of the native terminator or intron did not alleviate the leakiness. However, the P_AP1 was successfully switched off when high concentrations of phosphate were supplied in the media. The P_METE was shown to drive strong expression and can be modulated by varying the concentration of B₁₂ in the growth media. The P_METE was characterised relative to the P_EfTu and the widely used P_LHCF1 and shown to drive stronger expression. Transformants harbouring a lethal expression cassette encoding barnase under the regulation of the P_METE were obtained indicating that the P_METE could be effectively turned off. These lines were unable to grow when subcultured in B₁₂ deplete media. Including the METE terminator (T_METE) in reporter constructs drove 2 fold higher Venus expression than was observed when using the standard LHCF3 terminator (T_LHCF3). Truncating the P_METE resulted in decreased expression but no loss of B₁₂ regulation. The P_METE was most strongly expressed during exponential phase with expression dropping off through stationary phase. By simulating semi-continuous culturing, it was possible to maintain cells in log phase and as a result high activity of the P_METE.

To demonstrate the industrial relevance of this work the P_METE was used to regulate heterologous production of the diterpene casbene. Casbene synthase (CBS) catalyses the conversion of geranylgeranyl pyrophosphate (GGPP) to casbene and represents the first step in the synthesis of medicinally important derivatives such as lathyranes and jatropholanes. Transgenic lines harbouring an expression cassette containing CBS from Jatropha curcas under the the regulation of the P_METE produced casbene at titres of 100 µg/l (10 fg/cell). Higher casbene titres were observed, 750 µg/l (20 fg/cell), when lines were grown in F/2 supplemented with 10x nitrate, phosphate and trace elements. Varying the concentration of B_{12 in the media enabled tuneable casbene production with titres ranging from 0 to 180 µg/l when 1 µg/l or no was included in the media. Tagging CBS with Venus facilitated the identification of higher casbene producing transformants with multiple lines identified that produced approximately 2 mg/l. In contrast to Venus fluorescence assays casbene production was not improved by substituting the standard TLHCF3 with the TMETE.}