{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/362641"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/362641","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Expanding the molecular tools for the microalgae Chlorella vulgaris and Phaeodactylum tricornutum","abstract":"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 B12 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 T*LHCF3* with the T*METE*.","abstract_html":"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<sub>*AP1*</sub>), upregulated under conditions of phosphate stress, and the METE promoter (P<sub>*METE*</sub>), upregulated in the absence of vitamin B<sub>12</sub> (B<sub>12</sub>), 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<sub>*AP1*</sub>::Venus lines suggested that it suffered from leaky expression. Incorporation of the native terminator or intron did not alleviate the leakiness. However, the P<sub>*AP1*</sub> was successfully switched off when high concentrations of phosphate were supplied in the media. The P<sub>*METE*</sub> was shown to drive strong expression and can be modulated by varying the concentration of B<sub>12</sub> in the growth media. The P<sub>*METE*</sub> was characterised relative to the P<sub>*EfTu*</sub> and the widely used P<sub>*LHCF1*</sub> and shown to drive stronger expression. Transformants harbouring a lethal expression cassette encoding barnase under the regulation of the P<sub>*METE*</sub> were obtained indicating that the P<sub>*METE*</sub> could be effectively turned off. These lines were unable to grow when subcultured in B<sub>12</sub> deplete media. Including the METE terminator (T<sub>*METE*</sub>) in reporter constructs drove 2 fold higher Venus expression than was observed when using the standard *LHCF3* terminator (T<sub>*LHCF3*</sub>). Truncating the P<sub>*METE*</sub> resulted in decreased expression but no loss of B<sub>12</sub> regulation. The P<sub>*METE*</sub> 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<sub>*METE*</sub>. To demonstrate the industrial relevance of this work the P<sub>*METE*</sub> 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<sub>*METE*</sub> 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<sub>12<sub> in the media enabled tuneable casbene production with titres ranging from 0 to 180 µg/l when 1 µg/l <sub> or no <sub> 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 T<sub>*LHCF3*</sub> with the T<sub>*METE*</sub>.","abstract_has_math":false,"creators":["Hickland, Patrick"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Smith, Alison"],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-07-31","date_published":"2023-07-31","updated_at":"2026-07-22T22:24:27Z","subjects":["microalgae"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f3f238da-b0c3-4250-add1-09df28989e9e/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.104724","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Smith, Alison"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Algenuity"]},{"key":"dc:creator","label":"Author","values":["Hickland, Patrick"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2023-07-31"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/362641"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["microalgae"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f3f238da-b0c3-4250-add1-09df28989e9e/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.104724"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/a28ae4b9-2580-43b7-b2c3-e37b5ebfaf1d/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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 B12 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. 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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 B12 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. 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