Metabolic Engineering for Efficient Ketocarotenoid Accumulation in the Green Microalga Chlamydomonas reinhardtii

Sofia Amendola; Jacob S. Kneip; Florian Meyer; Federico Perozeni; Stefano Cazzaniga; Kyle J. Lauersen; Matteo Ballottari; Thomas Baier

doi:10.1021/acssynbio.2c00616

Metabolic Engineering for Efficient Ketocarotenoid Accumulation in the Green Microalga Chlamydomonas reinhardtii

Sofia Amendola, Jacob S. Kneip, Florian Meyer, Federico Perozeni, Stefano Cazzaniga, Kyle J. Lauersen, Matteo Ballottari, Thomas Baier

Research output: Contribution to journal › Article › peer-review

18 Scopus citations

Abstract

Astaxanthin is a valuable ketocarotenoid with various pharmaceutical and nutraceutical applications. Green microalgae harbor natural capacities for pigment accumulation due to their 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Recently, a redesigned ß-carotene ketolase (BKT) was found to enable ketocarotenoid accumulation in the model microalga Chlamydomonas reinhardtii, and transformants exhibited reduced photoinhibition under high-light. Here, a systematic screening by synthetic transgene design of carotenoid pathway enzymes and overexpression from the nuclear genome identified phytoene synthase (PSY/crtB) as a bottleneck for carotenoid accumulation in C. reinhardtii. Increased ß-carotene hydroxylase (CHYB) activity was found to be essential for engineered astaxanthin accumulation. A combined BKT, crtB, and CHYB expression strategy resulted in a volumetric astaxanthin production of 9.5 ± 0.3 mg L–1 (4.5 ± 0.1 mg g–1 CDW) in mixotrophic and 23.5 mg L–1 (1.09 mg L–1 h–1) in high cell density conditions, a 4-fold increase compared to previous reports in C. reinhardtii. This work presents a systematic investigation of bottlenecks in astaxanthin accumulation in C. reinhardtii and the phototrophic green cell factory design for competitive use in industrial biotechnology.

Original language	English (US)
Journal	ACS Synthetic Biology
DOIs	https://doi.org/10.1021/acssynbio.2c00616
State	Published - Feb 23 2023

Bibliographical note

KAUST Repository Item: Exported on 2023-02-27
Acknowledgements: This research was supported by the ERC Starting Grant SOLENALGAE (679814) and the University of Verona grant no. JPVR2018BALLOTTARI to M.B. Funding to K.J.L. was provided by the King Abdullah University of Science and Technology (KAUST) baseline research funding. F.M. acknowledges funding by BMBF project KaroTec (grant number: 03VP09460). The authors would like to express their thanks to Prof. Dr. Ralph Bock for providing strain UVM4 and to Tim Prausner for performing TLC.

ASJC Scopus subject areas

Biomedical Engineering
Biochemistry, Genetics and Molecular Biology (miscellaneous)

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10.1021/acssynbio.2c00616

https://repository.kaust.edu.sa/bitstream/10754/688169/1/Amendola%2c%20S%2c%20et%20al%202023%20ACS%20Synthetic%20Biology.pdf

Cite this

@article{d4425c50139445718181e1100b2838ae,

title = "Metabolic Engineering for Efficient Ketocarotenoid Accumulation in the Green Microalga Chlamydomonas reinhardtii",

abstract = "Astaxanthin is a valuable ketocarotenoid with various pharmaceutical and nutraceutical applications. Green microalgae harbor natural capacities for pigment accumulation due to their 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Recently, a redesigned {\ss}-carotene ketolase (BKT) was found to enable ketocarotenoid accumulation in the model microalga Chlamydomonas reinhardtii, and transformants exhibited reduced photoinhibition under high-light. Here, a systematic screening by synthetic transgene design of carotenoid pathway enzymes and overexpression from the nuclear genome identified phytoene synthase (PSY/crtB) as a bottleneck for carotenoid accumulation in C. reinhardtii. Increased {\ss}-carotene hydroxylase (CHYB) activity was found to be essential for engineered astaxanthin accumulation. A combined BKT, crtB, and CHYB expression strategy resulted in a volumetric astaxanthin production of 9.5 ± 0.3 mg L–1 (4.5 ± 0.1 mg g–1 CDW) in mixotrophic and 23.5 mg L–1 (1.09 mg L–1 h–1) in high cell density conditions, a 4-fold increase compared to previous reports in C. reinhardtii. This work presents a systematic investigation of bottlenecks in astaxanthin accumulation in C. reinhardtii and the phototrophic green cell factory design for competitive use in industrial biotechnology.",

author = "Sofia Amendola and Kneip, {Jacob S.} and Florian Meyer and Federico Perozeni and Stefano Cazzaniga and Lauersen, {Kyle J.} and Matteo Ballottari and Thomas Baier",

note = "KAUST Repository Item: Exported on 2023-02-27 Acknowledgements: This research was supported by the ERC Starting Grant SOLENALGAE (679814) and the University of Verona grant no. JPVR2018BALLOTTARI to M.B. Funding to K.J.L. was provided by the King Abdullah University of Science and Technology (KAUST) baseline research funding. F.M. acknowledges funding by BMBF project KaroTec (grant number: 03VP09460). The authors would like to express their thanks to Prof. Dr. Ralph Bock for providing strain UVM4 and to Tim Prausner for performing TLC.",

year = "2023",

month = feb,

day = "23",

doi = "10.1021/acssynbio.2c00616",

language = "English (US)",

journal = "ACS Synthetic Biology",

issn = "2161-5063",

publisher = "American Chemical Society",

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TY - JOUR

T1 - Metabolic Engineering for Efficient Ketocarotenoid Accumulation in the Green Microalga Chlamydomonas reinhardtii

AU - Amendola, Sofia

AU - Kneip, Jacob S.

AU - Meyer, Florian

AU - Perozeni, Federico

AU - Cazzaniga, Stefano

AU - Lauersen, Kyle J.

AU - Ballottari, Matteo

AU - Baier, Thomas

N1 - KAUST Repository Item: Exported on 2023-02-27 Acknowledgements: This research was supported by the ERC Starting Grant SOLENALGAE (679814) and the University of Verona grant no. JPVR2018BALLOTTARI to M.B. Funding to K.J.L. was provided by the King Abdullah University of Science and Technology (KAUST) baseline research funding. F.M. acknowledges funding by BMBF project KaroTec (grant number: 03VP09460). The authors would like to express their thanks to Prof. Dr. Ralph Bock for providing strain UVM4 and to Tim Prausner for performing TLC.

PY - 2023/2/23

Y1 - 2023/2/23

N2 - Astaxanthin is a valuable ketocarotenoid with various pharmaceutical and nutraceutical applications. Green microalgae harbor natural capacities for pigment accumulation due to their 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Recently, a redesigned ß-carotene ketolase (BKT) was found to enable ketocarotenoid accumulation in the model microalga Chlamydomonas reinhardtii, and transformants exhibited reduced photoinhibition under high-light. Here, a systematic screening by synthetic transgene design of carotenoid pathway enzymes and overexpression from the nuclear genome identified phytoene synthase (PSY/crtB) as a bottleneck for carotenoid accumulation in C. reinhardtii. Increased ß-carotene hydroxylase (CHYB) activity was found to be essential for engineered astaxanthin accumulation. A combined BKT, crtB, and CHYB expression strategy resulted in a volumetric astaxanthin production of 9.5 ± 0.3 mg L–1 (4.5 ± 0.1 mg g–1 CDW) in mixotrophic and 23.5 mg L–1 (1.09 mg L–1 h–1) in high cell density conditions, a 4-fold increase compared to previous reports in C. reinhardtii. This work presents a systematic investigation of bottlenecks in astaxanthin accumulation in C. reinhardtii and the phototrophic green cell factory design for competitive use in industrial biotechnology.

AB - Astaxanthin is a valuable ketocarotenoid with various pharmaceutical and nutraceutical applications. Green microalgae harbor natural capacities for pigment accumulation due to their 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Recently, a redesigned ß-carotene ketolase (BKT) was found to enable ketocarotenoid accumulation in the model microalga Chlamydomonas reinhardtii, and transformants exhibited reduced photoinhibition under high-light. Here, a systematic screening by synthetic transgene design of carotenoid pathway enzymes and overexpression from the nuclear genome identified phytoene synthase (PSY/crtB) as a bottleneck for carotenoid accumulation in C. reinhardtii. Increased ß-carotene hydroxylase (CHYB) activity was found to be essential for engineered astaxanthin accumulation. A combined BKT, crtB, and CHYB expression strategy resulted in a volumetric astaxanthin production of 9.5 ± 0.3 mg L–1 (4.5 ± 0.1 mg g–1 CDW) in mixotrophic and 23.5 mg L–1 (1.09 mg L–1 h–1) in high cell density conditions, a 4-fold increase compared to previous reports in C. reinhardtii. This work presents a systematic investigation of bottlenecks in astaxanthin accumulation in C. reinhardtii and the phototrophic green cell factory design for competitive use in industrial biotechnology.

UR - http://hdl.handle.net/10754/688169

UR - https://pubs.acs.org/doi/10.1021/acssynbio.2c00616

U2 - 10.1021/acssynbio.2c00616

DO - 10.1021/acssynbio.2c00616

M3 - Article

C2 - 36821819

SN - 2161-5063

JO - ACS Synthetic Biology

JF - ACS Synthetic Biology

ER -

Metabolic Engineering for Efficient Ketocarotenoid Accumulation in the Green Microalga Chlamydomonas reinhardtii

Abstract

Bibliographical note

ASJC Scopus subject areas

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