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Phytoene production utilizing the isoprenoid biosynthesis capacity of Thermococcus kodakarensis

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Abstract

Phytoene (C40H64) is an isoprenoid and a precursor of various carotenoids which are of industrial value. Archaea can be considered to exhibit a relatively large capacity to produce isoprenoids, as they are components of their membrane lipids. Here, we aimed to produce isoprenoids such as phytoene in the hyperthermophilic archaeon Thermococcus kodakarensis. T. kodakarensis harbors a prenyltransferase gene involved in the biosynthesis of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, which are precursors of squalene and phytoene, respectively. However, homologs of squalene synthase and phytoene synthase, which catalyze their condensation reactions, are not found on the genome. Therefore, a squalene/phytoene synthase homolog from an acidothermophilic archaeon Sulfolobus acidocaldarius, Saci_1734, was introduced into the T. kodakarensis chromosome under the control of a strong promoter. Production of the Saci_1734 protein was confirmed in this strain, and the generation of phytoene was detected (0.08–0.75 mg L−1 medium). We then carried out genetic engineering in order to increase the phytoene production yield. Disruption of an acetyl-CoA synthetase I gene involved in hydrolyzing acetyl-CoA, the precursor of phytoene, together with the introduction of a second copy of Saci_1734 led to a 3.4-fold enhancement in phytoene production.

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Abbreviations

ACS:

Acetyl-CoA synthetase

ASW:

Artificial sea water

YT:

Yeast extract and tryptone

AA:

20 Amino acids

S0 :

Elemental sulfur

Pyr:

Pyruvate

Mdx:

Maltodextrin

5-FOA:

5-Fluoroorotic acid

Ura:

Uracil

FPP:

Farnesyl pyrophosphate

GGPP:

Geranylgeranyl pyrophosphate

IPP:

Isopentenyl pyrophosphate

GPP:

Geranyl pyrophosphate

csg:

Cell surface glycoprotein

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Acknowledgements

The authors are grateful to Ms. Karin Nishimura for LC–MS analysis and to Ms. Rikako Fujimoto for constructing the expression plasmid in T. kodakarensis, pPcsgChiA1. This study was funded by the Core Research for Evolutional Science and Technology program of the Japan Science and Technology Agency to H.A. within the research area ‘Creation of Basic Technology for Improved Bioenergy Production through Functional Analysis and Regulation of Algae and Other Aquatic Microorganisms’.

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Authors and Affiliations

  1. Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, 615-8510, Japan

    Tsubasa Fuke, Takaaki Sato, Savyasachee Jha & Haruyuki Atomi

  2. JST, CREST, 7 Gobancho Chiyoda-ku, Tokyo, 102-0076, Japan

    Takaaki Sato & Haruyuki Atomi

  3. Environment and Biotechnology Division, Industrial Technology Development Institute, Department of Science and Technology, Bicutan, Taguig, Metro Manila, Philippines

    Myra L. Tansengco

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  1. Tsubasa Fuke
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  2. Takaaki Sato
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  3. Savyasachee Jha
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  4. Myra L. Tansengco
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  5. Haruyuki Atomi
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Correspondence to Haruyuki Atomi.

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Communicated by A. Driessen.

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Fuke, T., Sato, T., Jha, S. et al. Phytoene production utilizing the isoprenoid biosynthesis capacity of Thermococcus kodakarensis. Extremophiles 22, 301–313 (2018). https://doi.org/10.1007/s00792-018-0998-7

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