Phototrophic production of heterologous diterpenoids and a hydroxy-functionalized derivative from Chlamydomonas reinhardtii
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Phototrophic production of heterologous diterpenoids and a hydroxy-functionalized derivative from Chlamydomonas reinhardtii. / Lauersen, Kyle J.; Wichmann, Julian; Baier, Thomas; Kampranis, Sotirios C.; Pateraki, Irini; Møller, Birger Lindberg; Kruse, Olaf.
In: Metabolic Engineering, Vol. 49, 2018, p. 116-127.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Phototrophic production of heterologous diterpenoids and a hydroxy-functionalized derivative from Chlamydomonas reinhardtii
AU - Lauersen, Kyle J.
AU - Wichmann, Julian
AU - Baier, Thomas
AU - Kampranis, Sotirios C.
AU - Pateraki, Irini
AU - Møller, Birger Lindberg
AU - Kruse, Olaf
PY - 2018
Y1 - 2018
N2 - Photosynthetic microalgae harbor enormous potential as light-driven green-cell factories for sustainable bio-production of a range of natural and heterologous products such as isoprenoids. Their capacity for photosynthesis and rapid low-input growth with (sun)light and CO 2 is coupled to a robust metabolic architecture structured toward the generation of isoprenoid pigments and compounds involved in light capture, electron transfer, and radical scavenging. Metabolic engineering approaches using eukaryotic green microalgae have previously been hampered mainly by low-levels of nuclear transgene expression. Here, we employed a strategy of optimized transgene design which couples codon optimization and synthetic intron spreading for the expression of heterologous plant enzymes from the algal nuclear genome. The diterpenoids casbene, taxadiene, and 13R(+) manoyl oxide were produced after expressing heterologous diterpene synthases and enzymes participating in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway which were all targeted to the algal chloroplast. Additionally, a truncated and soluble plant microsomal cytochrome P450 monooxygenase was functionally expressed and able to hydroxylate 13R(+) manoyl oxide when directed into the chloroplasts. The heterologous diterpenoids were found to be excreted from the cells and accumulate in dodecane solvent-culture overlays. It was shown that the algal cell could tolerate significant metabolic pull towards diterpenoids without loss of native pigments. Using an algal strain producing 13R(+) manoyl oxide as a model, diterpenoid production was shown to be highest in photoautotrophic cultivations using CO 2 as the sole carbon source and day:night illumination cycles. Up to 80 mg 13R(+) manoyl oxide per gram cell dry mass (CDM) could be produced from C. reinhardtii in a 7 day batch cultivation with a sustained maximal productivity of 22.5 mg g cdm -1 d -1 over 3 consecutive days. Collectively the results presented here suggest that green algal cells have remarkable potential for the heterologous production of non-native isoprenoids and support the use of these hosts for (sun)light driven bioproduction concepts.
AB - Photosynthetic microalgae harbor enormous potential as light-driven green-cell factories for sustainable bio-production of a range of natural and heterologous products such as isoprenoids. Their capacity for photosynthesis and rapid low-input growth with (sun)light and CO 2 is coupled to a robust metabolic architecture structured toward the generation of isoprenoid pigments and compounds involved in light capture, electron transfer, and radical scavenging. Metabolic engineering approaches using eukaryotic green microalgae have previously been hampered mainly by low-levels of nuclear transgene expression. Here, we employed a strategy of optimized transgene design which couples codon optimization and synthetic intron spreading for the expression of heterologous plant enzymes from the algal nuclear genome. The diterpenoids casbene, taxadiene, and 13R(+) manoyl oxide were produced after expressing heterologous diterpene synthases and enzymes participating in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway which were all targeted to the algal chloroplast. Additionally, a truncated and soluble plant microsomal cytochrome P450 monooxygenase was functionally expressed and able to hydroxylate 13R(+) manoyl oxide when directed into the chloroplasts. The heterologous diterpenoids were found to be excreted from the cells and accumulate in dodecane solvent-culture overlays. It was shown that the algal cell could tolerate significant metabolic pull towards diterpenoids without loss of native pigments. Using an algal strain producing 13R(+) manoyl oxide as a model, diterpenoid production was shown to be highest in photoautotrophic cultivations using CO 2 as the sole carbon source and day:night illumination cycles. Up to 80 mg 13R(+) manoyl oxide per gram cell dry mass (CDM) could be produced from C. reinhardtii in a 7 day batch cultivation with a sustained maximal productivity of 22.5 mg g cdm -1 d -1 over 3 consecutive days. Collectively the results presented here suggest that green algal cells have remarkable potential for the heterologous production of non-native isoprenoids and support the use of these hosts for (sun)light driven bioproduction concepts.
KW - Microalgae
KW - Chlamydomonas reinhardtii
KW - Terpenoids
KW - Isoprenoids
KW - Diterpenoids
KW - Cytochrome P450s
U2 - 10.1016/j.ymben.2018.07.005
DO - 10.1016/j.ymben.2018.07.005
M3 - Journal article
C2 - 30017797
VL - 49
SP - 116
EP - 127
JO - Metabolic Engineering
JF - Metabolic Engineering
SN - 1096-7176
ER -
ID: 204467247