Engineering the unicellular alga Phaeodactylum tricornutum for high-value plant triterpenoid production.

Plant triterpenoids constitute a diverse class of organic compounds that play a major role in development, plant defence and environmental interaction. Several triterpenes have demonstrated potential as pharmaceuticals. One example is betulin, which has shown promise as a pharmaceutical precursor for the treatment of certain cancers and HIV. Major challenges for triterpenoid commercialization include their low production levels and their cost-effective purification from the complex mixtures present in their natural hosts. Therefore, attempts to produce these compounds in industrially relevant microbial systems such as bacteria and yeasts have attracted great interest. Here, we report the production of the triterpenes betulin and its precursor lupeol in the photosynthetic diatom Phaeodactylum tricornutum, a unicellular eukaryotic alga. This was achieved by introducing three plant enzymes in the microalga: a Lotus japonicus oxidosqualene cyclase and a Medicago truncatula cytochrome P450 along with its native reductase. The introduction of the L. japonicus oxidosqualene cyclase perturbed the mRNA expression levels of the native mevalonate and sterol biosynthesis pathway. The best performing strains were selected and grown in a 550-L pilot-scale photobioreactor facility. To our knowledge, this is the most extensive pathway engineering undertaken in a diatom and the first time that a sapogenin has been artificially produced in a microalga, demonstrating the feasibility of the photo-bio-production of more complex high-value, metabolites in microalgae.

D'Adamo S ，Schiano di Visconte G ，Lowe G ，Szaub-Newton J ，Beacham T ，Landels A ，Allen MJ ，Spicer A ，Matthijs M ... -  《-》

CYP716A subfamily members are multifunctional oxidases in triterpenoid biosynthesis.

Fukushima EO ，Seki H ，Ohyama K ，Ono E ，Umemoto N ，Mizutani M ，Saito K ，Muranaka T ... -  《-》

Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana.

The ability to evolve novel metabolites has been instrumental for the defence of plants against antagonists. A few species in the Barbarea genus are the only crucifers known to produce saponins, some of which make plants resistant to specialist herbivores, like Plutella xylostella, the diamondback moth. Genetic mapping in Barbarea vulgaris revealed that genes for saponin biosynthesis are not clustered but are located in different linkage groups. Using co-location with quantitative trait loci (QTLs) for resistance, transcriptome and genome sequences, we identified two 2,3-oxidosqualene cyclases that form the major triterpenoid backbones. LUP2 mainly produces lupeol, and is preferentially expressed in insect-susceptible B. vulgaris plants, whereas LUP5 produces β-amyrin and α-amyrin, and is preferentially expressed in resistant plants; β-amyrin is the backbone for the resistance-conferring saponins in Barbarea. Two loci for cytochromes P450, predicted to add functional groups to the saponin backbone, were identified: CYP72As co-localized with insect resistance, whereas CYP716As did not. When B. vulgaris sapogenin biosynthesis genes were transiently expressed by CPMV-HT technology in Nicotiana benthamiana, high levels of hydroxylated and carboxylated triterpenoid structures accumulated, including oleanolic acid, which is a precursor of the major resistance-conferring saponins. When the B. vulgaris gene for sapogenin 3-O-glucosylation was co-expressed, the insect deterrent 3-O-oleanolic acid monoglucoside accumulated, as well as triterpene structures with up to six hexoses, demonstrating that N. benthamiana further decorates the monoglucosides. We argue that saponin biosynthesis in the Barbarea genus evolved by a neofunctionalized glucosyl transferase, whereas the difference between resistant and susceptible B. vulgaris chemotypes evolved by different expression of oxidosqualene cyclases (OSCs).

Khakimov B ，Kuzina V ，Erthmann PØ ，Fukushima EO ，Augustin JM ，Olsen CE ，Scholtalbers J ，Volpin H ，Andersen SB ，Hauser TP ，Muranaka T ，Bak S ... -  《-》

Engineering Critical Enzymes and Pathways for Improved Triterpenoid Biosynthesis in Yeast.

Guo H ，Wang H ，Huo YX 《ACS Synthetic Biology》

Extrachromosomal Genetic Engineering of the Marine Diatom Phaeodactylum tricornutum Enables the Heterologous Production of Monoterpenoids.

Fabris M ，George J ，Kuzhiumparambil U ，Lawson CA ，Jaramillo-Madrid AC ，Abbriano RM ，Vickers CE ，Ralph P ... -  《ACS Synthetic Biology》