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

Abstract

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.

Emerging Technologies in Algal Biotechnology: Toward the Establishment of a Sustainable, Algae-Based Bioeconomy

“…Cyanobacteria, for example, have been widely used for heterologous plant-derived terpenoid engineering, extensively reviewed in Chaves and Melis (2018) and Lin and Pakrasi (2019), and proof-of-concept works have unveiled the potential of engineered eukaryotic microalgae such as C. reinhardtii in producing high value terpenoids such as the food flavoring and aromas sesquiterpenoid patchulol and (E)α-bisabolene (Lauersen et al, 2016;Wichmann et al, 2018) and diterpenoids such as casbene, taxadiene, and 13R(+)manoylnyl oxide , as well as lambdane diterpenoids (Papaefthimiou et al, 2019), which are relevant precursors of plant-derived therapeutic and cosmetic products. Similarly, the diatom P. tricornutum is currently being explored for similar applications and demonstrated its potential in producing triterpenoid lupeol and traces of betulin, precursors of the topoisomerase inhibitor betulinic acid (D'Adamo et al, 2019), commonly used in anticancer and antiviral pharmaceutical preparations and naturally produced in trace amounts from the bark of plant species, such as the white birch tree (Pisha et al, 1995).…”

Potential and Challenges of Improving Photosynthesis in Algae

“…Thus, microalgae own specific isoprenoid biosynthetic pathways that could be manipulated by genetic engineering to increase the natural capacities for the generation of isoprenoids, or to promote heterologous isoprenoid production. These class of molecules, due to their intensive color, fragrance and antioxidant properties, are in high demand for various applications in animal feed, medicines and nutraceuticals, pest control agents, cosmetics and pigments [157].…”

“…These are localized in different cell compartments but generate the same 5-C precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The MVA pathway was lost during evolution in the Chlorophyta and Eustigmatophyceae [166], while it was conserved in other microalgae, e.g., P. Tricornutum maintained the cytosolic MVA pathway, thus representing a choice species for isoprenoid engineering and heterologous terpenoid production [157,166].…”

“…In Phaeodacthylum, lupeol synthase isoforms from A. thaliana and L. japonicus were expressed to produce lupeol, an anti-inflammatory triterpene [157]. Moreover, co-expression in the diatom of CYP716A12 from M. truncatula and its corresponding reductase MtCPR, was aimed to further functionalize lupeol in betulin for the production of the antiretroviral, antimalarial agent betulinic acid [157].…”

The Synthetic Biology Toolkit for Photosynthetic Microorganisms

“…A molecular toolbox was created for diatoms using the Gateway standard (Siaut et al, 2007). More recently, D'Adamo and colleagues used the MoClo (Weber et al, 2011) and its Plant standard (Patron et al, 2015) to build plasmids later successfully used to transform P. tricornutum (D'Adamo et al, 2019).…”