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.

Keywords: algal synthetic biology; betulin; blue biotechnology; diatoms; lupeol; microalgae; natural product; triterpenoid biosynthesis.

Figure 1

Engineering Phaeodactylum tricornutum by introducing a plant sapogenin pathway. Schematic representation of sterol metabolism in diatoms starting from the precursor isopentenyl pyrophosphate (IPP) and leading to brassicasterol. In the plant sapogenin pathway, the 2,3‐oxidosqualene precursor is cyclized to lupeol, by a lupeol synthase; the lupeol can be further modified to betulinic acid through a cytochrome P450 family enzyme, along with its coenzyme NADPH reductase. The plant sapogenin pathway enzymes introduced in P. tricornutum will, therefore, compete with the native cycloartenol synthase for the common precursor 2,3‐oxidosqualene.

Figure 2

Lupeol production in Phaeodactylum tricornutum transformant lines. GC‐MS analysis of transformant lines AtLUS‐6 (yellow) and LjLUS‐25 (red) with the wild type (blue). Lupeol standard in black. 1. Brassicasterol (major sterol in P. tricornutum). 2. Lupeol; 3. 3β, 20‐dihydroxylupane by‐product of AtLUS enzyme only. Additional peaks: a. campesterol; b. unidentified diterpene molecule; *unidentified terpenoid molecules, mass spectra for these peaks with their best database hits are shown in Figure S3.

Figure 3

Expression and copy number of lupeol‐producing strains. (a) qPCR analysis of six lines showing expression of the Lotus japonicus lupeol synthase (LjLUS) mRNA relative to the geometric average of the RP3a and UBQ reference genes (b) Copy number of integrated LjLUS expression cassettes (black) and the ble ^r resistance marker (grey). Error bars represent the standard deviation from three biological replicates. Identical letters denote groups where means are not statistically different according to a post hoc Tukey test with α = 0.01.

Figure 4

Lupeol productivity in 400‐mL batch cultures. Triplicate cultures were grown in F/2 medium in laboratory‐scale Algem^® photobioreactors for 5 days. (a) Cell density and productivity per cell of brassicasterol for the wild type (diamonds) and LjLUS‐25 (squares). Lupeol was only detected in the LjLUS‐25 line (triangles). (b) Brassicasterol extracted from cell pellet of WT (diamonds) and the LjLUS‐25 line (squares). Lupeol extracted from LjLUS‐25 is shown by the triangles in the cell (full line) and the medium (dashed line). No brassicasterol was detected in the medium of either the WT or LjLUS‐25. (c) Gene expression of LjLUS and ble ^r resistance marker for LjLUS‐25 transformant line (WT as negative control) monitored during culturing time and relative to the reference genes RP3a and UBQ. Red arrows indicate the day when maximum lupeol productivity is observed corresponding to mid‐exponential growth phase as described in the text. All error bars represent the standard deviation from three biological replicates. Identical letters denote groups where means are not statistically different according to a post hoc Tukey test with α = 0.01.

Figure 5

Schematic representation of proposed model for extraction procedure with a biorefinery approach of triterpenes from Phaeodactylum tricornutum and Saccharomyces cerevisiae biomass. The extraction procedure for triterpenoids from P. tricornutum simultaneously isolates more commercially relevant coproducts compared to the yeast S. cerevisiae. Cost of extraction is estimated to be assuming equal levels of extraction efficiency. Cost of biomass for P. tricornutum is higher (produced in Europe, Necton), but market sizes of lipids omega 3 and carotenoids (unique products in microalgae) create cost offsets. References for panel A: a: *W. Yongmanitchai and O. P. Ward Growth of and Omega‐3 Fatty Acid Production by Phaeodactylum tricornutum under Different Culture Conditions Appl. Env. Micro., Feb. 1991, p. 419–425 0099‐2240/91/020419‐07$02.00/0, b: Yu‐Hong Yang, Lei Du, Masashi Hosokawa, Kazuo Miyashita, Yume Kokubun, Hisayoshi Arai and Hiroyuki Taroda. Fatty Acid and Lipid Class Composition of the Microalga Phaeodactylum tricornutum J. Oleo Sci. 66, (4) 363–368 (2017) https://doi.org/10.5650/jos.ess16205, c: this paper, d: S. M. Tibbetts, J. E. Milley, P. Santosh and J. Lall Chemical composition and nutritional properties of freshwater and marine microalgal biomass cultured in photobioreactors J Appl Phycol (2015) 27:1109–1119. References for panel B: e: International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol‐2, Issue‐2, Mar–Apr 2017 https://doi.org/10.22161/ijeab/2.2.2 ISSN: 2456–1878 Page | 558, f: M. Lamac Ka and J. S Ajbidor Ergosterol determination in Saccharomyces cerevisiae. Comparison of different methods. Biotech. Tech. 11, 723–725 (1997), g: E. Czarnotta, M. Dianat, M. Korf, F. Granica, J. Merz, J. Maury, S. A. Baallal Jacobsen, J. Förster, B. E. Ebert, and L. M. Blank. Fermentation and purification strategies for the production of betulinic acid and its lupane‐ type precursors in Saccharomyces cerevisiae. Biotech. Bioeng. 114, 2528–2538 (2017) https://doi.org/10.1002/bit.26377

Figure 6

mRNA expression of sterol biosynthesis enzymes during a five‐day time course in the wild type (WT) and the LjLUS‐25 line. Schematic representation of sterol metabolism in Phaeodactylum tricornutum, indicating the gene selected for mRNA expression analysis and relative heat map for WT and LjLUS‐25 line. Values are the expression relative to the geometric average of the RP3a and UBQ reference genes. Transcripts not significantly different between wt and LjLUS are indicated in grey. Enzymes involved in the cytosolic MVA pathway: HMGS: hydroxymethylglutaryl‐CoA synthase; HMGR: 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase; IDI‐SQS: isopentenyl diphosphate isomerase‐squalene synthase. Enzymes involved in brassicasterol biosynthesis: PtOSC: oxidosqualene cyclase, methylsterol monooxygenase, sterol dehydrogenase, C24 sterol reductase, C22 sterol desaturase. Enzymes involved in the plastidial MEP pathway: ISPD2: 2‐C‐methyl‐d‐erythritol 4‐phosphate cytidylyltransferase, ISP‐E: 4‐diphosphocytidyl‐2‐C‐methyl‐d‐erythritol kinase. In the yellow box, the non‐native lupeol synthase from Lotus japonicus, introduced in our engineered strain LjLUS25. Dashed lines indicate multiple reactions. Gene accession numbers are mentioned in the results section. Precursor abbreviations: IPP: Isopentyl pyrophosphate, MEP: 2‐C‐methyl‐d‐erythritol 4‐phosphate, CDP‐ME: 4‐diphosphocytidyl‐2‐c‐methylerythritol, CDP‐MEP: 4‐diphosphocytidyl‐2‐c‐methyl‐d‐erythritol 2‐phosphate, DMAP: dimethylallyl pyrophosphate, G3P: glycerol 3‐phosphate, Pyr: pyruvate.

Figure 7

Betulin production in Phaeodactylum tricornutum transformant lines. (a) GC‐MS analysis of WT (blue), LjLUS‐25 containing only the Lotus japonicus lupeol synthase (LjLUS) (red); transformant line with constructs harbouring L. japonicus lupeol synthase (LjLUS), Medicago truncatula cytochrome P450 (MtCYP716A12) and cytochrome P450 reductase (MtCPR) in WT background (purple); transformant line with construct harbouring fused P450‐reductase protein MtCYP716A12λΔ72CPR in LjLUS‐25 background (light blue); standards (black). Peak as indicated 1. Brassicasterol; 2. Lupeol; 3. Betulin. (b) Detail of CYP716A enzyme reaction. The oxidation at C28 leads to betulin intermediate, which is converted to betulin aldehyde, and betulinic acid.